THIRD&WORLD&CONGRESS&OF CHRONOBIOLOGY& ABSTRACTS.pdf · 2012. 3. 3. · 3" & & PRESIDENTMESSAGE& Chronobiology"has"become"awellZrecognized"discipline"in"contemporary"biomedical"research."The"

1

WFSC LAGC - SMRB

THIRD WORLD CONGRESS OF CHRONOBIOLOGY

Program and Abstracts Puebla, México. May 5 to 9, 2011

2

WE THANK OUR SPONSORS

Universidad Nacional Autónoma de México Coordinación de la Investigación científica

Instituto de Fisiología Celular Instituto de Neurobiología Instituto de Investigaciones Biomédicas

Facultad De Ciencias

DGAPA, PAPIIT IN 204811

Benemérita Universidad Autónoma de Puebla

Universidad Autónoma de Nuevo León

Universidad Autónoma Metropolitana

Universidad Veracruzana

Consejo Nacional de Ciencia y Tecnología Fondo de Cooperación Internacional en Ciencia y Tecnología, financiado por Unión Europea y CONACyT

Instituto de Ciencia y Tecnología del D.F. International Brain Research Organization

Latin America Regional Committee.

Alta Tecnología en Laboratorio s (Olympus)

Japanese Society for Chronobiology

Anonimous Sponsor

3

PRESIDENT MESSAGE

Chronobiology has become a well-‐recognized discipline in contemporary biomedical research. The study of biological rhythms, the mechanisms supporting them and their relevance for our daily life is now widespread at universities and clinical institutions worldwide. More and more students interested in scientific topics, are now incorporated into laboratories aimed at characterizing the molecular and physiological mechanisms that underlie the function of biological clocks. Increasingly more scientists are involved in establishing the relevance of chronobiological studies for preserving the environment and improving human health and life quality. It is in this context that we welcome our colleagues and friends to the Third World Congress in Chronobiology (3rd WCC) that takes place in the city of Puebla, Mexico.

Puebla is a city that has played an outstanding role in the history of Mexico. It was founded by the Spaniards on April 16th in 1531, only a few years after their arrival to Mexico, as a transit post to communicate Mexico City (the Capital) with the port of Veracruz in the Gulf of Mexico. Puebla is the city where an invading army was defeated in May 5 1867, and where the Mexican revolution started in 1910. Puebla was a village where native Mexicans and Spaniards learned to peacefully live together and developed a cultural mixture known as “mestizaje”. Such is the foundation from which our nation arose with its characteristic idiosyncrasy. On this occasion our meeting makes, again, Puebla a melting pot as scientists from different countries, with diverse cultural and ethnical backgrounds, but with the common aim of gaining knowledge from each other, will share experiences and, thus, strengthen our scientific community.

This 3rd WCC is organized by the Latin American Group of Chronobiology and the Mexican Society of Biological Rhythms on behalf of the World Federation of Societies on Chronobiology. The Latin American Group of Chronobiology was initiated in 1991 through the initiative of an enthusiastic group of chronobiologists from the University of Sao Paulo, Brazil, that invited colleagues from Argentina and Mexico to hold the First Latin American Symposium on Chronobiology (LASC). Since then the LASC has been held every other year and has grown to include colleagues not only from Latin America such as Chile, Cuba and Uruguay, but also from Canada, France, Germany, Japan, Spain, the United Kingdom and USA. This year the XI LASC was held just before the 3rd WCC in the nearby city of Cholula. For chronobiologists in Mexico, the challenge of hosting the 3WCC has been critical to fostering the formalization of the Mexican Society for Biological Rhythms (SMRB), which held its first meeting in August 4 to 6, 2010 in Juriquilla, Queretaro.

Colleagues and friends from all over the world: you are welcome to Mexico. The organizing committee wishes that the 3rd WCC fulfills your expectations of scientific exchange and networking.

Dr. Raúl Aguilar-‐Roblero President of the Organizing Committee Third World Congress of Chronobiology

4

5

Table of Contents

General Information

Organizing committee 7

Maps 8

Emergency phone numbers 11

Registration 13

Symposia and Plenary Lectures 13

Poster Presentations 14

Social Program 15

Program May 6 19

May 7 22

May 8 25

May 9 28

Poster program May 6 33

May 7 37

May 8 41

Abstracts Lectures 45

Symposia 49

Poster 121

Indexes Author 239

Keyword 245

6

7

General Information

Organizing Committee Raúl Aguilar-‐Roblero. Universidad Nacional Autónoma de México, MÉXICO. President

International Program Committee

John Araujo. Universidade Federal do Rio Grande du Norte, BRAZIL. Israel Ashkenazi. Sackler School of Medicine, ISRAEL. Jose Ramón Eguibar. Benemerita Universidad Autónoma de Puebla, MÉXICO. Russell Foster. University of Oxford, UK. Diego Golombek. Universidad Nacional de Quilmes, ARGENTINA. Mario Guido. Centro de Investigación en Química Biológica de Córdoba, ARGENTINA. Ken-‐ichi Honma. Hokaido University, Graduate School of Medicine, JAPAN. Takao Kondo. Nagoya University, JAPAN. Mirian Marques. Universidade de São Paulo, BRAZIL. Luiz Menna-‐Barreto. Universidade de São Paulo, BRAZIL. Paul Pevet. University of Strasbourg, FRANCE. Francesco Portaluppi. University Hospital of Ferrara, ITALY. William Schwartz. University of Massachusetts, Medical School, USA. Shigenobu Shibata. Waseda University, JAPAN. Rae Silver. Columbia University, USA.

Local Organizing Committee

Mario Caba. Universidad Veracruzana. Ivette Caldelas. Universidad Nacional Autónoma de México. Mauricio Díaz-‐Muñoz. Universidad Nacional Autónoma de México. Porfirio Carrillo. Universidad Veracruzana. Ma. Luisa Fanjul. Universidad Nacional Autónoma de México. Pablo Valdez. Universidad Autónoma de Nuevo León. Javier Velázquez. Universidad Autónoma Metropolitana. María del Carmen Cortés. Benemérita Universidad Autónoma de Puebla. Candelaria Ramírez. Universidad Autónoma de Nuevo León. Arturo Vega-‐González. Universidad de Guanajuato.

8

9

10

Arriving to the opening ceremony and welcome cocktail from down

town hotels

11

BUAP CULTURAL COMPLEX

1 Auditorium

2 Theater 3 South Building 2

4 North Building 2

5 South Building 1

6 North Building 1 7 Parking

8 School of Communication Sciences

9 Cultural Walkway

10 School of Arts 11 Cumulo de Virgo Roads

TERRACE

MAIN HALL POSTER HALL

VIRTUAL ROOM

12

Emergency Phone Numbers Fire Department Central 245-‐8001 245-‐7392 245-‐6965 245-‐7799 Red Cross (motor vehicle accidents) 234-‐0000 213-‐7704 213-‐7703 Transit Office 228-‐7768 228-‐7722 228-‐7769 Police 288-‐1863 288-‐1884

County Medical Emergencies 066 HOSPITALS: Betania 213-‐0524 Beneficencia Española de Puebla 242-‐4958 229-‐3700 Puebla 594-‐0600 Ángeles 303-‐6600 303-‐8300 303-‐ 2068

13

14

REGISTRATION

Registration Desk

The Registration Desk will be set up on the lobby of the Hotel “San Leonardo” (May 5th) and the entrance of the Seminary Hall (May 6 to 9) in the 1st floor of the BUAP Cultural Complex.

Opening Hours:

May 5 (Thu) 15:00-‐19:00 May 6 (Fri) 8:00-‐18:00 May 7 (Sat) 8:00-‐18:00 May 8 (Sun) 8:00-‐18:00 May 9 (Mon) 8:00-‐14:00

Credit Cards and Exchange

Only Mexican currency and credit cards (VISA, MasterCard, American Express) will be accepted.

Congress Kit

A Congress Kit will be handed to participants at the Registration Desk.

Contents: Program/Abstracts Badge Receipt Certificate

Badges

Participants must wear their badges at all times while at the Congress venue.

Banquet Tickets

Tickets for the banquet (May 9th 20:00 h) can be purchased at the Registration Desk until May 7th at 17:00 h. The banquet admission fee is 50 USD.

Tour Desk

The Tour Desk will be set up next to the Registration Desk, where travel agency staff will be stationed to welcome you.

ABOUT SYMPOSIA AND PLENARY LECTURES

The chairpersons and speakers should report at the assigned conference room 20 minutes prior to the beginning of each session.

1. Simposium presentations and time for debate

Speakers, please follow chairpersons' directions regarding timing.

2. Receipt of presentations

PCs (OS: Windows7, PowerPoint 2007) will be used for the presentations. Slide and video presentations will not be possible. The reception desk at each conference room will accept your data, and your presentation can be previewed there. (Macintosh users should bring their own PCs).

15

File names must include the number of the presentation, a space and your name in that order.

e.g. "S-‐1-‐1 Rae Silver"

Since the PCs may have problems with moving images or particular animated cartoons, please use your own PC for your presentation. All look-‐up files should be kept'in the same folders as the files in the PowerPoint to be presented.

3. Speaker desk

You may preview your presentation at the speakers' Desk, located at the Poster Hall (May 6 – 8: 8:00-‐17:00 h; May 9: 8:00-‐11:00).

Please note that Macintosh users should bring their own PCs. In addition, please bring your own associated display-‐conversion cable. Make sure in advance that the stored data opens without any garbled characters when opened on a PC other than your own.

7) A resolution of XGA (1024X768) can be achieved. The only equipment provided for presentations will be a single-‐screened PC projector.

ABOUT POSTER PRESENTATIONS

Usable area: 90 cm (width) x 120 cm (height).

Presenters should mount their posters the designated day of presentation (either May 6, 7 or 8) from 8:00 to 10:30 h and should be removed at 19:30 h.

Use the designated board indicated by poster numbers placed on the top left corner of the panel. Use double side adhesive tape or masking-‐tape. These will be provided at the entrance of the Posters Hall

Please note that posters that have not been removed by the end of the removal time will be removed and disposed of by the staff.

Poster Discussion

Authors are requested to stand in front of their panels between 17:00 and 19:00 of their designated day of presentation. Discussion of posters during the coffee breaks and lunch times are encouraged.

16

17

SOCIAL PROGRAM

Get-‐Together party (Free)

May 5 (Thu), 19:30-‐21:00, Carolino Building, Downtown Puebla.

AlI participants are welcome to attend.

Banquet (50 USD)

May 9 (Mon), 20:00-‐23:00, the location will be announced.

WFSC Council Bussines Meeting

May 7 (Sat) 18:00-‐19:00 h: Virtual room

18

19

Program

20

21

May 6 (Fri)

Main Hall

8:30 -‐ 10:30

Symposium 1. Molecular and Network Properties of the Suprachiasmatic Nucleus. Chairpersons: Sato Honma (Japan) and Rae Silver (USA).

S1.1 Cellular rhythms and neural networks in the mouse suprachiasmatic nucleus. Sato Honma.

S1.2 Phosphatase in suprachiamatic nucleus, PHLPP1/SCOP, controls period change after light-‐induced phase shift, ‘After-‐Effect of Phase-‐Shift’. Satoru Masubuchi.

S1.3 Explorations of the circuit structure of the SCN. Rae Silver.

S1.4 Regional period difference that generates a phase gradient in the mammalian circadian center. Yasufumi Shigeyoshi.

S1.5 From Nodes to Networks in the Mammalian Circadian System. Erik D. Herzog.

Movie Theater

8:30 -‐ 10:30

Symposium 2. New insights in the circadian mechanisms regulating food anticipation. Chairperson: Etienne Challet (France).

S2.1 Circadian clocks for all mealtimes: Anticipation of multiple daily meals in rats and mice. Ralph Mistlberger.

S2.2 It is possible to anticipate to pheromones? A study in newborn rabbits fed by enteral nutrition. Ivette Caldelas.

S2.3 Relationship between the Food-‐Entrainable Oscillator (FEO) and Methamphetamine Sensitive Circadian Oscillator (MASCO). Michael Menaker.

S2.4 Food entrainment of peripheral clocks evaluated by in vivo imaging. Yu Tahara.

Virtual Room

8:30 -‐ 10:30

Symposium 3. Role of circadian clocks in fitness and adaptation. Chairperson: Amita Shegal (USA).

S3.1 Circadian clocks support health by orchestrating removal of oxidative damage. Jaga Giebultowicz.

S3.3 Brood-‐related plasticity in circadian rhythms of bumble bee queens. Guy Bloch.

22

S3.2 The SCN neuronal network and its adaptation to temporal challenges. Horacio de la Iglesia.

S3.4. Diurnal activity in a small desert rodent: mechanisms, adaptations and constraints. Noga Kronfeld-‐Schor.

10:30 -‐ 11:00 Coffee Break (Terrace).

Main Hall 11:00 -‐ 11:30

Historical Lecture. Localization of Function: A Brief History of the Suprachiasmatic

Nucleus. Robert Y. Moore.

11:30 -‐ 12:30

Plenary Lecture. Intramolecular feedback of KaiC ATPase is the basic pacemaker of

circadian clock in cyanobacteria. Takao Kondo.

12:30 -‐ 15:00 Lunch.

Virtual Room

15:00 -‐ 17:00

Symposium 4. Molecular and cellular mechanisms of the Drosophila circadian system. Chairpersons: Norio Ishida and Kenji Tomioka (Japan).

S4.1 NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator. Paul Hardin.

S4.2 Circadian rhythm of Drosophila behavior; different brain-‐sites required for locomotor and courtship. Norio Ishida.

S4.3 A possible synaptic transmission involved in the Drosophila circadian clock. Kenji Tomioka.

S4.4 The consequence of arrhythmicity in the German cockroach. How-‐Jing Lee.

Movie Theater

15:00 -‐ 17:00

Symposium 5. Nonphotic Entrainment in Mammals. Chairperson: Roberto Refinetti (USA).

S5.1 On the Social Regulation of Circadian Timekeeping. William J. Schwartz.

23

S5.2 Entrainment by food restriction. Jorge Mendoza.

S5.3 Entrainment of circadian rhythms by cycles of ambient temperature in mammals. Roberto Refinetti.

S5.4 BRAIN REWARD: STIMULATION FOR NONPHOTIC PHASE REGULATION OR JUST A GOOD TIME? J. David Glass.

Main Hall

15:00 -‐ 17:00

Symposium 6. A time to heal: cross-‐talk between the immune and the circadian systems. Chairperson: Ruud Buijs (México).

S6.1 Photoperiodic control of innate and adaptive immunity. Brian Pendergast.

S6.2 Dysregulation of Inflammatory Responses by Chronic Circadian Disruption. Alec Davidson.

S6.3 Laborious Phase Shifting (LPS): following the pathways of immune-‐circadian communication. Diego Golombek.

S6.4 The circadian clock controls the response of T cells to antigen.Nicolas Cermakian.

S6.5 Time of infection determines the response of the innate and adaptive immune system. Natali Nadia Guerrero.

17:00 -‐ 19:00 Poster Session

24

May 7 (Sat)

Movie Theater

8:30 -‐ 10:30

Symposium 7. Neuropeptides and Circadian Rhythms. Chairperson: Hugh Piggins (UK).

S7.1 Role of the neuropeptide VIP in the mammalian circadian system. Chris Colwell.

S7.2 Neuropeptide modulation of the intracellular calcium concentration of suprachiasmatic nucleus neurons. Charles Allen.

S7.3 Heterogeneous and novel electrophysiological actions of orexin-‐A on circadian clock neurons in mice. Mino Belle.

S7.4 Neuropeptides derived from clock-‐controlled genes as mediating signals for the output of suprachiasmatic clock. Qun-‐Yong Zhou.

Virtual Room

8:30 -‐ 10:30

Symposium 8. Entrainment of the Circadian Clocks during Development. Chairperson: Alena Sumova (Czech Republic).

S8.1 Entrainment of the circadian clocks along ontogenesis. Alena Sumova.

S8.2 Maternal melatonin, a chronobiotic for fetal circadian clocks. Maria Seron-‐Ferre.

S8.3 The developing pacemaker: sensitive but resilient. Fred Davis.

S8.4 The rabbit pup, a natural model of food entrainment. Mario Caba.

Main Hall

8:30 -‐ 10:30

Symposium 9. Clock control of glucose metabolism. Chairperson: Andries Kalsbeek (The Netherlands).

S9.1 Circadian system and its disruption; effects on glucose metabolism in humans. Frank Scheer.

S9.2 The metabolic clockwork. Akhilesh Reddy.

25

S9.3 Circadian clocks in adipose tissue. Jeffrey Gimble.

S9.4 Hypothalamic neuropeptides involved in the SCN control of hepatic glucose production. Andries Kalsbeek.


Main Hall 11:00 -‐ 11:30

Historical Lecture. History of Chronobiological Societies‐Chronobiologists are always

looking for the best friend. Ken-‐ichi Honma.

11:30 -‐ 12:30

Plenary Lecture. Physiological and metabolic adaptations associated to daytime food

synchronization in rat. Mauricio Díaz-‐Muñoz.

12:30 -‐ 15:00 Lunch.

Virtual Room

15:00 -‐ 17:00

Symposium 10. Retina and Peripheral Clocks. Chairpersons: Mario Eduardo Guido (Argentina) and Ana Maria Castrucci (Brazil).

S10.1 Light responses in peripheral tissues: how do things change following evolution in perpetual darkness? David Whitmore.

S10.2 Early-‐stage retinal melatonin synthesis impairment in streptozotocin-‐induced diabetic Wistar rats. Daniella C. Buonfiglio.

S10.3 Circadian Oscillators in Retinal Ganglion Cells. Light and Dopamine Regulation and Intrinsic Photoreceptive Capacity. Mario Eduardo Guido.

S10.4 Fish, amphibian and avian cells as peripheral clocks: a comparative approach to study melanopsin signaling and regulation of clock genes. Ana Maria de Lauro Castrucci.

Main Hall

15:00 -‐ 17:00

Symposium 11. Seasonal timing of Reproduction in Vertebrates. Chairperson: Valerie Simonneaux (France).

S11.1 Tanycytes and RFamides, the new players in seasonal reproduction. Paul Klosen.

26

S11.2 CNS Sites of Melatonin Action for Reproductive and Body Fat Responses in Siberian Hamsters. Timothy J. Bartness.

S11.3 Acute Induction of Eya3 by late-‐night light stimulation triggers Tshβ expression in photoperiodism. Hiroki R. Ueda.

S11.4 Photoperiod and the male effect can be used to control the reproductive activity in subtropical goats. Jose Alberto Delgadillo.

Movie Theater

15:00 -‐ 17:00

Symposium 12. The pathology of desynchronization. Chairperson: Roberto Salgado-‐Delgado (México).

S12.1 Desynchrony as a tool to investigate the role of the human circadian system in physiology and pathophysiology. Frank Scheer.

S12.2 Desynchronized Circadian Rhythms: Bow to the Master. Horacio de la Iglesia.

S12.3 Food as chronotherapy to ameliorate the adaptation to a new time zone. Manuel Angeles-‐Castellanos.

S12.4 Nightwork leads to obesity and diabetes: a rat model of nightwork uncovers internal desynchrony at the level of the hypothalamus and within the liver. Roberto Salgado-‐Delgado.


Virtual Room

18:00 -‐ 19:00 WFSC Business Meeting

27

May 8 (Sun)

Movie Theater

8:30 -‐ 10:30

Symposium 13. Physiological correlates and mechanisms of circadian brain oscillators beyond the SCN. Chairperson: Oscar Castanon (EUA).

S13.1 Daily rhythms in olfactory discrimination depend on clock genes, but not the suprachiasmatic nucleus. Daniel Granados.

S13.2 The cerebellum; a circadian oscillator synchronized by food. Jorge Mendoza.

S13.3 Dopaminergic modulation of clock mechanisms. Suzanne Hood.

S13.4 Putative circadian oscillators in the epithalamus and hypothalamus. Hugh Piggins.

Main Hall

8:30 -‐ 10:30

Symposium 14. The Importance of Being Entrained. Chairperson: Martha Merrow (The Netherlands).

S14.1 The consequences of dys-‐entrainment. Till Roenneberg.

S14.2 On the evolution of Drosophila blue light photoreceptor CRYPTOCHROME and its relation to the visual system. Gabriella M. Mazzotta.

S14.3 The early worm catches the light (and the heat): circadian entrainment in C. elegans. Diego Golombek.

S14.4 Protein phosphatase 1 (PP1) regulates period length and phase resetting of the mammalian circadian clock. Urs Albrecht.

Virtual Room

8:30 -‐ 10:30

Symposium 15.The mammalian circadian timing system: Hormonal key mechanisms involved in organization and coordination of central and peripheral clocks. Chairperson: Paul Pévet (France).

S15.1 HSD3B1: a new enzyme linking circadian clock and hypertension. Hitoshi Okamura.

28

S15.2 Physiology of the adrenal circadian clock. Henrik Oster.

S15.3 Glucocorticoid regulation of clock gene expression in the mammalian brain. Lauren Segall.

S15.4 Melatonin, an endocrine output of the central clock involved in the regulation of human circadian rhythms. Bruno Claustrat.


Main Hall

11:00 -‐ 11:30

Historical Lecture. Persistent, Endogenous, Innate, Precise: On the early history of some

key concepts in circadian rhythms. Serge Dann.

11:30 -‐ 12:30

Plenary Lecture. Circadian control of output in Drosophila.

Fernanda Ceriani.

12:30 -‐ 15:00 Lunch.

Main Hall

15:00 -‐ 17:00

Symposium 16. Circadian regulation of behaviors in mammals. Chairpersons: Ken-‐ichi Honma (Japan) and Horacio de la Iglesia (USA).

S16.2 The master and slave oscillators for behavioral rhythms in mice. Yujiro Yamanaka.

S16.3 A functional property of circadian pacemakers which control behavioral rhythms. Wataru Nakamura.

S16.4 Temporal nice switching in the house mouse: why and how? Roelof A. Hut.

S16.5 The role of the habenula in the regulation of locomotor activity. Mathew J. Paul.

Movie Theater

15:00 -‐ 17:00

Symposium 17. Nutrients and palatable snacks the dissection of brain and peripheral oscillators. Chairperson: Carolina Escobar (México).

S17.1 DMH-‐SCN interaction permits food anticipatory behavior during the rest phase. Ruud Buijs.

29

S17.2 Role of food components in entrainment of mouse liver clock. Shigenobu Shibata.

S17.3 “Sweet transition” of neuronal activity induced by entrainment on palatable food. Elena Timofeeva.

S17.4 Gut peptides are not necessary for food anticipatory activity induced by a highly palatable meal.Megan Dailey.

Virtual Room

15:00 -‐ 17:00

Symposium 18. Roles for Glial Cells in the Circadian Neural Circuitry. Chairperson: Rob Jackson (USA).

S18.1 Circadian Rhythms in Glial Calcium Signaling Mediate Rhythmic Extracellular ATP Accumulation in the Suprachiasmatic Nucleus. Mark Zoran.

S18.2 Astrocyte-‐astrocyte communication and connexin 43 regulate mammalian circadian rhythms. Luiciano Marpegan.

S18.3 Adult glial cells modulate the circadian neuronal circuit. Rob Jackson.

S18.4 The involvement of glia in the circadian regulation of neuronal morphology. Elżbieta Pyza.


30

May 9 (Mon)

Virtual Room

8:30 -‐ 10:30

Symposium 19. Circadian regulation of sensory processing. Chairperson: Erik Herzog (USA).

S19.1 Circadian regulation of sensory processing. Erik Herzog.

S19.2 Circadian Regulation of Olfactory Sensitivity and Olfactory Behavior in the Cockroach, Leucophaea maderae. Terry Page.

S19.3 Circadian rhythm in olfaction in a subterranean organism. Martha Merrow.

S19.4 Circadian gating of photic inputs in plants. Andrew Millar.

S19.5 Circadian Modulation of Simple and Selective Attention in Humans. Ken Wright.

Movie Theater

8:30 -‐ 10:30

Symposium 20. Quantitative analysis of rhythmic data. Chairperson: Mary Harrington (USA).

S20.1 Tracking phase in a network of heterogeneous SCN cells. Stephanie Taylor.

S20.2 Applying Wavelet Transforms to Circadian Data. Tanya Leise.

S20.3 New methods of analysis for Chronobiology: complexity, fractals, dimensions, recurrence plots. Díez-‐Noguera.

S20.4 Modeling entrainment of the mammalian circadian clock. Adrián Granada.

Main Hall

8:30 -‐ 10:30

Symposium 21. Inter-‐Individual Variability in Circadian rhythms and Sleep homeostasis. Chairperson: Antoine Viola (Switzerland).

S21.1 Effects of PERIOD3 polymorphism on circadian rhythmicity and sleep homeostasis in healthy older individuals. Antoine Viola.

S21.2 Involvement of clock genes in sleep homeostasis in mice. Valerie Mongrain.

31

S21.3 Functional genetic polymorphisms of BDNF and ADA modulate sleep and neurobehavioral performance during. Valérie Bachmann.

S21.4 Interaction of non-‐circadian effects of light with circadian rhythm and sleep homeostasis. Patrice Bourgin


Main Hall

11:00 -‐ 11:30

Historical Lecture. Serendipity or "the prepared mind": the true history of some

discoveries in the Menaker lab. Michael Menaker.

11:30 -‐ 12:30

Plenary Lecture. Sociobiology of human timing.

Till Roenneberg.

20:00 -‐ 23:00 Closing Dinner

32

33

Poster program May 6 – 8

17:00 – 19:00

34

35

May 6 P001 WINTER LIGHTING CONDITIONS ACCELERATE RE-‐ENTRAINMENT TO SIMULATED TIME

ZONE TRAVEL IN HAMSTERS Elizabeth M. Harrison, Michael R. Gorman.

P002 AFTEREFFECTS OF ENTRAINMENT IN THE FIELD PROVIDE NEW INSIGHTS INTO THE SWITCH FROM DIURNALITY TO NOCTURNALITY IN THE SUBTERRANEAN RODENT TUCO-‐TUCO (Ctenomys Cf. Knight) Barbara Mizumo Tomotani, Danilo Eugênio de França Laurindo Flôres, Patricia Tachinardi, José Demetrio Paliza, Gisele Akemi Oda, Verónica Sandra Valentinuzzi.

P003 OVERWEIGHT REDUCES ENTRAIMENT EFICIENCY OF LOCOMOTOR ACTIVITY IN THE VOLCANO MOUSE NEOTOMODON ALSTONI Vania Patricia Carmona Alcocer, Manuel Miranda Anaya.

P004 WAVELENGTH DEPENDENCE FOR EFFECTS OF DIM SCOTOPHASE ILLUMINATION ON CIRCADIAN WAVEFORM AND REENTRAINMENT IN SIBERIAN HAMSTERS Michael R. Gorman, Jeffry A. Elliott.

P005 SHORT-‐DAY RESPONSE IN DJUNGARIAN HAMSTERS DISPLAYING DIFFERENT CIRCADIAN PHENOTYPES Dietmar Weinert, Konrad Schöttner, Maren Schmidt.

P006 REORGANIZATION OF THE MAMMALIAN PACEMAKER UNDER ULTRA LONG DAY LENGTHS PROVIDES AN ASSAY FOR OSCILLATOR COUPLING Jennifer A. Evans, Tanya L. Leise, Oscar Castanon-‐Cervantes, Alec J. Davidson.

P007 LOCOMOTOR ACTIVITY AND SLEEP RHYTHMS IN THE MONGOLIAN GERBIL MERIONES UNGUICULATUS ARE DIFFERENTIALLY AFFECTED BY SHORT AND LONG PHOTOPERIOD Cinthia Rocío Juárez-‐Tapia, Pilar Duran, Manuel Miranda-‐Anaya.

P008 PHOTOPERIODIC WAVEFORM AND AMPLITUDE REGULATION OF MAMMALIAN PHASE RESPONSE CURVES Jeffrey A Elliott, Michael R Gorman.

P009 EFFECT OF DIFFERENT PHOTOPERIODS ON THE RHYTHMIC INDUCTION OF HYPOXIA-‐INDUCIBLE FACTOR-‐1 AND HEAT SHOCK PATHWAY Rosa María Velázquez-‐Amado, Elsa G. Escamilla-‐Chimal, Ana Gabriela García-‐Tecpan, María Luisa Fanjul-‐Moles.

P010 PERIOD 1 GENE EXPRESSION IN THE BRAIN OF A DUAL PHASING RODENT, THE OCTODON DEGUS Beatriz B Otalora, Megan H Hagenauer, Maria Angeles Rol, Juan Antonio Madrid, Theresa M Lee.

P011 LIGTH/DARK CYCLE AND CLOCK GENES Mario Pedrazzoli.

36

P012 EFFECT OF EXPERIMENTAL GLAUCOMA ON THE NON-‐IMAGE FORMING VISUAL SYSTEM Nuria M. A. de Zavalía, Santiago A Plano, Diego C Fernandez, María Florencia Lanzani, Ezequiel Salido, Nicolás A Belforte, María Inés Keller Sarmiento, Diego A Golombek, Ruth E Rosenstein.

P013 PINEAL CHRONOBIOLOGY, SUNSPOTS AND GEOMAGNETIC STORMS José Luis Bardasano Rubio, José Luis Aldeanueva Aguirre, José Luis Arranz, Juan Álvarez-‐U de la Torre, Miguel Raposo Sánchez.

P014 POSTEMBRYONIC MATURATION OF THE DIURNAL RHYTHM OF PINEAL MELATONIN BIOSYNTHESIS PATHWAY IS SEASON-‐DEPENDENT IN CHICKEN KEPT IN CONTROLLED LABORATORY CONDITIONS Aneta Piesiewicz, Urszula Kedzierska, Krystyna Zuzewicz, Krystyna Skwarlo-‐Sonta, Pawel Marek Majewski.

P015 THE DAILY MELATONIN PATTERN IN DJUNGARIAN HAMSTERS IS DEPENDING ON THE CIRCADIAN PHENOTYPE Dietmar Weinert, Konrad Schöttner, Valérie Simonneaux, Patrick Vuillez, Stephan Steinlechner, Paul Pévet.

P016 ENVIRONMENTAL FACTORS AFFECTING THE ROOSTING BEHAVIOR OF INDIAN CLIFF SWALLOW HIRUNDO FLUVICOLA Arati Singh, Shrutika Kankariya, Arti Parganiha, A. K. Pati.

P017 ROOSTING BEHAVIOR OF INDIAN HOUSE SWIFT, APUS AFFINIS WITH REFERENCE TO ENVIRONMENTAL FACTORS Shrutika Kankariya, Arati Singh, A. K. Pati, Arti Parganiha.

P018 THE CIRCADIAN BODY TEMPERATURE RHYTHMS OF DJUNGARIAN HAMSTERS REVEAL DIFFERENT CIRCADIAN PHENOTYPES Dietmar Weinert, Konrad Schöttner, Jim Waterhouse.

P019 MOTOR ACTIVITY OF MANATEE (Trichechus manatus manatus) IN SEMI-‐CAPTIVITY CONDITIONS: PRELIMINARY RESULTS Victoria Eugenia Holguín, Victor Manuel Alcaraz, Jairo Ignacio Muñoz.

P020 YAWNING AND PENILE ERECTION OSCILLATES ALONG THE CIRCADIAN CYCLE IN THE HY SUBLINE José R. Eguíbar, Ma. Del Carmen Cortés, Araceli Ugarte.

P021 CRONOECOLOGY OF REST-‐ACTIVITY RHYTHM AND THE BEHAVIORS OF THE SPIDER MONKEY Ateles geoffroyi IN SEMI-‐LIBERTY: A ELECTRIFIED ENCLOSURE José Carlos Sánchez-‐Ferrer, Domingo Canales-‐Espinoza, Ana María Santillán-‐Doherty, Jairo Muñoz-‐Delgado.

P022 CIRCADIAN VARIATION IN SHORT -‐TIME ESTIMATION AND THYROID HORMONES LEVELS IN YOUNG HUMANS DURING 30 H CONSTANT ROUTINE Babita Pande, Atanu Kumar Pati, Arti Parganiha, Pradeep Kumar Patra.

37

P023 THE CIRCADIAN SYSTEM INLFUENCES SHORT-‐TIME PERCEPTION Patricia V. Agostino, Ivana L. Bussi, Micaela do Nascimento, Diego A. Golombek.

P024 DIURNAL VARIATIONS IN THE LEARNING OF A MOTOR SKILL Alejandra Galeana Garcia, Georgina Almeida Rosas, Maribel Delgado Herrera, Raúl Aguilar Roblero, Alejandra Ruiz Contreras.

P025 CIRCADIAN VARIATIONS IN COGNITIVE INHIBITION AND FLEXIBILITY Benito Martínez, Aída García, Javier Talamantes, Candelaria Ramírez, Pablo Valdez.

P026 CIRCADIAN VARIATIONS IN ALERTNESS Javier Talamantes López, Aida García García, Candelaria Ramírez Tule, Pablo Valdez Ramírez.

P027 THE VALUE OF MONITORING AMBULATORY BLOOD PRESSURE IN OLD PEOPLE Zi Yan Zhao, Zhi Hui Yan, Yue Rong Fu.

P028 ASSOCIATION OF CIRCADIAN ACTIVITIES, SLEEP, AND QUALITY OF LIFE IN CHINESE CENTENARIANS Zi Yan Zhao, Zhi Hui Yan, Yue Rong Fu, Feng Zhang.

P029 SLEEP-‐WAKE CYCLE AND ACTIVITY-‐REST CYCLE IN ELDERLY PEOPLE Anahí Flores, Xóchitl Angélica Ortiz, Minerva Aída García, Candelaria Ramírez, Pablo Valdez.

P030 SERUM FACTORS CHANGE CELLULAR CLOCK PROPERTIES IN OLDER INDIVIDUALS Lucia Pagani, Karen Schmitt, Fides Meier, Jan Izakovic, Ermanno Morriggi, Konstanze Romer, Antoine Viola, Christian Cajochen, Anna Wirz-‐Justice, Anne Eckert, Steven A. Brown.

P031 CIRCADIAN RHYTHMS AND TIME ESTIMATION IN HEALTHY YOUNG AND ELDERLY HUMANS Mario Leocadio Miguel, Luiz Menna-‐Barreto.

P032 CIRCADIAN BLOOD PRESSURE VARIABILITY AMONG SOUTHEAST INDIAN POPULATION AS FUNCTION OF AGE, GENDER AND DIPPING PATTERN Nishtha Vaidya, Atanu Kumar Pati, Arti Parganiha.

P033 EFFECTS OF A 30-‐MINUTES ADVANCE IN SCHOOL SCHEDULE ON SUSTAINED ATTENTION IN CHILDREN Juventino Cortez, Diana Juárez, Jorge Borrani, Aida García Candelaria Ramírez, Pablo Valdez.

P034 CHRONOTYPE ASSESSMENT: VALIDATION OF THE MCTQ IN A SAMPLE OF UNIVERSITY STUDENTS FROM BRAZIL Marilene Farias Alam, Karla V. Allebrandt, Mabel Mascarenhas Wiegand, Giovana Dantas, Rosa Maria Levandovski, Rosana Mendonça de Souza, Maria Paz Hidalgo.

P035 CHRONOTYPES: A CORRELATION WITH EATING BEHAVIOR Ana Beatriz Harb, Wolnei Calmo, Rosa Maria Levandovski, Ceres Oliveira, Kelly Allison, Albert Stunkard, Maria Paz Hidalgo.

38

P036 LIGHTING AND HEALTH: CASE STUDY IN RETAIL STORES Betina Tschiedel Martau, Maria Paz Hidalgo.

P037 SLEEP PATTERNS AND RISK FACTORS FOR METABOLIC SYNDROME AMONG PROFESSIONAL PUBLIC TRANSPORT DRIVERS Ma. Elena Durán Lizárraga, Juan Manuel Ponce, Miguel Ángel Palomino Garibay.

P038 MORNINGNESS–EVENINGNESS, USE OF STIMULANTS, AND MINOR PSYCHIATRIC DISORDERS AMONG UNDERGRADUATE STUDENTS Giovana Dantas, Marcia L. M. Schneider, Denise C. Vascincelos, Rosa Levandovski, Wolnei Caumo, Karla V. Allebrandt, Marlene Doring, Maria Paz L. Hidalgo.

P039 CIRCADIAN CLOCK GENE EXPRESSION IN BRAIN REGIONS OF ALZHEIMER'S DISEASE PATIENTS AND CONTROL SUBJECTS Nicolas Cermakian, Elaine Waddington Lamont, Philippe Boudreau, Diane B. Boivin.

P040 DO NOT MESS WITH YOUR BIOLOGICAL CLOCK: SEIZURE AGGRAVATION AFTER THE PHASE SHIFT Magdalena Kinga Smyk, Anton Coenen, Marian Henryk Lewandowski, Gilles van Luijtelaar.

P041 6-‐SULFATOXIMELATONIN AS A PREDICTOR OF CLINICAL OUTCOME IN DEPRESSIVE PATIENTS TREATED WITH INHIBITORS Maria Paz Hidalgo, Wolnei Caumo, Giovana Dantas, Daiane Franco, Iraci Trres, Regina P Markus.

P042 CIRCADIAN RHYTHM OF ENERGY EXPENDITURE AND CONSUMPTION OF OXYGEN BY INDIRECT CALORIMETRY IN ENTERAL NUTRITION THERAPY Marlene Pooch Leuck, Rosa Maria Levandovski, Claudia Will Machado, Maria Paz Loayza Hidalgo.

P043 PRETERM INFANTS HAVE IMPROVED GROWTH IN LIGHT/DARK CYCLE COMPARED WITH CONTINUOUS BRIGHT LIGHT Samuel Vásquez, Manuel Ángeles-‐Castellanos, Carolina Escobar.

P044 CHRONOBIOLOGIC VISCUM ALBUM STUDY IN BREAST CANCER Abuín A. Abuin Landín, Luis Menna Barreto.

P045 DISCUSSION ABOUT THE INTERACTION BETWEEN DRUGS AND HUMAN BODY BIOLOGICAL CLOCK Feng Xiu Jie, Zhuang Hong Yan.

39

May 7 P046 CUSTOM-‐MADE SOFTWARE FOR DIGITAL SIGNAL ANALYSIS ON CHRONOBIOLOGY

Arturo Vega-‐González, Raúl Aguilar-‐Roblero.

P047 THEORETICAL INVESTIGATION FOR THE ORIGIN OF THE ASYMMETRIC RESYNCHRONIZATION AFTER AN ABRUPT SHIFT OF THE LIGHT: DARK CYCLE Takeshi Asakawa, Satoshi Koinuma, Koh-‐hei Masumoto, Mamoru Nagano, Yasufum Shigeyoshi.

P048 FLYING MICE: INTERNAL FORCED DESYNCHRONIZATION IN A MURINE MODEL OF CHRONIC JET-‐LAG Leandro Pablo Casiraghi, Gisele Akemi Oda, Juan José Chiesa, Wolfgang Otto Friesen, Diego Andrés Golombek.

P049 A SUBTERRANEAN RODENT’S “NATURAL ENTRAINMENT” 1Danilo Eugênio de França Laurindo Flôres, 1Barbara Mizumo Tomotani, 1Patricia Tachinardi, 1Gisele Akemi Oda, 2Verónica Sandra Valentinuzzi.

P050 PERIOD-‐2 INTEGRATES BOTH CIRCADIAN AND HOMEOSTATIC ASPECTS OF SLEEP REGULATION Thomas Curie, Valérie Mongrain, Stephane Dorsaz, Yann Emennegger, Paul Franken.

P051 NON-‐LINEAR DYNAMICS OF HEART RATE VARIABILITY SHOW SLEEP-‐WAKE HOMEOSTATIC PREDOMINANCE DURING SUSTAINED WAKEFULNESS Antoine U. Viola.

P052 CHANGES OF DISTAL SKIN TEMPERATURE AND SUBJECTIVE SLEEPINESS UNDER MIDDAY BRIGHT LIGHT EXPOSURE Nana N. Takasu, Hisayo Nishida, Akiko Nitta, Kana Nishimura, Tomoko Wakamura.

P053 ACTIGRAPHY AND SLEEP LOGS IN NEWBORNS: DIFFERENT RESULTS ACCORDING TO AGE Clarissa Bueno, Luiz Menna-‐Barreto.

P054 CHARACTERIZATION OF THE SLEEP-‐WAKE CYCLE IN KINDERGARTEN CHILDREN THAT ATTENDED SCHOOL IN THE MORNING IN NATAL, BRAZIL Aline Silva Belísio, Fernanda Fernandes Kolodiuk, Jane Carla Souza, Zoélia Moura Bessa, Deyse Silva Bezerra, Geilson Lima Araújo, Ivanise Sousa Guimarães, Fernando Mazzilli Louzada, Carolina Macedo Azevedo.

P055 CHARACTERIZATION OF SLEEP-‐WAKE CYCLE, DAYTIME SLEEPINESS AND SLEEP QUALITY OF BRAZILIAN HIGH-‐SCHOOL TEACHERS Carolina Virginia Macédo de Azevedo, Jane Carla Souza, Aline Silva Belãsio, Zoília Camila Moura Bessa, Ivanise Cortez de Sousa Guimarães.

P056 EFFECTS OF 24-‐H SLEEP DEPRIVATION ON ATTENTION Juventino Cortez Diana Juárez, Aida García, Candelaria Ramírez, Pablo Valdez.

40

P057 EFFECTS OF SLEEP REDUCTION ON THE COMPONENTS OF ATTENTION Diana Juárez, Martha Guerrero, Layla Arroyo, Juventino Cortez, Aida García, 6) Candelaria Ramírez, Pablo Valdez.

P058 EFFECTS OF SLEEP DEPRIVATION ON WORKING MEMORY Jacqueline Del Ángel, Ana Gabriela Iracheta, Diana Juárez, Juventino Cortez, Aida García, Candelaria Ramírez, Pablo Valdez.

P059 APPLY TCM CHRONOBIOLOGY TO REGULATE SLEEP RHYTHMS Feng Xiu Jie, Zhuang Hong Yan.

P060 SLEEP HABITS, CHRONOTYPES AND OBESITY/OVERWEIGHT IN MEDICAL STUDENTS AT UNAM Eduardo González, Donají Heredia, Carolina Escobar.

P061 DETECTION OF CHRONO PREMETABOLIC SYNDROME IN DIFERENT MEXICAN POPULATIONS Salvador Sánchez-‐de la Peña, Franz Halberg, Jonathán Levi Rito-‐Medina, Irene Mendoza-‐Lujambio, Germaine Cornélissen, Alfonso López-‐Fiesco.

P062 SLEEP COMPLAINTS AND ITS RELATIONSHIP TO DAYTIME SLEEP AND ACTIGRAPHIC PARAMETERS IN CIRRHOTIC PATIENTS: AN EXPLORATORY STUDY Montserrat Concepción Reséndiz Garcìa, Aldo Torre, Violeta Alejandra Castaño Meneses, Andrés Duarte Rojo, Maria Victoria Santiago Ayala, Demian Gil Aldeco, Arturo Vega González, Guillermo Garcìa Ramos, Matilde Valencia Flores.

P063 DETERIORATION OF SLEEP QUALITY AMONG IN HABITANTS OF CHHATTISGARH Ms. Chaynika Nag, Dr. R. K. Pradhan.

P064 RELATIONSHIP BETWEEN SOCIAL RHYTHM, SLEEP PHASE AND MINOR PSYCHIATRIC SYMPTOMS IN HEALTHY WORKERS Regina Lopes Schimitt, Rosa Levandovski, Maria Paz Loayza Hidalgo.

P065 DECREASE OF EVENING PLASMA PROLACTIN IN THE DELAYED SLEEP PHASE SYNDROME. IS A HYPERTONIC DOPAMINERGIC SYSTEM INVOLVED? Bruno Claustrat, Helène Bastuji, Laure Peter-‐derex, Thierry Petitjean, Françoise Borson-‐Chazot, Jocelyne Brun.

P066 PHARMACOLOGICAL TREATMENT ASSESSMENT WITH MARTAZAPINE ON SLEEP QUALITY OF GERIATRIC PATIENTS WITH MAJOR DEPRESSION: AN ACTIGRAPHY TEST Betty Marjorie Rothschild-‐Fuentes, Jairo Muñoz-‐Delgado, Alejandro Jiménez-‐Genchi, José Carlos Sánchez-‐Ferrer, Andrés Roche-‐Bergua.

P067 GLYCEMIC CONTROL AFFECTS SLEEP INITIATION AND SLEEP QUALITY IN T1DM Mark Thomas Ugliara Barone, D R Franco, M K Carra, Fabiola Schorr, Geraldo Lorenzi, Luiz Menna-‐Barreto.

P068 THE ASSOCIATION AMONG SLEEP QUALITY AND MID-‐SLEEP PHASE WITH USE OF ANTIHYPERTENSIVE DRUGS

41

Rosa Maria Levandovski, Till Roenneberg, Karla Viviani Allebrandt, Maria Paz Loayza Hidalgo.

P069 THE IMMOBILITY EPISODES OF TAIEP RATS HAVE ULTRADIAN EXPRESSION Ma. del Carmen Cortés, Lara, José R. Eguíbar.

P070 THE SPIKE-‐WAVE DISCHARGES IN THE MYELIN MUTANT TAIEP RAT HAVE A CIRCADIAN RHYTHM Jackeline Corona, Ma. del Carmen Cortés, José R. Eguíbar.

P071 DEPENDENCE ON TIMING OF 2-‐AG ADMINISTRATION INTO THE LATERAL HYPOTHALAMUS TO MODIFY THE SLEEP WAKING CYCLE OF RATS Mauricio Marcel Pérez-‐Morales, Seraid Caynas, Ilia Alvarado, Mónica Méndez-‐Díaz, Oscar Prospero-‐García.

P072 DIFFERENCES IN SLEEP AND DAILY METABOLIC PROFILES BETWEEN OVERWEIGHED AND NORMAL MICE NEOTOMODON ALSTONI Citlalli Fuentes Granados, Pilar Durán, Manuel Miranda Anaya.

P073 EFFECT OF TOTAL SLEEP DEPRIVATION ON THE EXPRESSION OF NEUROGLOBIN IN THE ADULT RAT BRAIN Montserrat Alheli Melgarejo, Eva Acosta Peña, Arturo Venebra Muñoz, Fabio García García.

P074 CEREBRAL ELECTROPHYSIOLOGICAL ACTIVITY INDUCED BY SYSTEMIC INFLAMMATORY RESPONSE SYNDROME (SIRS) Francisco Nachón García, Juan Santiago García, Alberto de la Herrán Arita, Armando Martínez Chacón, René Drucker Colín, Fabio García García.

P075 CIRCADIAN CLOCK AND CELL CYCLE COUPLING IS REQUIRED FOR CORRECT WOUND HEALING Elzbieta Kowalska, Pascal Bruegger, Dominik Hoegger, Juergen Ripperger, Thorsten Buch, Anke Mueller, Achim Kramer, Urs Albrecht, Thomas Birchler, Claudio Contaldo, Steven A. Brown.

P076 ACUTE COCAINE IMPAIRS ADULT CIRCADIAN CLOCK PHASE REGULATION: INVOLVEMENT OF THE PER2 CLOCK GENE Adam C. Stowie, Allison J. Brager, Rebecca A. Prosser, Dave J. Glass.

P077 DAILY TIMING OF CHRONIC INTERMITTENT ALCOHOL EXPOSURE AND WITHDRAWAL DIFFERENTIALLY DISRUPTS CIRCADIAN BODY TEMPERATURE RHYTHMS IN C57BL/6 MICE Susan Amanda Sinning, Michael R. Gorman.

P078 CIRCADIAN GENE MODULATION AND BRAIN SITES OF ACTION IN ACAMPROSATE SUPPRESSION OF ALCOHOL INTAKE AND CRAVING Allison Joy Brager, Rebecca A Prosser, J. David Glass.

P079 HYPOTHALAMIC CONTROL OF BLOOD PRESSURE: A ROLE FOR THE BIOLOGICAL CLOCK Frederik Buijs, Mari Carmen Basualdo, Carolina Escobar, Ruud Buijs.

42

P080 EXPERIMENTAL CHRONIC JET LAG PROMOTES GROWTH AND LUNG METASTASIS OF LEWIS LUNG CARCINOMA IN C57BL/6 MICE Mingwei Wu, Jing Zeng, Zhaolei Zeng, Lijian Xian.

P081 EFFECT OF LIGHT/DARK CYCLE ON SEIZURE SEVERITY AND HIPPOCAMPAL NEURONAL CELL DEATH INDUCED BY STATUS EPILEPTICUS IN THE DEVELOPING RAT Dulce Mariely Álvarez-‐Croda, María Leonor López-‐Meraz.

P082 EFFECTS OF PRENATAL EXPOSURE TO VALPROIC ACID ON ULTRASONIC VOCALIZATIONS OF RAT DURING LACTANCY PERIOD Paul Saft, Jorge Manzo, Consuelo Morgado-‐Valle, Luis Beltrán-‐Parrazal, María Elena Hernández, Brenda Brug, Luis Isauro García, Rebeca Toledo.

P083 DIURNALLY OR NOCTURNALLY ELICITED SYSTEMIC INFLAMMATION DOWN REGULATES AANAT GENE EXPRESSION IN THE CHICKEN PINEAL GLAND Krystyna Skwarlo-‐Sonta, Urszula Kedzierska, Aneta Piesiewicz, Alicja Olesiejuk, Maria Waloch, Pawel Marek Majewski.

P084 A CIRCADIAN CLOCK REGULATES SWIMMING DIRECTION OF ERETMOCHELYS IMBRICATA HATCHLINGS (HAWKSBILL MARINE TURTLE) Kristel Lorena Gopar-‐Canales, Manuel Miranda-‐Anaya.

P085 SIXTH ABDOMINAL GANGLION MODULATES THE CIRCADIAN RHYTHMS OF LOCOMOTOR ACTIVITY AND ABDOMINAL POSTURE IN CRAYFISH Leonardo Rodríguez Sosa, Gabina Calderon Rosete.

P086 SCN DRIVEN CHANGES IN THE ACTIVATION OF ARCUATE NUCLEUS ΑMSHNEURONS Mara Alaide Guzmán Ruiz, Guadalupe Acosta Galván, Daniela Herrera-‐Moro Chao, Ma. Carmen Basualdo Sigales, Carolina Escobar Briones, Ruud Buijs.

P087

P088 HOW CORTICO – BASAL GANGLIA – THALAMOCORTICAL NETWORK CAN INFLUENCE RATE OF "INTRINSIC CLOCK” Izabella G. Silkis.

P089 THE PARTICIPATION OF THE VAGUS NERVE IN THE REGULATION OF OVARIAN FUNCTIONS DEPENDS ON DAY THE ESTROUS CYCLE AND TIME OF THE DAY Pamela María Everardo, María Guadalupe Gúzman, Carlos Abraham García, Angélica Flores, María Esther Cruz, Roberto Domínguez.

P090 THE ROLE OF KISS1 NEURONS IN CIRCADIAN-‐TIMED LUTEINIZING HORMONE SURGES IN SHORT DAY FEMALE HAMSTERS Azim R Khan, Eric C Ku, R. Gorman, Alexander S Kauffman.

43

May 8 P091 MODULATION OF THE CIRCADIAN RHYTHM OF BODY TEMPERATURE BY INTENSE ACTIVITY

IN A SUBTERRANEAN RODENT (CTENOMYS CF. KNIGHTI) Patricia Tachinardi, Barbara Mizumo Tomotani, Danilo Eugénio de França Laurindo Flôres José Eduardo Pereira Wilken Bicudo, Verónica Sandra Valentinuzzi, Gisele Akemi Oda.

P092 SOCIAL INTERACTION ALTERS CIRCADIAN RHYTHM MANIFESTATION IN RATS SUBMITTED TO CONSTANT LIGHT Antoni Diez-‐Noguera, Lucia Castejon, Trinitat Cambras.

P093 COMPARATIVE ANALYSIS OF CIRCADIAN RHYTHMS OF THE MOSQUITOES AEDES AEGYPTI AND CULEX QUINQUEFASCIATUS UNDER TEMPERATURE CYCLES Gustavo Bueno Rivas, Carla Gentile, Alexandre Afranio Peixoto.

P094 RAT LIVER GABAERGIC SYSTEM CHARACTERIZACTION DURING FOOD ENTRAINABLE OSCILLATOR EXPRESSION Olivia Vázquez Martínez, Mónica Villalobos Leal, Isabel Mendez, Diego Hernández Saavedra, Mauricio Díaz Muñoz.

P095 BEHAVIORAL AND TEMPERATURE CHANGES THAT PRECEDE FOOD ENTRAINMENT Dulce María Palomares-‐Vázquez, Estefa Espitia, Rodrigo Ivan Osnaya, Carolina Escobar.

P096 EXPECTATION FOR PALATABLE FOOD. A PROCESS THAT IS GENERATED GRADUALLY IN THE BRAIN Aurea Susana Blancas Velázquez, Katia Rodríguez Gonzales, Carolina Escobar Briones.

P097 REGULATION OF HEPATIC MITOCHONDRIAL BETA-‐OXIDATION DURING THE EXPRESSION OF THE FOOD ENTRAINED OSCILLATOR Julieta Rivera, Mauricio Díaz.

P098 STUDY OF THE ZONAL DISTRIBUTION OF LIVER PER1 PROTEIN DURING THE EXPRESSION OF THE FEEDING ENTRAINABLE OSCILLATOR Dalia Luz De Ita, Mauricio Diaz.

P099 SUB CELLULAR LOCALIZATION AND RELEASE OF THE MITOCHONDRIAL ENZYME ORNITHINE TRANSCARBAMYLASE, IN RAT LIVER DURING THE EXPRESSION OF THE FOOD ENTRAINED OSCILLATOR Braulio Ayala-‐García, Mauricio Díaz-‐Muñoz.

P100 DIFFERENTIAL INVOLVEMENT OF OREXIN CELLS IN FOOD ENTRAINMENT Angeles Jiménez, Mario Caba, Carolina Escobar.

P101 NURSING TIME SYNCHRONIZES THE EXPRESSION OF PER1 PROTEIN IN THE PREOPTIC AREA AND BED NUCLEUS OF THE STRIA TERMINALIS OF THE FEMALE RABBIT José Enrique Meza, Rossana Zepeda, Mercedes Acosta, Stefan Walisewski, Mario Caba.

P102 FUNCTIONAL ADAPTATION OF THE GHRELIN-‐GROWTH HORMONE-‐IGF-‐1 AXIS DURING THE EXPRESSION OF THE FOOD ENTRAINED OSCILLATOR Elvira Arellanes-‐Licea, Martha Carranza, Maricela Luna, Mauricio Díaz-‐Muñoz.

44

P103 SCHEDULED FEEDING DOES NOT REENTRAIN THE ESTROUS CYCLE IN FEMALE WISTAR RATS MAINTAINED IN CONSTANT LIGHT Donají Heredia García, Mariana Pliego Caballero, Isabel García-‐Peláez, Carolina Escobar Briones.

P104 DAYTIME RESTRICTED FOOD SCHEDULE CHANGED THE HEPATIC ZONATION OF THE PHOSPHOENOLPYRUVATE CARBOXYKINASE Moisés Pérez-‐Mendoza, Mauricio Díaz-‐Muñoz.

P105 GLUCOCORTICOID RECEPTOR SIGNALING IS MODIFIED BY RESTRICTED FEEDING Alma Dalia Luna, Mauricio Díaz.

P106 INDUCTION OF FOS AND PER1 PROTEINS IN THE MAIN OLFACTORY BULB OF RABBIT PUPS IN RELATION TO SCHEDULED NURSING Nahum Nolasco, Enrique Meza, Mario Caba.

P107 REFEEDING AFTER FASTING ELICITS INSULIN-‐DEPENDENT REGULATION OF PER2 AND REV-‐ERBA WITH SHIFTS IN LIVER CLOCK Yu Tahara, Makiko Otsuka, Yuta Fuse, Akiko Hirao, Shigenobu Shibata.

P108 RABBIT NURSING SHOWS CIRCADIAN PERIODICITY AND IS MODULATED BY SUCKLING STIMULATION CHARACTERISTICS Gabriela González-‐Mariscal, Ana Celia Lemus, Raúl Aguilar-‐Roblero.

P109 THE SUPRACHIASMATIC NUCLEUS IS NECESSARY FOR CHOCOLATE ENTRAINMENT Katia Rodríguez González, Aurea Susana Blancas, Manuel Ángeles Castellanos, Carolina Escobar.

P110 DAILY RHYTHM OF BLOOD GLUCOSE INCREASE BY HIGH OR LOW DIGESTIBLE STARCH INTAKE IN NORMAL MICE AND MICE WITH RESTRICTED FEEDING PARADIGM Akiko Hirao, Misa Itokawa, Hiroki Nagahama, teiji Outsu, Takuma Imanishi, Ayako Shinozaki, Yu Tahara, Shigenobu Shibata.

P111 DO NEWBORN RABBITS BEARING SUPRACHIASMATIC NUCLEI LESIONS ANTICIPATE TO NURSING? Oscar Hernández-‐Campos, Rodrigo Montúfar-‐Chaveznava, Ivette Caldelas.

P112 DOES THE OLFACTORY BULB OF NEWBORN RABBITS EXHIBIT TIME DEPENDENT RESPONSES TO THE MATERNAL PHEROMONE 2MB-‐2? Lucero Anabel Trejo-‐Muñoz, Rodrigo Montúfar-‐Chaveznava, Ivette Caldelas.

P113 PHOSPHORYLATION-‐DEPENDENT DEGRADATION OF CRY2 REGULATES CLOCK GENE EXPRESSION AND CIRCADIAN PERIOD Arisa Hirano, Nobuhiro Kurabayashi, Yoshitaka Fukada.

P114 DETERMINATION OF INTRON MOVEMENT IN PER GENE Gabina Calderón Rosete, Francisco Martínez Pérez, Juan A. González Barrios, Luis Kameyama, Jorge L. Fuentes Lorenzo, Leonardo Rodríguez Sosa

45

P115 EXPLORATION OF THE HUMAN MOLECULAR CLOCK IN FIELD STUDIES Marta Novakova, Martin Sladek, Alena Sumova.

P116 DAILY FLUCTUATIONS IN THE BIOSYNTHESIS OF PHOSPHATIDYLCHOLINE AND ACTIVITY OF ITS KEY ENZYME CTP: PHOSPHOCHOLINE CYTIDYLYLTRANFERASE IN SYNCHRONIZED CULTURES OF FIBROBLASTS Victoria América Acosta Rodríguez, Sebastián Márquez, Mario Eduardo Guido.

P117 OPSIN EXPRESSION AND LIGHT RESPONSES IN RGC-‐5 CELLS Paula Sofía Nieto, Diego Javier Valdez, Victoria América Acosta-‐Rodríguez, Mario Eduardo Guido.

P118 CIRCADIAN REGULATION OF CYTOPLASMIC MRNA-‐GRANULES Juan Ignacio Lescano, Mario Eduardo Guido, Eduardo Garbarino Pico.

P119 JNK PHOSPHORYLATES BMAL1-‐CLOCK COMPLEX AND CONTROLS OSCILLATION SPEED AND PHOTIC REGULATION OF THE CIRCADIAN CLOCK Yoshitaka Fukada, Hikari Yoshitane, Shin-‐ya Nishide, Hiroto Nakajima, Daisuke Ono, Hiroshi Kiyota, Kiyomichi Imamura, Naoya Shinozaki, Hirokazu Matsuki, Naoya Wada, Hirofumi Doi, Toshiyuki Hamada, Ken-‐ichi Honma, Sato Honma.

P120 PHOTOTRANSDUCTION IN ZEM-‐2S EMBRYONIC CELLS OF ZEBRAFISH DANIO RERIO Bruno Ribeiro Ramos, Leonardo Ribeiro Lima, Maria Nathália Magalhães Moraes, Maristela Oliveira Poletini, Ana Maria Lauro Castrucci.

P121 EFFECTS OF LIGHT ON PER1 AND PER2 TRANSCRIPTION: MODULATION AND SYNCHRONIZATION IN Xenopus laevis MELANOPHORES Maria Nathália Carvalho Moraes, Bruno Ribeiro Ramos, Maristela Oliveira Poletini, Leonardo Ribeiro Lima, Ana Maria Lauro Castrucci.

P122 CRUSTACEAN HYPERGLYCEMIC HORMONE mRNA OSCILATION IN THE CRAYFISH PROCAMBARUS CLARKII EYESTALK Janikua Nelson-‐Mora, Julio Prieto-‐Sagredo, María Luisa Fanjul-‐Moles.

P123 GENOMIC DISSECTION OF TASK-‐RELATED PLASTICITY IN CIRCADIAN RHYTHMS IN HONEY BEES Guy Bloch, Sandra L. Rodríguez-‐Zas, Bruce R. Southey, Mira Cohen, Gene E. Robinson.

P124 MEASURE OF THE mRNA OF RYANODINE 2 RECEPTOR AND GENES PER1 AND PER2 IN UNIQUE CELL OF THE SUPRAQUIASMATIC NUCLEUS AT TWO DIFFERENT HOURS (ZT12 AND ZT23) José Luis Chávez Juárez, Daniel Quinto Muñoz, Raúl Aguilar Roblero.

P125 DIFFERENTIAL DEVELOPMENT OF THE DIURNAL CLOCK GENE EXPRESSION IN THE OLFACTORY BULB AND SUPRACHIASMATIC NUCLEUS OF EUROPEAN RABBIT Oscar Hernández Campos, Rodrigo Montúfar Chaveznava, Ivette Caldelas.

P126 PARTICIPATION OF INTRACELLULAR CALCIUM-‐RELEASE CHANNELS AND CALCIUM PUMPS IN THE CIRCADIAN RHYTHMICITY OF THE SUPRACHIASMATIC NUCLEUS Adrian Báez-‐Ruiz, Raúl Aguilar-‐Roblero, Gabriella S. Lundkvist, Mauricio Díaz-‐Muñoz.

46

P127 HISTOCHEMICAL CHARACTERIZATION OF ENDOPLASMIC RETICULUM PROTEINS THAT MOVILIZE INTRACELLULAR CALCIUM IN THE SCN Clara Mercado López, Mauricio Díaz Muñoz.

P128 THE EFFECT OF GATING RYANODINE RECEPTORS ON THE INTRACELULAR CALCIUM CONCENTRATION IN SCN NEURONS IN MICE Daniel Quinto-‐Muñoz, Stephan Michel, Raúl Aguilar-‐Roblero.

P129 MECHANISMS OF RESPIRATORY RHYTHM GENERATION IN VITRO: EFFECT OF INCREASING CA2+ BUFFERING CAPABILITY IN PREBÖTZINGER COMPLEX INSPIRATORY NEURONS Consuelo Morgado-‐Valle, Jorge Manzo, Luis Isauro García, Luis Beltrán-‐Parrazal.

P130 MODULATION OF APOPTOTIC ACTIVITY IN LIVER CELLS BY THE FOOD ENTRAINABLE OSCILLATOR Christian Molina Aguilar, Olivia Vázquez Martínez and Mauricio Díaz Muñoz.

Instituto de Neurobiología, UNAM Campus Juriquilla, Querétaro, México.

47

ABSTRACTS

Lectures

48

49

SERENDIPITY OR "THE PREPARED MIND": THE TRUE HISTORY OF SOME DISCOVERIES IN THE MENAKER LAB Michael Menaker, Pinar Pezuk, Michael Sellix. University of Virginia, USA. Who is conducting the circadian orchestra? The circadian orchestra is composed of autonomous circadian oscillators in most cells, tissues and organs of the body. As in a musical orchestra the individual players must either be temporally coordinated in order to make either recognizable music or adaptively appropriate responses to the environment. In both cases temporal coordination is achieved by the action of a “conductor,” employing either a baton or a set of (poorly understood) neurochemical signals. The classical answer to the question: “Who is conducting the circadian orchestra?” is “the suprachiasmatic nucleus (SCN).” This is correct but not complete since there are other central oscillators that can take over in the absence of the SCN. In SCN lesioned rodents these other conductors can be evoked by either temporally restricted feeding regimens (FEO) or chronic exposure to methamphetamine (MASCO). Neither the anatomical location(s) nor the molecular mechanism(s) that generate their rhythmicity are known at present. Until recently the evidence for their existence rested primarily on behavioral data. I will present new data demonstrating that non-‐SCN conductors regulate the phases of circadian oscillators in individual organs. I will briefly discuss two additional questions: What is the relationship if any, between SCN and non-‐SCN conductors? Are FEO and MASCO one or two distinct conductors?

50

HISTORY OF CHRONOBIOLOGICAL SOCIETIES‐CHRONOBIOLOGISTS ARE ALWAYS LOOKING FOR THE BEST FRIEND.

Ken-‐ichi Honma, MD, Ph.D. Hokkaido University, Graduate School of Medicine, Sapporo Japan

In the late 1940’ to early 1950’, the researches in the field of biological rhythms shifted from the descriptive to analytical stage. A substantial number of scientists became interested in this field, and in due course got together. To my knowledge, the first academic society for biological rhythm research was founded in 1937 under the German name, ‘International Gesellschaft fur Biologishe Rhythmusforschung’, and the first international congress was held in Ronneby, Sweden in 1938. The society has changed its name several times and is now known as the International Society for Chronobiology (ISC). ISC holds an international meeting every second year and recently the International Congress of Applied Chronobiology and Chronomedicine. Since 1937, more than 15 societies related to biological rhythm research were founded. But some of them were rearragened or mereged, and some vanished.

In the early 1980’, the Society of Reserch for Biological Rhythms (SRBR) was established by scientists maily in USA and Europe and holds a meeting in every second year. At almost the same time, two study groups for biological rhythm research were founded in Japan, which were later-‐on merged into the Japanese Society for Chronobiology (JSC) in 1994. With practical purposes, the Society for Light Treatment and Biological Rhythms (SLTBR) was founded in 1988, and an international meeting is held annually. In 2005, the European Biological Rhythms Society (EBRS) was established as a successor to the European Pineal and Biological Society and the European Pineal Study Group. In addtion, we have several other societies such as the latin American Chronobiology Group and the American Association of Medical Chronobiology and Chronotherapeutics, which have regular scientific meetings.

In 2001, 13 chronobiological societies in the world got togetehr and founded the World Federation of Societies for Chronobiology (WFSC). In 2003, the first World Congress for Chronobiology (WCC) of WFSC was held in Sapporo, Japan WFSC is a society-‐based organization and aims to promote mutual interactions among the chronobiological societies wtih various purposes and principles. The WCC was expected to be held every fourth years. The 2nd one in 2007 was originally planned to be hosted by SRBR, but it was finally held by JSC in Tokyo, Japan. I am very pleased to attend the thrid one in Puebra Mexico this year.

51

ABSTRACTS

Simposia

52

53

S1.1 CELLULAR RHYTHMS AND NEURAL NETWORKS IN THE MOUSE SUPRACHIASMATIC NUCLEUS

Sato Honma, Daisuke Ono, Tomoko Yoshikawa, Ken-‐ichi Honma. Department of Physiology, Hokaido University Graduate School of Medicine Sapporo, 060-‐8036, Japan.

The master circadian clock of mammals in the hypothalamic suprachiasmatic nucleus (SCN) is a hierarchical multi-‐oscillatory system in which individual cell-‐autonomous rhythms are coupled to exhibit coherent tissue rhythms. Currently, a cell autonomous circadian clock is thought to be composed of a transcriptional and translational feedback loop involving several clock genes and their protein products. The bioluminescence reporter technique for monitoring the clock gene expression enabled us to know the tick of the circadian clock in vitro. Neuronal activity and interactions can also be monitored in real time from enzymatically isolated neurons as well as single neurons from the SCN slices cultured on a multi-‐electrode array dish. We monitored gene expression and neural activity from single cells of SCN explants and found that the phase relation between the two rhythms were different within a single SCN explants. The phase relation between Per1-‐luc and PER2::LUC rhythms also differed depending on the regions in the SCN and ambient light conditions. In mice lacking CRY1 and CRY2, in which behavioral rhythms became arrhythmic in DD, single SCN neurons exhibited robust and synchronized circadian rhythms in the spontaneous firing whereas desynchronized Per1 expression rhythms with widely varied circadian periods. These results indicate that networks within the SCN can dynamically change in response to various inputs. Different coupling mechanisms are suggested for Per1 expression and neural rhythms. Supported by grants from the Ministry of Education, Science, Culture and Sports Japan, Nos. 21390064 and 20249010.

S1.2 PHOSPHATASE IN SUPRACHIAMATIC NUCLEUS, PHLPP1/SCOP, CONTROLS PERIOD CHANGE AFTER LIGHT-‐INDUCED PHASE SHIFT, "AFTER-‐EFFECT OF PHASE-‐SHIFT" 1Satoru Masubuchi, 2Tianyan Gao, 2Audrey O Neill, 1Kristin Eckel-‐Mahan, 2Alexandra C. Newton, 1Paolo Sassone-‐Corsi. 1Department of Pharmacology, School of Medicine, University of California, Irvine, CA, USA. 2Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA.

Protein phosphorylation in the suprachiasmatic nucleus (SCN) controls circadian period or clock resetting. The pleckstrin homology domain leucine-‐rich repeat protein phosphatase 1, PHLPP1 (also known as suprachiasmatic nucleus circadian oscillatory protein, SCOP) expression oscillates in a circadian fashion in the SCN. To know the role of PHLPP1 in the clock function, we analyzed the circadian behavior of null-‐mutant mice (PHLPP1-‐/-‐). When animals were moved from light-‐dark 12h : 12h to constant darkness, free running period (tau) was not changed in PHLPP1-‐/-‐. Next, a light pulse in the early subjective night which

54

delays the clock phase was applied. The magnitude of phase delay, estimated by the first 10 days of activity onsets, was not different between genotypes. However, by using next 10 days of activity onsets, the phase delay was much larger in PHLPP1-‐/-‐. This discrepancy came from the irregular change of tau after resetting. A light pulse in early subjective night delays the clock phase accompanied by extension of the circadian period. This period change is known as ‘After-‐Effect of Phase-‐Shift’ (J. comp. physiol. 106:223 (1976)). Interestingly, phase delaying light did not extend the period of PHLPP1-‐/-‐ in the first 10 days and the period became shorter in next 10 days. Delayed shortening (10 days later; ~0.3h) of tau made a difference in final phase delay by ~3h (= 10 days x ~0.3h). Acute mPer1 and mPer2 induction by light pulse and following one cycle of mPer2 oscillation were not changed in the SCN, which indicated the SCN was stimulated similarly by light and shifted equally. This SCN clock gene expression profile was compatible with the behavioral data of PHLPP1-‐/-‐, delayed period change induced final large phase shift. ‘After-‐Effect of Phase-‐Shift’ occurs also by phase advancing light pulse in the subjective late night. Light in the late night shortened following period in wild type mice but not in PHLPP1-‐/-‐. This evidence indicates that PHLPP1 contributes the proper response to resetting light, ‘After-‐Effect’. PHLPP1-‐/-‐ is the first animal model of ‘After-‐Effect’. In classical model, coupling of multioscillatory systems causes After-‐Effect. PHLPP1 may contribute to neural network for After-‐Effect. PHLPP1 differentially attenuates Akt/PKB, PKC, and ERK1/2 signaling, thereby controlling the duration and amplitude of responses evoked by these kinases. By analyzing mutants of these target molecules, the molecular pathway of the After-‐Effect phenotype may be clarified. Supported by Uehara Memorial Foundation Research Fellowship, Mochida Memorial Foundation for Medical and Pharmaceutical Research Fellowship (to S.M.), National Institutes of Health CA 113265, T32 (to K.E.-‐M), Institut National de la Sante et de la Recherche Medicale (P.S.-‐C.), and NIH GM 67946 (to A.C.N.); R01-‐GM081634-‐01, R21 AG033888 (to P.S.-‐C.).

S1.3 EXPLORATIONS OF THE CIRCUIT STRUCTURE OF THE SCN 1,2,3,4Rae Silver, 1Nicholas C. Foley, 2Tina Y. Tong, 5Duncan Foley, 6,7David K. Welsh, 2Joseph LeSauter. 1Department of Cognitive and Neural Systems, Boston University, Boston, MA 02215, USA. 2Department of Psychology, Barnard College of Columbia University, New York, NY, USA. 3Dept. of Psychology Columbia University, New York, NY, USA. 4Department of Pathology and Cell Biology, Columbia University Health Sciences New York, NY, USA. 5Department of Economics, New School for Social Research, New York NY, USA. 6Department of Psychiatry, University of California, La Jolla, CA, USA. 7Veterans Affairs San Diego Healthcare System, San Diego, CA, USA.

Because we can observe oscillation within individual cells and in the tissue as a whole, the suprachiasmatic nucleus (SCN) presents a unique system in the mammalian brain for the

55

analysis of cellular elements and the networks of which they are a part. While dispersed cells of the SCN sustain circadian oscillations in isolation, they appear to be unstable oscillators that require network interactions for robust cycling (see Herzog, this session). Using cluster analysis to assess bioluminescence in acute brain slices from PERIOD2::Luciferase (PER2::LUC) knockin mice, and immunochemistry of SCN from animals harvested at various circadian times, we assessed the spatiotemporal activation patterns of PER2 to explore the emergence of a coherent oscillation at the tissue level. The results indicate that circadian oscillation is characterized by a stable daily cycle of PER2 expression involving orderly serial activation of specific SCN subregions, followed by a silent interval, with substantial symmetry between left and right side of the SCN. The biological significance of the clusters identified in slices was confirmed by co-‐expression of LUC and PER2 in immunochemically stained brain sections with the spatiotemporal pattern of LUC expression resembling that revealed in the cluster analysis of bioluminescent slices. We conclude that the precise timing of PER2 expression within individual neurons is dependent on their location within the nucleus, and that small groups of neurons within the SCN give rise to distinctive and identifiable subregions. We propose that serial activation of these subregions is the basis of robustness and resilience of the SCN’s daily rhythm. The next goal is first, to understand the circuits that form the basis of SCN oscillation at the level of the tissue and second to examine how these confer robustness and precision on the oscillation of individual component cells. The challenge is to explain how stability and plasticity in these neural circuits can result from alterations in synaptic strength or connectivity, and from changes in the excitability of the individual neurons. Because plasticity and novel emergent properties in brain circuitry occur at so many levels of organization and over so many timescales, we enlist theoretical and computational methods, along with biologically significant experimental work to organize empirical data and to interpret the results. To this end, we have developed novel computational methods to assess network structure. By linking theoretical and experimental studies, we are starting to reveal SCN network dynamics. Supported by NIH grants RO1 NS37919 and MH075045 (RS); R01 MH082945 (DKW) and a V.A. Career Development Award (DKW).

S1.4 REGIONAL PERIOD DIFFERENCE THAT GENERATES A PHASE GRADIENT IN THE MAMMALIAN CIRCADIAN CENTER 1Yasufumi Shigeyoshi, 1Satoshi Koinuma, 2Takeshi Asakawa, 1Koh-‐hei Masumoto, 1Kei-‐ichi Furukawa, 1Mamoru Nagano, 3Kazuhiro Yagita. 1Department of Anatomy and Neurobiology, School of Medicine, Kindai University, Osaka, Japan. 2System Technologies Laboratories, SONY Corporation, Tokyo, Japan. 3Department of Neuroscience and Cell Biology, Kyoto Prefectural University of Medicine, Kyoto, Japan.

The suprachiasmatic nucleus (SCN) is the center of the mammalian circadian rhythm. The molecular machinery of the circadian rhythm composed of integrated feedback and forward

56

loops lies in the each individual neurons in the SCN. The SCN is equipped with elaborate plasticity to generate appropriate physiological responses to the alternating environmental light conditions. Individual oscillating neurons have diverse endogenous circadian periods but usually they are synchronized by intercellular coupling mechanism. The diversity in the period of each oscillators neurons have been well investigated, however, the clustering of the oscillators of the similar periods has not been reported. We disrupted the intercellular coupling and observed the regional differences of the period of the oscillating small-‐latticed regions on the SCN by using a transgenic rat bearing a luciferase reporter gene driven by regulatory elements from a per2 clock gene (Per2::dluc rat). The analysis divided the SCN into two regions; one region showing periods less than 24h (SPR) and another region showing periods longer than 24 hours (LPR). The SPR lies in the medial smaller region of the dorsal SCN and the LPR occupied the remaining larger region. Interestingly, the SPR corresponds well with the region in which the phase wave in the SCN is launched. Further, we observed the dissociation of SPR and LPR when we advanced the light: dark (12h:12h) cycle for eight hours. Then, we constructed a mathematical model of the synchronized oscillators in the SCN based on the present findings. The model reproduced the phase gradient and the kinetics of the synchronized and desynchronized oscillators in the SCN faithfully. Acknowledgments: We thank Dr. Sei-‐ichi Hashimoto for kindly providing us the Per2::dluc rat.

S1.5 FROM NODES TO NETWORKS IN THE MAMMALIAN CIRCADIAN SYSTEM Erik D. Herzog, Alexis B. Webb, Sungwon An, Sara J. Aton, Rebecca M. Krock, G. Mark Freeman, Jr. Dept. Biology, Washington University, St. Louis, MO, USA.

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is required for daily rhythms including sleep-‐wake behavior, hormone release, and metabolism. Individual neurons in the SCN have the intrinsic capacity to generate circadian rhythms in firing rate and gene expression. Cell-‐cell interactions stabilize and amplify these rhythms to produce a coordinated output. Circadian synchrony between oscillators is critical for coherent output from the SCN, and thus for behavioral rhythmicity. How these neurons remain synchronous is unclear. We have found that the neuropeptide vasoactive intestinal polypeptide, VIP, and its cognate receptor, VPAC2R, are required for synchrony among SCN cells. We have found that VIP entrains SCN cells in a dose-‐ and time-‐of-‐day dependent manner through parallel adenylate cyclase and phospholipase C signaling. To reveal the underlying functional connectivity, we cross-‐correlate neuronal spike trains recorded on multielectrode arrays. We find evidence for significant connectivity between individual neurons. These correlations peak within 10 ms of firing by a reference neuron, and can be classified as excitatory or inhibitory. We find that the strength and number of connections between neurons oscillate over the day. Application of specific receptor antagonists allows us to determine which neurotransmitters mediate these correlations. Furthermore, we can

57

determine whether this millisecond-‐resolution communication is necessary for circadian synchrony by applying specific receptor antagonists to SCN slices and imaging the expression of a bioluminescent reporter driven by the clock gene Period2. We find, for example, that blockade of GABA signaling with bicuculline, gabazine or picrotoxin decreases the number of inhibitory correlations by ~90 % and the number of excitatory correlations by ~ 0 -‐ 50%. GABA blockade did not, however, reduce circadian synchrony in SCN slices. Blockade of ionotropic glutamate receptors with APV and CNQX also did not affect the Per2::Luc rhythm in SCN slices. We conclude that whereas neuropeptide signaling is critical for synchrony among SCN neurons, fast neurotransmission is critical to distinct functions which may include entrainment to environmental cues and determination of precision in the SCN. Supported by NIMH grant 63104.

S2.1 CIRCADIAN CLOCKS FOR ALL MEALTIMES: ANTICIPATION OF MULTIPLE DAILY MEALS IN RATS AND MICE 1Ralph E Mistlberger, 1Danica F Patton, 2Mathew Luby, 2Cynthia T Hsu, 1Brianne A. Kent, 1Sofina Chan, 1Maksim Parfyonov, 1Elliott G. Marchant, 2Andrew D. Steele. 1Simon Fraser University, Canada. 2California Institute of Technology, USA. Food anticipatory activity rhythms induced by restricting rats and mice to a single daily mealtime exhibit formal properties consistent with control by food-‐entrainable circadian oscillators. Few studies have examined anticipation of multiple daily mealtimes. These studies indicate that rats readily anticipate 2 daily mealtimes, but fail to exhibit anticipatory wheel running to a third daily meal. Formal properties of 2-‐meal anticipation suggest mediation by separate entrained oscillators. We have conducted behavioral studies to further characterize the formal properties of 2-‐meal anticipation in rats, and to reassess anticipation of 3 or more daily meals using behavioral measures other than wheel running in rats and mice. Meal omission and meal shift tests in rats anticipating 2 daily meals in the light period (at 7h or 10h intervals) produced results consistent with entrainment of separate oscillators by each meal, and that rule out mediation by hourglass and interval timers. The results could also be accommodated by a continuously consulted clock model (discrimination of multiple phases of one entrained clock). Rats provided 3 daily meals at 6-‐h intervals exhibited anticipatory food-‐bin activity to each meal and these rhythms persisted during meal omission tests. Mice provided 3 or 6 daily meals exhibited anticipatory activity measured using an automated, video-‐based behavior recognition system. Mealtime associated activity persisted during meal omission tests in constant dark, ruling out hourglass and interval timing. Anticipation of 3 or more daily meals challenges multiple entrained oscillator models, and may involve phase discrimination or entrainment of non-‐circadian oscillators. In parallel with behavioral studies, clock gene and immediate

58

early gene correlates of 2-‐meal anticipation in rats are being mapped, as a first approach to determine how and where multiple meal timing is represented in the brain.

S2.2 IT IS POSSIBLE TO ANTICIPATE TO PHEROMONES? A STUDY IN NEWBORN RABBITS FED BY ENTERAL NUTRITION Ivette Caldelas. Depto. Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México. During early development the European rabbit lives in complete darkness in subterranean burrows and isolated from potentially entraining environmental signals other than the vital, once-‐daily visit of their mother to nurse the young rabbits. Several studies demonstrated that newborn anticipate to the regular arrival of the lactating female with an increase in general arousal, in body temperature, and other parameters. The anticipatory changes persist even if the newborn rabbits are isolated from the mother. The once-‐daily nursing is an effective non-‐photic synchronizing signal for rabbit newborn circadian system; previous studies of our group demonstrate that shifts in the nursing schedule produce phase shifts at behavioural, physiological and molecular level. However, it is not clear which sensory information during nursing has influence on the newborn circadian system. In this study we evaluate the participation of the female rabbit pheromone 2-‐metyl-‐2but-‐enal (2MB-‐2) contained in the mother’s milk, as a non-‐photic synchronizing signal. We determined the effect of once-‐daily exposure to 2MB-‐2 on the temporal pattern of locomotor activity, core body temperature, and serum metabolites in newborn rabbits fed by enteral nutrition. For this reason newborn rabbits (n=125) were separated from their mothers at birth and maintained in L:L, from the postnatal day 1 (P1) to P8. Rabbits were randomly assigned to one of three conditions: fed by their mother (NAT); exposed to a 3min pulse of the pheromone 2MB-‐2 and fed artificially (2MB-‐2), or exposed to 3 min pulse of water and fed artificially (CON), once every 24 hours. On P8 the rabbits were sacrificed at different times of the day and serum glucose, triacylglycerides and free fatty acids levels were determined. At behavioural and physiological level, the NAT group showed clear diurnal rhythmicity, with a conspicuous rise approximately two hours before nursing. The newborn exposed to 2MB-‐2 showed similar temporal patterns to those observed in the NAT group, in addition this group displayed the anticipatory component two ours previous the 2MB-‐2 exposition. In contrast, the CON group exhibited atypical temporal patterns of activity and temperature, lacking the anticipatory component. At metabolic level, three groups exhibited a diurnal pattern with similar phases in serum glucose, triacylglycerides and free fatty acids. The behavioural and physiological data indicate that the daily exposure to the maternal pheromone 2MB-‐2 had an influence on the expression of these patterns, suggesting that this maternal volatile has an effect on the central oscillators of the rabbit pups, presumably as a non-‐photic synchronizing signal. On the other hand, the metabolic

59

data indicate that the exposure to the maternal pheromone 2MB-‐2 had no effect at peripheral level. Supported by ICYTDF/261/2009, PAPIIT IN219510-‐3 and CONACyT 131314. Keywords: Anticipatory activity, Development, Rabbits

S2.3 RELATIONSHIP BETWEEN THE FOOD-‐ENTRAINABLE OSCILLATOR (FEO) AND METHAMPHETAMINE SENSITIVE CIRCADIAN OSCILLATOR (MASCO) Michael Menaker, Pinar Pezuk, Michael Sellix. University of Virginia, USA. Who is conducting the circadian orchestra? The circadian orchestra is composed of autonomous circadian oscillators in most cells, tissues and organs of the body. As in a musical orchestra the individual players must either be temporally coordinated in order to make either recognizable music or adaptively appropriate responses to the environment. In both cases temporal coordination is achieved by the action of a “conductor,” employing either a baton or a set of (poorly understood) neurochemical signals. The classical answer to the question: “Who is conducting the circadian orchestra?” is “the suprachiasmatic nucleus (SCN).” This is correct but not complete since there are other central oscillators that can take over in the absence of the SCN. In SCN lesioned rodents these other conductors can be evoked by either temporally restricted feeding regimens (FEO) or chronic exposure to methamphetamine (MASCO). Neither the anatomical location(s) nor the molecular mechanism(s) that generate their rhythmicity are known at present. Until recently the evidence for their existence rested primarily on behavioral data. I will present new data demonstrating that non-‐SCN conductors regulate the phases of circadian oscillators in individual organs. I will briefly discuss two additional questions: What is the relationship if any, between SCN and non-‐SCN conductors? Are FEO and MASCO one or two distinct conductors? Keywords: suprachiasmatic nucleus, food entrainable oscillators, circadian organization

S2.4 FOOD ENTRAINMENT OF PERIPHERAL CLOCKS EVALUATED BY IN VIVO IMAGING Yu Tahara, Shigenobu Shibata. Department of Physiology & Pharmacology, School of Advanced Science and Engineering, Waseda University, Japan. Food entrainable oscillator (FEO), it could be inducible by daily restricted feeding (RF) paradigm to nocturnal mice, can elicit the behavioral rhythm of food anticipatory activity

60

(FAA) before the restricted feeding time, and also entrain the peripheral circadian clocks to equip the food metabolism by inducing the clock-‐regulated metabolic genes. Recently we investigated the mechanism of food entrainment of clock gene expression rhythms in the liver, and found the different responses of clock gene to nutrients signal and also to insulin (Tahara et al., 2011). We also demonstrated that interaction of food volume and starvation interval is key factor for entrainment of peripheral clock by RF (Hirao et al., 2010). There are no reports evaluating peripheral clock by in vivo imaging. In comparison with in vitro or ex-‐vivo systems, monitoring clock gene expression in vivo has several merits, for example, monitoring same animal with different experimental conditions, simultaneously monitoring clock gene expression rhythm from several peripheral organs, and reducing the artifact of animal scarification and medium change. In this symposium, we address the basic data for monitoring Per2 gene expression rhythm of Per2::luc KI mouse using IVIS in vivo monitoring system, and RF-‐induced entrainment of peripheral clocks including liver, kidney and salivary glands. In clinical experiments, outputs from suprachiasmatic nucleus-‐dependent light entrainable oscillator (LEO) are examined under constant routine feeding schedule to reduce FEO. First, we examined Per2 gene expression rhythm under 4 or 6 mealtimes per day with same amount of food for each mealtime, and total food volume per day was reduced to 80, 70, or 60% of free feeding schedule. Although we do not know the mechanism, bigger phase angle was observed in kidney comparing among kidney, liver and salivary gland. Peripheral clocks were entrained by LEO not by FEO, and phase angle was advanced with negative dependency of daily food volume. Under 2 meals per day conditions (80% food), phase of peripheral clock was slightly and insignificantly advanced in ZT6/ZT18 RF group than in ZT0/ZT12 group. Under the condition of one meal per day, phase of peripheral clock was strongly advanced to daytime. Now 3 meals per day (ZT2, ZT10, ZT18 group or ZT6, ZT14, ZT20 group), or (ZT0 ZT6, ZT12 group or ZT12, ZT18, ZT0 group) are under experiments. We are also interesting in T=25 cycle experiment for 2 meals and 5 meals per day. We will discuss the usefulness and limitation of in vivo imaging system as a tool for evaluating circadian system. Keywords: imaging, Per2::luc, restricted feeding

S3.1 CIRCADIAN CLOCKS SUPPORT HEALTH BY ORCHESTRATING REMOVAL OF OXIDATIVE DAMAGE Jadwiga Maria Giebultowicz, Natraj Krishnan, Paura Michelle Beaver. Oregon State University, USA. Circadian clocks control a large number of biological processes that are vital to maintaining health, including daily sleep/activity patterns, oscillations in neuronal, physiological, and metabolic functions. We reported that the levels of endogenous reactive oxygen species (ROS) fluctuate in a daily rhythm in heads of wild type Drosophila. In mutants with

61

disrupted clocks, ROS levels are non-‐rhythmic and significantly higher. Clock mutant flies show increased accumulation of ROS and oxidative damage over adult lifespan. In particular, they show increased levels of protein carbonyls, which are major products of protein oxidation. These data suggest that the circadian system may be involved in protein homeostasis by regulating repair or removal of damaged proteins. Indeed, we determined that enzymes responsible for the rate limiting step of glutathione biosynthesis, the catalytic and modulatory subunits of glutamate cysteine ligase (GCLc and GCLm), show coordinated circadian expression in fly heads. In addition, the important protein repair enzyme methionine sulfoxide reductase A (MSRA) also showed rhythmic expression profiles. These rhythms were abolished in flies with a mutated clock further supporting their regulation by the circadian system. Some protein modifications, such as carbonyl formation cannot be repaired, yet we observed distinct circadian fluctuations in the levels of carbonylated proteins. To address the mechanism of these fluctuations, we measured activity of proteasome around the clock and revealed strong circadian oscillations in proteolytic activity of the proteasome complex. Proteasomal activity oscillations were abolished in mutants with disrupted clock mechanism. To understand how rhythms of proteasomal activity are generated, we are investigating the relationships between levels of locomotor activity, oxidative damage and proteasome function. Several lines of evidence suggest that proteasomal rhythms persist when activity levels or protein carbonylation levels are altered, leading to a conclusion that proteasome activity is directly controlled by the clock rather than a secondary response to other rhythms. In summary, our data show that circadian clocks play important roles in protein homeostasis by coordinating pathways involved in the repair and removal of oxidatively damaged proteins. Keywords: Drosophila, Glutathione biosynthesis, proteasome activity.

S3.2 THE SCN NEURONAL NETWORK AND ITS ADAPTATION TO TEMPORAL CHALLENGES Horacio O. de la Iglesia. Department of Biology, University of Washington, Seattle, USA. Communication between neuronal oscillators within the suprachiasmatic nucleus is critical for the integrity of the circadian system and for the synchronization of the system to environmental light. The mechanisms underlying this communication are only now beginning to be understood. Here we report that mice carrying a heterozygous mutation of the NaV1.1 voltage-‐gated sodium channel, which is the primary channel in GABAergic interneurons, have longer circadian period than their wildtype littermates. Heterozygous mice (HETs) also lack light-‐induced phase delays and although they entrain to a 12:12 light-‐dark (LD) cycles they have slow resetting in response to abrupt delays or advances of the LD cycle. In contrast to these impaired photic responses, HETs have exacerbated masking

62

behavior by nocturnal light exposure and a normal electroretinogram. Analysis of light-‐induced gene expression in response to nocturnal light pulses revealed that although HETs show normal induction of cFos and Per1 within the ventral SCN they show blunted increased gene expression within the dorsal SCN, which is characteristic in their wildtype littermates. Intracellular calcium imaging in SCN slices confirmed that electrical stimulation of optic chiasm elicits increased calcium responses in ventral and dorsal neurons but these responses are diminished in HETs and almost absent in homozygote KOs for the channel. Remarkably, the abnormal behavioral circadian phenotype in HETs can be rescued by the systemic administration of GABA-‐transmission enhancing drugs. Our data indicate that impaired GABA-‐mediated SCN interneuronal communication leads to major changes in the function of the master circadian pacemaker and to a failure of the clock to properly respond to temporal challenges such as jetlag. Keywords: SCN, entrainment, GABA

S3.3 BROOD-‐RELATED PLASTICITY IN CIRCADIAN RHYTHMS OF BUMBLE BEE QUEENS Guy Bloch, Ada D. Eban-‐Rothschild. Department of Ecology, Evolution and Behavior, The Hebrew University of Jerusalem, Israel. Unlike most animals in which activity with no circadian rhythms is pathological or linked to deteriorating performance, worker bees and ants naturally care for their sibling brood around the clock with no apparent ill effects. Here we tested whether bumble bee queens who care alone for their first batch of offspring are also capable of a similar chronobiological plasticity. We monitored locomotor activity of Bombus terrestris queens at various life cycle stages, and queens for which we manipulated the presence of brood or removed the ovaries. We found that gynes typically emerged from the pupae with no circadian rhythms, but after several days showed robust rhythms that were not affected by mating or diapauses. Colony-‐founding queens with brood showed attenuated circadian rhythms, irrespective of the presence of ovaries. By contrast, queens that lost their brood switched again to activity with strong circadian rhythms. The discovery that circadian rhythms in bumble bee queens are regulated by the life cycle and the presence of brood suggests that plasticity in the circadian clock of bees is ancient and related to maternal behaviour, and is not a derived trait that evolved with the evolution of the worker caste. Keywords: social behavior, plasticity, maternal behavior

63

S3.4 DIURNAL ACTIVITY IN A SMALL DESERT RODENT: SELECTIVE FORCES, ADAPTATIONS AND CONSTRAINS Noga Kronfeld-‐Schor, Levy Ofir, Dayan Tamar. Tel Aviv University, Israel. In most mammals, activity is limited to a specific part of the diel cycle. Because anatomical, physiological, and behavioral adaptations that have evolved for diurnal or nocturnal activity differ, they may restrict animals to a certain activity phase. However infrequently, species may overcome these evolutionary constraints and alter their activity patterns. The temporal shift in activity patterns may evolve as a response to more suitable climatic conditions, as a consequence of biotic interactions, or in response to a combination of both abiotic and biotic pressures. Climate, biotic interactions, and evolutionary constraints may have similar, different, or even conflicting effects on activity patterns. An excellent model system for the study of the selective forces affecting activity patterns occurs in Israel, where two ecologically similar congeneric species of spiny mice have a unique temporal partitioning in activity patterns: the common spiny mouse, Acomys cahirinus, is nocturnal, while the golden spiny mouse, A. russatus, is diurnally active. Studies suggested that the golden spiny mouse was competitively excluded into diurnality by the common spiny mouse. Under field experimental conditions, the removal of common spiny mice from the joint habitat enabled golden spiny mice to be active also during the night, although their activity remains largely diurnal. Moreover, most golden spiny mice are nocturnal under controlled laboratory conditions, and field individuals turn nocturnal within a day when transferred to the laboratory. Evolution is cumulative, and the conditions favoring the initial evolution of a trait and its subsequent maintenance need not be identical. There are two major nonexclusive hypotheses to explain the current diurnal activity of golden spiny mice. The genus Acomys evolved as a savanna rodent of tropical Africa; hence it could be speculated that Acomys species developed adaptations to high ambient temperatures that preceded their adaptation to water shortage. These adaptations enabled the golden spiny mouse to switch its activity into the thermoregulatory challenging diurnal niche upon encountering the common spiny mice. Over evolutionary time, golden spiny mice underwent further adaptations to activity during this phase of the diel cycle, and consequently, in absence of common spiny mice, golden spiny mice remain primarily diurnal. Thus their current diurnal activity reflects past selective forces to which they are now highly adapted; in the case of its interaction with the common spiny mouse one might invoke the ‘ghost of competition past’. However, results of a model we have recently developed show that golden spiny mouse physiology and habitat structure make diurnal activity energetically advantageous, suggests that climate driven diurnality may serve as an alternative hypothesis. Thus, if golden spiny mice were already adapted to high ambient temperatures when the species

64

arrived the Judean desert, the diurnal activity of golden spiny mice in this region may have evolved regardless of competition. Keywords: Temporal partitioning, Activity patterns, Evolution

S4.1 NEMO KINASE CONTRIBUTES TO CORE PERIOD DETERMINATION BY SLOWING THE PACE OF THE DROSOPHILA CIRCADIAN OSCILLATOR Paul E. Hardin, Wangjie Yu, Jerry H Houl. Texas A&M University, Department of Biology and Center for Biological Clocks Research, United States. The Drosophila circadian oscillator is comprised of transcriptional feedback loops that are activated by CLOCK (CLK) and CYCLE (CYC) and repressed by PERIOD (PER) and TIMELESS (TIM). The timing of CLK-‐CYC activation and PER-‐TIM repression is regulated post-‐translationally, in part through rhythmic phosphorylation of CLK, PER and TIM. Although kinases that control PER and TIM levels and subcellular localization have been identified, additional kinases are predicted to target PER, TIM and/or CLK to promote time-‐specific transcriptional repression. We screened for kinases that alter circadian behavior via clock cell directed RNA interference (RNAi) and identified the proline-‐directed kinase nemo (nmo) as a novel component of the circadian oscillator. Both nmo RNAi knock down and a nmo hypomorphic mutant shorten circadian period, whereas nmo overexpression lengthens circadian period. CLK levels increase when nmo expression is knocked down in clock cells, whereas CLK levels decrease and PER and TIM accumulation is delayed when nmo is overexpressed in clock cells. These data suggest that nmo slows the pace of the circadian oscillator by altering CLK, PER and TIM expression, thereby contributing to the generation of a ~24-‐hour circadian period. Keywords: phosphorylation, clock proteins, Drosophila

S4.2 CIRCADIAN RHYTHM OF DROSOPHILA BEHAVIOR; DIFFERENT BRAIN-‐SITES REQUIRED FOR LOCOMOTOR AND COURTSHIP Norio Ishida. National Institute of Advanced Industrial Science and Technology, Japan.

65

S4.3 A POSSIBLE SYNAPTIC TRANSMISSION INVOLVED IN THE DROSOPHILA CIRCADIAN CLOCK 1Kenji Tomioka, 1Yujiro Umezaki, 1Hideki Nakagoshi, 2Koouji Yasuyama. 1Graduate School of Natural Science and Technology, Okayama University, Japan. 2Department of Natural Science, Kawasaki Medical School, Japan. Circadian locomotor rhythms of Drosophila melanogaster are controlled by a neuronal circuit composed of approximately 150 cerebral clock neurons that are roughly classified into seven groups; three are located between the optic lobe and the protocerebral lobe and called s-‐LNv, l-‐LNv and LNd, other three groups called DN1, DN2 and DN3, are located in the dorsal part of the brain, and the remaining one called LPN is located in the lateral posterior protocerebrum. In the circuit, s-‐LNv cells expressing pigment-‐dispersing factor (PDF) play an important role in organizing the circadian clock network. Recent studies imply that unknown chemical neurotransmitter(s) (UNT) other than PDF is also expressed in the PDF-‐positive neurons. To explore its role in the clock network, we examined the circadian locomotor rhythms of transgenic flies in which chemical synaptic transmission in PDF-‐positive neurons was blocked by tetanus toxin light chain (TNT) selectively expressed in the PDF-‐expressing neurons. In constant darkness (DD), the flies showed a clear free-‐running rhythm, which was significantly different from pdf null mutants. Under constant light conditions (LL), however, they showed complex rhythms with a short and a long period component. The UNT is thus likely involved in the synaptic transmission in the clock network and its release caused by LL leads to arrhythmicity. Immunocytochemistry revealed that LL induced phase separation in TIMELESS (TIM) cycling among some of the PDF-‐positive and PDF-‐negative clock neurons in the transgenic flies. These results suggest that in addition to PDF, UNT plays an important role in the Drosophila circadian clock network, and activation of PDF pathway alone by LL leads to expression of the complex locomotor rhythm through desynchronized oscillations among some of the clock neurons. Keywords: Drosophila, circadian, synaptic transmission

S4.4 THE CONSEQUENCE OF ARHYTHMICITY IN THE GERMAN COCKROACH How-‐Jing Lee. Dept. Entomology, National Taiwan University, TAIWAN. The arhythmicity can be defined as dismantle of clockwork, interruption of time signal pathway, or transition state of phase shifting. The function of a circadian clock is to mediate physiological condition in order to perform behavior to synchronize with environmental

66

cycle. It has a detrimental effect once the arhythmicity is caused by the malfunction of clockwork. However, the effects of arhythmicity in another two conditions are unclear. When the output signal pathway is blocked, two results might be occurred. If this interruption happens at the first level of signal cascade, this individual has the same consequence as the one with dysfunction of clockwork. It would be complicated if the signal is blocked at the second level or first level of another pathway. Female adults of the German cockroach express arrhythmic locomotion, but their ERG show rhythmic. Under this situation, the arhythmicity in locomotion represents an adaptation for certain environmental condition or life stage. The third type of arhythmicity indicates a temporary chaotic physiological condition due to unsynchronized clock with Zeitgeber during phase shifting. If a constant and rapid phase shifting occurs, this arhythmicity can shorten lifespan and decrease fecundity. The underlying mechanisms will be discussed. Keywords: Blattella germanica, circadian clcok, phase shifting

S5.1 ON THE SOCIAL REGULATION OF CIRCADIAN TIMEKEEPING William J. Schwartz, Premananda Indic, Matthew J. Paul. Univ. Massachusetts Medical School, USA. Our knowledge of the circadian system of mammals at the molecular, cellular, tissue, and organismal levels is growing, mostly based on experiments using singly-‐housed rodents in plastic cages with temperature, humidity, and access to food rigidly controlled. Of course, in nature many species do not live out their lives in seclusion but instead within communities with highly developed social structures. Social cues may play an important role in modifying activity rhythms by enabling animals to synchronize their behaviors to achieve common goals or, alternatively, to avoid each other to lessen competition for a limited resource. Relatively little is known about the mechanisms for such inter-‐individual temporal adaptations, or how the circadian system might be involved. There are reasons to believe that the efficacy of social cues might require that cohabitants be in direct physical contact for a relatively long period of time, effects not easily reproduced by indirect or pulsatile social contacts. Historically, however, the problem has been that the usual methods of recording daily activity fail to distinguish the activity of individual animals housed together in the same cage. We have begun to study the conditions under which cohabitation effects might be revealed in the laboratory by cohousing hamsters and mice with their conspecifics, exploiting miniature implantable devices (ibuttons) for longitudinally recording body temperature and mathematical tools (wavelet transforms) for rigorously analyzing such non-‐stationary datasets. Social regulation of the rhythmicity of individuals may be a critical factor for a group's adaptation to the natural habitat (including for blind individuals), and a better understanding of this level of biological organization will likely generate a more complex, but ultimately more comprehensive, view of clocks and rhythms.

67

S5.2 ENTRAINMENT BY FOOD RESTRICTION Jorge Mendoza. Institute of Cellular and Integrative Neurosciences, Centre National de la Recherché Scientifique UPR3212, University of Strasbourg, France. The circadian clock, contained in the hypothalamic suprachiasmatic nucleus (SCN), aligns behavioural and physiological processes with the solar cycle and orchestrates peripheral oscillators in the rest of the body. Whereas peripheral circadian oscillators (including, of course, the mysterious unknown food-‐entrainable oscillator) can be entrained by feeding signals, the SCN appears to be refractory to these stimuli. However, under particular feeding conditions, the SCN clock may also receive and respond to food/metabolic cues, modulating the circadian gene expression and behaviour. Homeostatic and hedonic aspects of food seem to be relevant for these effects on the clock, indicating that the circadian system is thus intimately linked to the central pathways which appear to be involved in some kind of feedback loop to bond feeding, metabolic and reward state to the SCN. Supported by CNRS, University of Strasbourg and NEUREX Keywords: suprachiasmatic, feeding, reward

S5.3 ENTRAINMENT OF CIRCADIAN RHYTHMS BY CYCLES OF AMBIENT TEMPERATURE IN MAMMALS Roberto Refinetti. University of South Carolina, USA. A master circadian pacemaker located in the suprachiasmatic nuclei (SCN) controls circadian rhythms in mammals. This pacemaker receives monosynaptic input from the eyes, which allows it to be entrained by environmental cycles of light and darkness. In several mammalian species, it has been demonstrated that the rhythm of locomotor activity can be entrained not only by cycles of light and darkness but also by other cycles of environmental stimuli, such as food availability, exercise/arousal, and ambient temperature. Entrainment by ambient temperature cycles has been documented in a few (homeothermic) mammalian species. It has generally been found that the light-‐dark cycle is a stronger entraining agent than the ambient-‐temperature cycle. However, in very few studies were animals exposed simultaneously to congruous or conflicting environmental cycles of illumination and temperature. In these few studies, little attention was given to the distinction between masking effects and truly entraining effects of the environmental cycles. Studies still ongoing in the author’s laboratory have investigated the rhythm of locomotor activity in

68

mice exposed simultaneously to light-‐dark cycles and cycles of ambient temperature. Mice have been kept in individual cages with access to running wheels. Their rhythms of locomotor activity have been consistently shown to free-‐run in constant darkness and to be entrained separately by environmental cycles of light and darkness and of ambient temperature. When the two environmental cycles are presented simultaneously with different cycle lengths (for example, 23 h and 24 h) with only a few hours of stimulation per cycle, the light-‐dark cycle does not act as a stronger synchronizer than the temperature cycle. The behavior of the mice often shows a dissociation into two components that seems to reflect the separate entrainment of two pacemakers or a complex mixture of masking and entrainment of a single pacemaker. At least in mice, ambient temperature seems to be as strong an entraining stimulus as light. It is likely that previous observations of the predominance of photic stimuli in entrainment were in fact observations of the predominance of photic masking (and not of photic entrainment). The studies with competing environmental cycles described here constitute an early effort to understand the concerted action of multiple environmental synchronizers, thus providing a more ecologically-‐realistic laboratory setting for the study of the circadian organization of behavior. Keywords: behavior, circadian, non-‐photic entrainment

S5.4 BRAIN REWARD: STIMULATION FOR NONPHOTIC PHASE REGULATION OR JUST A GOOD TIME? J. David Glass, Jessie Guinn, Allison J. Brager. Kent State University, USA. Circadian clock timing is regulated by various nonphotic stimuli, including social interaction, being awakened, drugs, exercise, novelty, temperature and arousing or motivating states accompanying behavioral and other manipulations. Given the role of hedonic (reward-‐based) drive in motivating survival-‐dependent overt activity, it heuristically seems reasonable that central reward signaling also could contribute to the circadian timing of such behaviors. However, reports that presumably rewarding stimuli in rodents, including sex and palatable food presentation have little circadian clock-‐shifting effect argue against this idea. Reward stimuli act on a network consisting of the mesolimbic dopamine pathway (including dopaminergic neurons in the ventral tegmental area [VTA]) and its interaction with the mesopontine system (including cholinergic neurons of the pedunculopontine tegmentum [PPT] and the laterodorsal tegmentum [LDT]). Pathways conveying phase-‐resetting signaling to the SCN clock are the intergeniculate leaflet (IGL) and midbrain raphe nuclei. Notably, the IGL area receives cholinergic input from the mesopontine system, and cholinergic stimulation of the IGL induces nonphotic phase-‐shifts. Here we present evidence that the SCN circadian clock indirectly receives phase-‐resetting input from the brain systems

69

that register the reinforcing effects of natural and drug-‐related reward. In experiment 1, a phase-‐resetting effect of VTA activation was examined in hamsters using electrical VTA stimulation at ZT 6-‐7, immediately followed by release to DD (Aschoff type II procedure). This stimulation produced phase-‐advance shifts of 57±30 min (range; 23-‐148 min). In experiment 2, the effects of voluntary wheel-‐running (a rewarding phase-‐resetting stimulus) at ZT 6-‐7 on the activity of VTA, PPT and LDT cells was estimated by Fos expression. This treatment increased the number of Fos-‐immunoreactive cells in these areas by 454±46%, 491±95% and 582±100%, respectively vs. nonrunning controls. In experiment 3, direct stimulation of the VTA with cocaine (that advances circadian phase when injected systemically at midday) induced phase-‐advance shifts of 78±12 min. Collectively, these observations suggest a possible scheme whereby natural and drug activation of the mesolimbic/pontine reward system modulates circadian phase via a cholinergic projection to the IGL, with subsequent activation of geniculohypothalamic phase-‐resetting input to the SCN. Understanding such processes is important for improving knowledge of how drugs of abuse and nonphotic signaling pathways interact to produce the circadian dysfunction associated with addiction. NIH grant NS35229 to JDG Keywords: reward, exercise, cocaine

S6.1 PHOTOPERIODIC CONTROL OF INNATE AND ADAPTIVE IMMUNITY Brian J. Prendergast. University of Chicago, USA. Many organisms exhibit annual cycles in reproductive physiology and in immune function. Seasonal changes in day length figure prominently as a proximate cue capable of driving these cycles. Whereas mechanisms by which day length affects reproduction are well elaborated, far less is known about how the photoperiod engages seasonal changes in immunocompetence. And although humans are largely aseasonal breeders, we exhibit robust seasonal rhythms in disease, morbidity and mortality. In Siberian hamsters and laboratory (Wistar) rats, day length manipulations alter blood leukocyte phenotypes, and result in markedly different immune responses to a variety of antigen/pathogen challenges. Our research has focused on whether common or distinct neural and endocrine mechanisms mediate effects of day length on the reproductive and immune systems in these species. Work to be presented will address the respective roles of the circadian system, pineal melatonin, thyroid hormone catabolism, and gonadal hormone production in the genesis of seasonal rhythms in immunocompetence. Keywords: immune system, photoperiod,

70

S6.2 DYSREGULATION OF INFLAMMATORY RESPONSES BY CHRONIC CIRCADIAN DISRUPTION 1Oscar Castanon-‐Cervantes, 1Chris Ehlen, 1Ketema Paul, 2Karen Gamble, 1Alec Davidson, 1Morehouse School of Medicine, USA. 2University of Alabama at Birmingham, USA. Chronic disruption of circadian timing in shift work or during chronic jet lag in animal models leads to a higher risk of several pathologies. Many of these conditions in both shift workers and experimental models share the common risk factor of inflammation. In this study, we show that experimentally induced circadian disruption altered innate immune responses. Endotoxemic shock induced by LPS was magnified, leading to hypothermia and death after four consecutive weekly 6-‐h phase advances of the light/dark schedule, with 89% mortality compared with 21% in unshifted control mice. This may be due to a heightened release of proinflammatory cytokines in response to LPS treatment in shifted animals. Isolated peritoneal macrophages harvested from shifted mice exhibited a similarly heightened response to LPS in vitro, indicating that these cells are a target for jet lag. Sleep deprivation and stress are known to alter immune function and are potential mediators of the effects we describe. However, polysomnographic recording in mice exposed to the shifting schedule revealed no sleep loss, and stress measures were not altered in shifted mice. In contrast, we observed altered or abolished rhythms in the expression of clock genes in the central clock, liver, thymus, and peritoneal macrophages in mice after chronic jet lag. We conclude that circadian disruption, but not sleep loss or stress, are associated with jet lag-‐related dysregulation of the innate immune system. Such immune changes might be a common mechanism for the myriad negative health effects of shift work. Keywords: Immune, Jet lag

S6.3 LABORIOUS PHASE SHIFTING (LPS): FOLLOWING THE PATHWAYS OF IMMUNE-‐CIRCADIAN COMMUNICATION. Diego A Golombek, José M. Duhart, María A. Juliana Leone, Natalia Paladino. Universidad Nacional de Quilmes / CONICET, Argentina. Bidirectional interactions between the immune and the circadian systems have been under intensive study in recent years. We have previously reported that peripheral immune stimuli are capable of altering behavioral circadian outputs such as locomotor activity rhythms. We are now assessing which are the molecular and cellular mediators between the immune system and the mammalian biological clock. Previous results from our laboratory showed that low dose administration of LPS at CT-‐15 induced photic-‐like phase

71

delays in wheel running rhythms. We now show that this response is mediated by TNF-‐alpha since i.c.v. administration of soluble TNF-‐alpha receptor (but not IL-‐1β] antagonism) prior to LPS stimulation inhibited phase shifts. Moreover, TLR4 (Toll-‐Like Receptor 4)-‐null mice exhibited significantly smaller phase delays as well as absence of inhibition of wheel-‐running activity and c-‐Fos and Per1 induction in the PVN (but not the SCN) after LPS stimulation, confirming a role for this receptor in the effects of immune stimuli on the circadian clock. Since we have previously reported that SCN astroglia is an interface in immune-‐circadian modulation, we also analyzed the effects of cytokine administration on clock gene expression of these cells, and found that TNF-‐alpha reduces per1-‐luc expression in SCN astrocytes. Also, conditioned media from immune-‐challenged SCN astrocytes affected per1-‐luc expression in NIH-‐3T3 cells. In summary, we have characterized some of the pathways that mediate the effects of immune stimuli on the mammalian circadian clock. We show that the molecular pathways involve the LPS receptor TLR4 and, at least at central level, the action of TNF-‐alpha. Moreover, we found new evidences that place astrocytes as mediators of the immune-‐circadian communication. Keywords: : lipopolysaccharide (LPS), proinflammatory cytokines, astroglia

S6.4 THE CIRCADIAN CLOCK CONTROLS THE RESPONSE OF T CELLS TO ANTIGEN 1,2Nicolas Cermakian, 1,2Erin E. Fortier, 3Julie Rooney, 1 Hugues Dardente, 3 Marie-‐Pierre Hardy, 3Nathalie Labrecque. 1Douglas Mental Health University Institute, 2McGill University, 3Maisonneuve-‐Rosemont Hospital Research Centre, Montreal, QC, Canada. Objectives: Circadian variations have been found in the immune system, including daily rhythms in circulating numbers of leukocytes and serum concentration of cytokines. Although the circadian clock is known to control various physiological systems, very little is known about the timing of events in the immune system. We hypothesized that the circadian clock controls T cell function. The aims of this study were: i) to identify clock gene expression in mouse lymph nodes (LNs); ii) to investigate T cell proliferation rhythms; and iii) to shed light on the molecular mechanisms underlying this rhythmic response. Methods: Adult WT and Clock mutant mice were entrained to a light-‐dark cycle and sacrificed at regular intervals over 24 hours. LNs were sampled and used to: i) Extract RNA and quantify clock gene expression by real-‐time PCR; ii) Measure T-‐cell proliferation following anti-‐CD3 stimulation; and iii) Examine the expression of T cell signaling proteins. Results: Our results show that LNs exhibit rhythmic clock gene expression. T cells show a robust circadian variation in proliferation after stimulation via the T cell receptor (TCR) that is lost in Clock gene mutant mice. In addition, the tyrosine kinase ZAP70, which is immediately downstream of the TCR in the T cell activation pathway, exhibits rhythmic expression. Conclusions: This is the first evidence for control of the T cell response by the molecular

72

clockwork. Our results have uncovered a novel mode of regulation of T-‐cell proliferation. This study linking circadian rhythms and the adaptive immune response may also provide cues for more efficient vaccination strategies. Funded by the Canadian Institutes of Health Research Keywords: T cells, immune response, clock genes

S6.5 TIME OF INFECTION DETERMINES THE RESPONSE OF THE INNATE AND ADAPTIVE IMMUNE SYSTEM 1Natali Nadia Guerrero, 1Roberto Carlos Salgado, 1Maricarmen Basualdo, 2Carolina Escobar, 1Rudol Marinus Buijs. 1Instituto De Investigaciones Biomédicas UNAM, México 2Facultad De Medicina, UNAM.

Most immune variables manifest daily fluctuations in their basal levels. Several studies have shown a relationship between changes in the light dark cycle and changes in the immune response. In order to examine the relationship between the biological clock and the immune system, we evaluated interleukin production, temperature response, brain activation and the antibody response after a LPS or DNP-‐OVA challenge respectively at two time points (ZT2 and ZT14) and during constant light conditions (L/L). Animals challenged during the night period (ZT14) showed a higher interleukin production that correlates with a higher antibody response and a more intense temperature response. Our results demonstrate that the intensity of the immune response is dependent on the time of the immune challenge and that both, the innate and the adaptive immune response are higher during the night period. This, we suggest is due to a higher inhibition of the SCN of the immune system during the day time than during the night in nocturnal rodents. This study was supported by Conacyt 79797 and PAPIIT IN215308

S7.1 ROLE OF THE NEUROPEPTIDE VIP IN THE MAMMALIAN CIRCADIAN SYSTEM Christopher S. Colwell. University of California, Los Angeles USA. In this talk, I will describe some of our work exploring the role of vasoactive intestinal peptide (VIP) in the mammalian circadian system. I will present evidence that VIP-‐deficient mice show profound deficits in their ability to entrainment to light. At the level of the SCN, these mice also show significant reduction in the magnitude of NMDA-‐evoked increase in the generation of action potentials. Furthermore, the light-‐induced increase in gene expression is reduced with a striking impact on the duration of the light-‐evoked Per1 signal

73

within the SCN observed in the mutant mice. On the output side, the VIP-‐deficient mice show profound disruptions in their circadian rhythms in locomotor activity, heart rate, and cortisol secretion. Interestingly, the loss of the peptide has much less impact on the body temperature rhythm that is normally considered a reliable measure of SCN output. As time allows, I will describe our understanding of the mechanisms underlying VIP on SCN physiology and emphasize the gaps in our knowledge. In summary, our evidence indicates that VIP is critical for the input and output of the circadian system. Keywords: vasoactive intestinal peptide, Neuropeptides, SCN

S7.2 NEUROPEPTIDE MODULATION OF THE INTRACELLULAR CALCIUM CONCENTRATION OF SUPRACHIASMATIC NUCLEUS NEURONS Charles N. Allen, Robert P. Irwin. Oregon Health & Science University, USA. Neuroactive peptides including vasoactive intestinal peptide (VIP), vasopressin (AVP) and nociceptin/orphanin FQ (OFQ) are expressed in the suprachiasmatic nucleus (SCN) and regulate the activity of the circadian clock. High-‐affinity OFQ receptors are functionally expressed in the SCN and OFQ reduces the magnitude of light-‐induced phase changes. Modulation of the intracellular calcium concentration ([Ca2+]i) is an essential component of the feedback loops that generate circadian rhythms and an important signal mediating photic entrainment. Increasing the [Ca2+]i is a critical link coupling excitatory synaptic neurotransmission to changes of gene expression. The [Ca2+]i may be increased by the opening of voltage-‐dependent Ca2+ channels due to membrane depolarization and Ca2+ uptake or release from intracellular stores. Neuropeptide regulation of Ca2+ homeostasis in SCN neurons may be important for synchronizing the phase of SCN neurons. However, the mechanisms by which neuropeptides act on [Ca2+]i to regulate circadian timing remain largely unknown. We, therefore, examined the effect of VIP, AVP, and OFQ on [Ca2+]i, by simultaneously imaging multiple SCN neurons maintained in hypothalamic slices. VIP reduced the [Ca2+]i of SCN neurons during the day, but had little effect at night. During the day, VIP lowered the [Ca2+]i to near nighttime levels. In contrast, AVP elevated [Ca2+]i during both the day and night. The data indicate that the VIP effects on [Ca2+]i were dependent, and the AVP effects independent of the action potential firing activity state of the SCN neuron. OFQ reduced the excitability of SCN neurons during the day with a reduction of the [Ca2+]i to levels observed in SCN neurons during the night. Stimulation of the retinohypothalamic tract at frequencies, which mimic environmental light signals evoked transient [Ca2+]i increases that were not altered by VIP. These data demonstrate that even when inhibited by VIP, SCN neurons still respond to light input. AVP slowly elevated the [Ca2+]i during both the day and night, without an apparent dependence on the

74

firing activity state of the neuron. These data are consistent with the induction of Ca2+-‐mediated transcriptional and translational changes to the clock, being at least in part dependent on the activity state of the neuron1 and modulation by neuropeptides2 via both external and internal sources of Ca2+. (1) Irwin, R.P. & Allen, C.N. (2007) Calcium response to retinohypothalamic tract synaptic transmission in suprachiasmatic nucleus neurons. J Neurosci, 27, 11748-‐11757. (2) Irwin, R.P. & Allen, C.N. (2010) Neuropeptide-‐mediated calcium signaling in the suprachiasmatic nucleus network. The European journal of neuroscience, 32, 1497-‐1506. Keywords: calcium, circadian rhythm, neuropeptide.

S7.3 HETEROGENEOUS AND NOVEL ELECTROPHYSIOLOGICAL ACTIONS OF OREXIN-‐A ON CIRCADIAN CLOCK NEURONS IN MICE Mino David Belle, Hugh David Piggins. Faculty of Life Sciences, University of Manchester. Endogenous near 24h (circadian) rhythms in mammals are generated by the main circadian clock in the hypothalamic suprachiasmatic nucleus (SCN). Here, the co-‐ordinated activity of cell autonomous oscillators communicates circadian phase information to the rest of the brain and body via neural and paracrine signals. Some key components of the SCN molecular clock (the so-‐called clock genes/proteins) are well characterized and include the period 1 (per1) gene and its protein product PER1. Synchronization of the SCN clock by environmental light (photic cues) and by stimuli that promote internal arousal (non-‐photic cues) results in daily rhythms in physiology and behaviour. The major light input pathway to the SCN neurons, and the neurotransmitters involved are well known. However, the neurochemical signals for non-‐photic information to the SCN are less understood, but there has been renewed interest in the arousal-‐promoting orexin/hypocretin neuropeptides. Orexins are synthesized by neurons localized mainly in the lateral hypothalamus (LH). Some LH neurons innervate the SCN, but it is unclear if and how orexins influence SCN neuronal activity, particularly those expressing Per1. In this study, we used immunocytochemistry to determine how orexin neurons innervate the mouse SCN, and electrophysiology to investigate the effects of orexin A (OXA) on SCN neurons expressing Per1::eGFP, as well as those neurons in which Per1::eGFP could not be detected (“Per1”, and “non-‐Per1” cells, respectively). Our results indicate that OXA immunoreactive axons were sparsely distributed in the SCN, but formed putative appositions onto Per1 neurons. Whole-‐cell recordings show that, overall, OXA evoked diverse actions in a large proportion of Per1 and non-‐Per1 cells, with clear time of day effects. In general, the effects of OXA were similar both in Per1 and non-‐Per1 cells, being predominantly inhibitory in most cells across the projected day-‐night cycle, with a small number of cells being excited by this neuropeptide

75

during the day. Our most overt finding is that duration and magnitude of the OXA-‐induced inhibition are larger at night. Investigation into the mechanisms underpinning OXA’s inhibitory actions revealed that during the day OXA acts in an indirect manner to suppress the activity of Per1 and non-‐Per1 cells, but at night direct mechanisms are recruited to strongly inhibit electrical activity in these cells. We conclude that OXA signalling in the SCN is complex and relies on intrinsic clock-‐controlled membrane properties of SCN cells to produce its diverse effects on SCN electrical activity. Supported by the BBSRC and the Wellcome Trust Keywords: Orexin A, Per1::eGFP, SCN

S7.4 NEUROPEPTIDES DERIVED FROM CLOCK-‐CONTROLLED GENES AS MEDIATING SIGNALS FOR THE OUTPUT OF SUPRACHIASMATIC CLOCK Qun Yong Zhou, University of California, USA. Circadian clocks drive daily rhythms in virtually all organisms. The suprachiasmatic nucleus (SCN) in the hypothalamus is the predominant circadian clock in mammals. To function as a pacemaker, the intrinsic timing signal from the SCN must be transmitted to different brain regions. Several secreted factors encoded by clock-‐controlled genes (prokineticin PK2, vasopressin, and cardiotrophin-‐like cytokine) have been implicated as output molecules. Gene disruption experiments have revealed that that PK2 and its receptor, PKR2 are important for the maintenance of robust circadian rhythms. PK2 and PKR2-‐null mice displayed significantly reduced rhythmicity for a variety of other physiological and behavioral parameters, including locomotor rhythms, sleep-‐wake cycle, body temperature, circulating glucocorticoid and glucose levels, as well as the expression of peripheral clock genes. A bacterial artificial chromosome transgenic mouse with EGFP reporter gene expression was driven by the PK2 promoter has allowed the mapping of an efferent projections map from the PK2-‐positive neurons in the SCN. The PK2-‐expressing neurons appear to project to many known SCN target areas, including the ventral lateral septum, medial preoptic area, subparaventricular zone, paraventricular nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamic area and paraventricular thalamic nucleus, supporting the role of PK2 as an output molecule. The altered estrus cycle and reduced fertility in PK2, PKR2 single and double heterozygous mice also indicates that PK2 signaling as a SCN output signal may also regulate reproductive behavior. Disruption of vasopressin-‐V1a signaling results in damped circadian rhythms in locomotor activity. Vasopressin as an output signal may also function within the SCN, regulating the expression level of PK2. Thus, there exists interaction between these two putative output molecules. It is likely that that

76

these output factors may play redundant or synergistic functions in eliciting the overall circadian rhythms. Keywords: output, prokineticin 2, vasopressin

S8.1 ENTRAINMENT OF THE CIRCADIAN CLOCKS ALONG ONTOGENESIS Alena Sumova, Marta Novakova, Kristyna Mateju, Rehab El-‐Hennamy, Lenka Polidarova, Martin Sladek. Department of Neurohumoral Regulations, Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic. In mammals, the central clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus is a complex structure composed of a web of mutually synchronized individual cell oscillators. The SCN clock entrains numerous peripheral clocks which drive tissue specific physiological functions in a time-‐programmed fashion. The present study addressed the question of when and how the molecular clockwork underlying circadian oscillations within the SCN and the peripheral circadian clocks develop during ontogenesis. Moreover, the potency of maternal non-‐photic and photic cues to entrain the central and peripheral clocks during prenatal and/or early postnatal ontogenesis was investigated. To test maternal entrainment of the fetal SCN clock, the pregnant mothers maintained under light/dark regime with 12 h of light of 12 h of darkness were exposed to a 6 h delay of the dark period, or they were maintained in constant light and subjected to daytime restricted feeding regime. 1-‐day-‐old pups were sampled throughout the 24 h cycle under conditions of constant darkness and light, respectively. To test photic entrainment during the early postnatal period, a light pulse was administered either during the subjective day or during the first or second part of the subjective night to pups at postnatal days 1, 3, 5 and 10 and the pups were sampled 30 min after each light pulse. In all studies, expression of clock and clock controlled genes and levels of their protein products were detected by in situ hybridization, RT-‐PCR and immunocytochemistry. The data revealed that development of synchronized molecular oscillations in the SCN and peripheral clocks is gradual and extends into postnatal period. The molecular oscillations develop earlier in the SCN than in the peripheral clocks. The maternal cues may phase-‐shift the SCN clock at the fetal stage when no or very faint synchronized molecular oscillations can be detected in population of the SCN neurons. The light sensitivity of the circadian clock develops gradually during postnatal ontogenesis. The results stress the importance of development of intercellular coupling among individual oscillators for development of the circadian SCN clock and its ability to synchronize the peripheral clocks. Sponsored by grants No. NT11474-‐4/2010 and 305090321

77

Keywords: circadian system, ontogenesis, entrainment

S8.2 MATERNAL MELATONIN, A CHRONOBIOTIC FOR FETAL CIRCADIAN CLOCKS 1María Josefa Seron-‐Ferre, 2Guillermo José Valenzuela, 1Claudia Lorena Torres-‐Farfán. 1ICBM, Facultad de Medicina, Universidad de Chile, Chile, 2Department of Women’s Health, Arrowhead Regional Medical Center, Colton, CA, USA, Preparation for the challenges to be experienced after birth is achieved during fetal life, including the circadian system. Regardless of the degree of maturity at birth, clock genes are present and oscillate in fetal organs in late gestation. In rodents, a number of experiments demonstrate that fetal circadian clocks are part of the offspring development program and develop in absence of a functional maternal suprachiasmatic nucleus (SCN) and by extension, of maternal circadian rhythms. However, fetal clocks require of maternal circadian signals for synchronization, which helps postnatal adaptation by setting the phase of overt postnatal rhythms. To date two chronobiotic signals: maternal feeding time and maternal melatonin have been studied. Our group has been interested in the study of maternal melatonin as homeostatic and chronobiotic hormone in the fetus and newborn. The fetus, that does not secrete melatonin, is exposed to clock time and photoperiod information through the maternal melatonin rhythm; since melatonin is one of the few maternal hormones crossing unaltered the placenta. Indeed, during fetal life melatonin receptors are widely distributed from nervous system to endocrine tissues, supporting that melatonin may act in different fetal clocks. In fetal primates a functional circadian oscillator is present in SCN and adrenal, and suppression of maternal melatonin during gestation (exposure to constant light) alters clock gene expression in the fetal SCN but not in the fetal adrenal gland. In addition, newborns from these mothers show a non entrained rhythm of body temperature. In rats, maternal pinealectomy during gestation results in desynchronization of the pup’s circadian rhythm of drinking. Both in the capuchin and the rat, daily replacement with exogenous melatonin restores entrainment of newborn circadian rhythms. An interpretation of the former result is that melatonin may entrain the phase of the fetal SCN, at this entrainment carries on to newborn rhythms. However, the lack of effect melatonin suppression on clock gene oscillation in the capuchin fetal adrenal, despite the presence of functional melatonin receptors suggests that the fetal SCN may not govern circadian function of the primate fetal adrenal. The possibility that the fetal adrenal is circadian clock independent from the fetal SCN was tested in the fetal rat. We found that at 18 days of gestation, age at which the fetal rat SCN does not show a rhythm of clock gene expression, in vivo the rat fetal adrenal is a peripheral oscillator, expressing clock genes and producing corticosterone in a circadian fashion. Oscillation of clock genes, steroidogenic protein StAR and melatonin receptor were maintained in vitro and responded directly to melatonin treatment. Thus in the rat, the fetal adrenal is a peripheral clock amenable to be entrained by melatonin. Whether maternal melatonin entrains the rat fetal adrenal in vivo

78

is currently being investigated. In adult mammals completeness of the circadian system, with a pacemaker (SCN) that commands peripheral oscillators, is an essential biological system involved in the regulation of almost all physiological functions. However, the data available suggest that circadian fetal organization differs with that of the adult and supports that from a circadian perspective the fetus may be considered as having autonomous peripheral circadian clocks entrained by the mother. Supported by Fondecyt 1090-‐381 and 1080649, Chile. Keywords: Fetal, melatonin, non-‐human primates

S8.3 THE DEVELOPING PACEMAKER: SENSITIVE BUT RESILIENT Fred C. Davis. Northeastern University, United States. The suprachiasmatic nucleus (SCN) differentiates over several days after its neurons become postmitotic. Early differentiation includes the expression of characteristic genes, the circadian cycling of genes, and the entrainment of cycles by maternal rhythms. Later differentiation includes synaptogenesis, afferent innervation, and responses to light. The product of differentiation is a structurally and functionally complex nucleus. Even before rhythms can be directly measured in the SCN, entrainment is possible. Part of the evidence for this is that, in hamsters, behavioral rhythms of pups can be entrained by conditions at or before E14, only a day after SCN neurogenesis is complete. These experiments show that incipient oscillations are especially sensitive to phase-‐setting perturbations. It is possible that such perturbations have long-‐lasting effects on the pacemaker if they occur during differentiation. An experiment to examine this found that the pacemaker is sensitive to repeated perturbations but that the effects on its functional properties are negligible. This and other experiments, such as SCN transplantation, suggest that SCN differentiation is remarkably stable and that functional properties arise through a resilient program of self-‐assembly. Keywords: development, SCN, entrainment

79

S8.4 THE RABBIT PUP, A NATURAL MODEL OF FOOD ENTRAINMENT 1Mario Caba, 1Elvira Morgado, 1Claudia Juárez, 1Enrique Meza, 2Angel I. Melo, 3Belisario Domínguez. 1Centro de Investigaciones Biomédicas, Universidad Veracruzana, Xalapa, Ver, México.2CIRA, CINVESTAV-‐UAT, México. 3Fac. Medicina Veterinaria, Universidad Veracruzana, Ver., Ver., México. Nursing in the rabbit occurs on a precise circadian schedule. Dams nurse their pups once daily for a brief period of less than 5 min every 24 h. Chronobiological studies demonstrates that rabbit pups have a daily anticipatory behavioral arousal just before the dam arrives to nurse either during the day or during the night. In addition to core body temperature, hormonal, metabolic and neural parameters are also entrained and shifted in parallel with daily nursing. During nursing, in addition to receiving milk, pups are exposed to several cues from the mother, and it is not clear which of these is the main entraining signal. In the present contribution we explored the effect of food on the entraining of locomotor behavior, plasma glucose, corticosterone and neural parameters in 7 day old rabbit pups. At postnatal day 1 an intragastric tube was placed by gastrotomy in the pups. Beginning the next day and for the rest of the experiment pups were fed with a milk substitute through the intragastric cannula either at 10:00 or 02:00. At postnatal days 5-‐7 pups exhibited behavioral arousal with an increase in locomotor behavior 60 min before milk formula infusion in comparison to values 90 and 60 min before and after feeding time. Glucose levels were lowest at the time of feeding (103 mg/dl) and four to 20 h after feeding oscillate between 120-‐180 mg/dl, similar to our previous experiments where pups were nursed by their mothers. Corticosterone was highest four h before feeding then decreased to trough concentrations 12-‐16 h after feeding, increasing again in anticipation of next feeding bout. In the brain, the suprachiasmatic nucleus has a rhythm of FOS and the clock protein, PER1, the product of the Per1 clock gene, that were not significantly affected by the feeding schedule. Conversely, supraoptic, paraventricular and tuberomammillar nuclei have a rhythm of both FOS and PER1 that shifts in parallel to scheduled feeding. Together with our previous and present contributions we conclude that rabbit pups are a natural model of food entrainment as food, in this case artificial milk, is a strong entraining signal for behavioral, hormonal, metabolic and neural parameters. Keywords: non-‐photic entrainment, lactation, corticosterone

80

S9.1 CIRCADIAN SYSTEM AND ITS DISRUPTION; EFFECTS ON GLUCOSE METABOLISM IN HUMANS Frank A. J. L. Scheer. Division of Sleep Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA. The dawn phenomenon “hyperglycemia upon awakening” in diabetic patients and healthy controls has awakened a great interest among clinicians and researchers in the daily fluctuation of glucose metabolism and its underlying mechanisms. There is clear evidence for a role of both the sleep/wake cycle and the endogenous circadian system in the daily rhythm in plasma glucose levels. The circadian system itself, thus independent of environmental or behavioral changes, has been reported to modulate glucose utilization, hepatic glucose production, insulin secretion, and insulin sensitivity. Multi-‐synaptic projections from the suprachiasmatic nucleus to liver, pancreas, and adipose tissue, humoral factors modulating uptake and/or output of glucose (such as cortisol and epinephrine which express robust endogenous circadian rhythms), and peripheral clocks in these tissues work in concert to optimally prepare the body for the daily fluctuations in feeding. Recently, it has been shown that disruption of this synchronization between feeding/fasting cycles and the central circadian pacemaker rapidly results in impaired glucose tolerance and decreased leptin concentrations in humans. These effects of desynchrony appear to be distinct from “just” eating at a different circadian time and cannot be explained by decreases in sleep quality. Together, this suggests these changes are due to disruption of the internal circadian system. The recent demonstration that variants in the melatonin receptor 1B gene (MTNR1B) and in the cryptochrome 2 gene (CRY2) are robustly associated with type 2 diabetes in humans provides compelling converging support that circadian pathways have a key role in glucose metabolism. Future studies are required to uncover the molecular and physiological consequences of circadian disturbances that underlie such changes in glucose metabolism and leptin secretion. These observations have led to a better understanding of the possible mechanisms underlying the increased risk of diabetes, obesity, and cardiovascular disease and may help in the development of countermeasures of circadian disruption and novel therapeutic strategies for the treatment of diabetes. Support: This work was supported by National Institutes of Health Grants R01-‐HL094806, R01-‐HL64815, K24-‐HL76446, and MO1-‐RR02635. Keywords: Circadian Rhythms, Glucose, Metabolism

81

S9.2 THE METABOLIC CLOCKWORK Akhilesh B. Reddy. University of Cambridge, UK. Our research is focussed on how the circadian (24 hour) clock works at the molecular level. We are particularly interested in non-‐transcriptional and biochemical oscillations that underlie circadian oscillations. We had originally observed that a peroxiredoxin (Prdx6) exhibited circadian rhythms of post-‐translational modification in mouse liver (Reddy et al. 2006; Current Biology). This finding came into play when we took an orthodox approach to determine if circadian rhythms could exist in eukaryotes in the absence of transcription. We used red blood cells, which have no DNA and therefore do not make new RNA, to answer this question. Remarkably, we found that circadian rhythms of oxidation and reduction of peroxiredoxins (and other metabolic oscillations) persisted for several days in red cells kept in cell culture (O’Neill & Reddy 2011; Nature). Once we had established this assay in Cambridge, we extended our observations to a primitive eukaryote, Ostreococcus tauri. Even though humans and these algae are separated by over 1000 million years of evolution, they share this basic mechanism (O’Neill et al. 2011; Nature). Keywords: Non-‐transcriptional, Peroxiredoxin, Algae

S9.3 CIRCADIAN CLOCKS IN ADIPOSE TISSUE 1Jeffrey M. Gimble, 1Gregory M. Sutton, 2Bruce A. Bunnell, 3Andrey A. Ptitsyn, 1Z. Elizabeth Floyd. 1Pennington Biomedical Research Center, Baton Rouge, LA, USA. 2Tulane University Medical Center, New Orleans, LA, USA. 3Whitney Laboratory-‐University of Florida, St. Augustine, FL, USA. Circadian rhythms are a critical factor underlying the endocrine and metabolic function of adipose tissue. The transcription factors and enzymes responsible for the maintenance of circadian rhythms have now been identified in adipose depots by multiple independent laboratories. Evidence of circadian mechanisms has been detected in adipose tissues at the stem cell, organ, and systemic levels. Using pre-‐adipocyte cell models in vitro, it has been established that Bmal1 and EPAS1 transcriptionally regulate adipogenesis. Likewise, dexamethasone and serum shock can be used to synchronize the circadian apparatus in primary cultures of human and murine adipose-‐derived stromal/stem cells. In vivo studies harvesting murine or human adipose tissues have demonstrated that at least 20% of the expressed genes in adipose tissues display an oscillatory rhythm based on transcriptomic

82

microarray analyses. The subset of genes displaying robust oscillations with similar phase and amplitude across all tissues includes the core of basic circadian and metabolic genes. However, the largest cohort of conservatively estimated rhythmic genes is distinct between metabolically active tissues. These transcriptomic analyses, along with serum measurements, have determined that glucose and lipid metabolism displays circadian regulatory features. Animal experiments and human epidemiological studies provide additional evidence that disturbances in circadian pathways are associated with an increased risk of diabetes mellitus and obesity. Thus, circadian mechanisms offer a putative target for the prevention and treatment of obesity and its related co-‐morbidities. Keywords: Metabolism, Clock Genes, Adipose tissue

S9.4 HYPOTHALAMIC NEUROPEPTIDES INVOLVED IN THE SCN CONTROL OF HEPATIC GLUCOSE PRODUCTION Andries Kalsbeek, Chun-‐Xia Yi, Ewout Foppen, Eric Fliers. Academic Medical Center (AMC), Dept Endocrinology & Metab, Meibergdreef 9, 1105 AZ Amsterdam & Neth Inst for Neuroscience, Dept Hypothalamic Integration Mechanisms, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands. The hypothalamic paraventricular nucleus (PVN) is an important target area of biological clock output as it harbors the neuro-‐endocrine neurons that control peripheral hormones such as corticosterone and thyroid-‐stimulating hormone, as well as the pre-‐autonomic neurons that control the sympathetic and parasympathetic branches of the autonomic nervous system (ANS). The master biological clock, located in the hypothalamic suprachiasmatic nuclei (SCN), uses its projections to these neuro-‐endocrine and pre-‐autonomic neurons in the hypothalamus to control daily hormone rhythms, e.g. adrenal corticosterone and pineal melatonin release. The SCN also plays an essential role in maintaining daily blood glucose concentrations. Using local intra-‐hypothalamic administration of GABA and glutamate receptor (ant)agonists we previously demonstrated how changes in ANS activity contribute to the daily control of plasma glucose and plasma insulin concentrations. Selective hepatic denervations evidenced that the ANS is also an important gateway for the SCN to transmit the (phase-‐shifting) effects of light to the glucoregulatory and clock gene machinery of the liver. Finally, using ICV administration of neuropeptides and/or their (ant) agonists we were able to delineate how the SCN may also “use” hypothalamic neuropeptide systems to control the daily plasma glucose rhythms. We found that the VIP-‐containing SCN outputs and the orexin-‐containing neurons in the lateral hypothalamus are important molecular links to modulate hepatic glucose production. On the other hand, PACAP release in the PVN turned out to be a strong stimulator of hepatic glucose production, but this effect does not seem to part of the circadian timing system. In our most recent experiments we investigated the possible involvement of vasopressin and

83

oxytocin in the circadian timing system. No evidence could be found for an involvement of the vasopressinergic SCN-‐projections. On the other hand, the oxytocin-‐containing PVN neurons do seem to be one of the targets for the SCN output. Another possible link between the circadian timing system and glucose homeostasis is the pineal release of melatonin. Despite the recent link of mutations in the melatonin receptor with an increased risk of 2 type 2 diabetes, our investigations so far only found minor effects of pinealectomy, with or without restitution of melatonin, on glucose metabolism. Clearly, further studies combining neuroanatomy and physiology are necessary to reveal the pathways used by the circadian timing system to enforce its rhythmic message on the glucose regulatory system. Keywords: Glucose, Vasopressin, Orexin

S10.1 LIGHT RESPONSES IN PERIPHERAL TISSUES: HOW DO THINGS CHANGE FOLLOWING EVOLUTION IN PERPETUAL DARKNESS? 1David-‐Whitmore, 1Andrew Beale, 2Christophe Guibal, 1Katherine Tamai, 2Victor Reynoso, 1Yoshiyuki Yamamoto. 1University College London, UK. 2UNAM, México. Fish tissues and cells not only contain circadian clocks, but are also directly light responsive. Studies using specific zebrafish mutants have shown that the presence of classical light responsive structures, such as the retina and pineal gland, are not required for this circadian light sensitivity. Furthermore, this light response can be found in the earliest stages of embryo development, way before the presence of differentiated photoreceptors. The Mexican blind cavefish, Astyanax mexicanus, represents another unique model system in which to study clock function, and also the circadian light input pathway. These animals have evolved in complete darkness for a minimum of 100,000 years in a series of isolated caves in North Eastern Mexico. Of greater significance is the fact that the founder, river or surface fish strains, still exist within the neighbouring rivers. This fact allows us to directly compare the molecular changes that have occurred in the circadian clock system between river and cave evolved strains. In this paper, we will focus on the evolutionary changes that have occurred relating to clock entrainment, and how the light input pathway has altered following evolution under constant dark conditions in these populations of Mexican cavefish. Keywords: light, Astyanax, entrainment

84

S10.2 EARLY-‐STAGE RETINAL MELATONIN SYNTHESIS IMPAIRMENT IN STREPTOZOTOCIN-‐INDUCED DIABETIC WISTAR RATS 1Daniella do Carmo Buonfiglio, 1Rodrigo A. Peliciari-‐Garcia, 1Rafael Peres, 1Fernanda Gaspar do Amaral, 1Tatiane Araujo Nogueira, 2Solange Castro Afeche, 1José Cipolla Neto. 1São Paulo, Institute of Biomedical Sciences, Department of Physiology and Biophysics, Brazil. 2Laboratory of Pharmacology, Butantan Institute, Brazil. Retinal melatonin synthesis occurs in the photoreceptor layer in a circadian manner controlling several physiologic rhythmic phenomena, besides being the most powerful natural free radical scavenger. The present work evaluated the diurnal profile of retinal melatonin content and the regulation of its synthesis in the retina of streptozotocin-‐induced diabetic rats. Male Wistar rats entrained to a 12h:12h light/dark cycle were diabetic-‐induced by streptozotocin. Control, diabetic and Insulin-‐treated diabetic animals were sacrificed every 3 hours throughout the light/dark cycle. Retinal melatonin content was measured by HPLC, AANAT activity was analyzed by radiometric assay, Bmal1 gene expression was determined by qPCR and cAMP content was accessed by ELISA. Control animals showed a clear retinal melatonin and AANAT activity daily rhythm with high levels in the dark period. Interestingly, diabetic rats had both parameters reduced and this impairment was prevented by immediate insulin treatment. In addition, Bmal1 daily expression profile was lost in the diabetic group related to control and the retinal cAMP level was reduced at ZT6 and ZT15 in diabetic rats. The present work shows a melatonin synthesis reduction in diabetic rats retinas associated to a reduction in AANAT activity that may be prevented by insulin treatment. The retinal cAMP reduction seems to be responsible for the AANAT activity decrease in diabetic animals. We can conclude that melatonin synthesis reduction observed in the pineal gland of SZT-‐induced diabetic rats is also observed in a local melatonin tissue-‐specific synthesizer, the retina. Keywords: Retina, Diabetes, Melatonin

85

S10.3 CIRCADIAN OSCILLATORS IN RETINAL GANGLION CELLS. LIGHT AND DOPAMINE REGULATION AND INTRINSIC PHOTORECEPTIVE CAPACITY Mario Eduardo Guido, Diego Javier Valdez, Paula Sofía Nieto, Eduardo Garbarino-‐Pico, Maria Ana Contin, Daniela Mariana Verra, Nicolas Maximiliano Diaz, Victoria America Acosta-‐Rodriguez. CIQUIBIC-‐Department of Biological Chemistry, Faculty of Chemical Sciences, National University of Cordoba-‐CONICET, 5000 Córdoba, Argentina. Retinal clocks induce changes in the visual system as a function of the time of day in anticipation of the illumination variations occurring at dawn and dusk. Retinal ganglion cells (RGCs) send visual and circadian information to the brain regarding the environmental illumination and a subset of cells was shown to be intrinsically photosensitive. In the chicken retina, RGCs contain autonomous circadian oscillators that synthesize melatonin with higher levels during the subjective day in constant darkness (DD), light (LL) or light phase of a 12:12 h LD cycle in antiphase to the nocturnal rhythm observed in photoreceptor cells (PRCs). RGCs also display the expression and activity of arylalkylamine N-‐acetyltransferase (AA-‐NAT), a key enzyme in melatonin biosynthesis, with the highest levels during the subjective day. Remarkably, in LL, the rhythm in AA-‐NAT mRNA expression is lost in PRCs but still observed in RGCs. To further investigate light responses, we assessed the presence of photopigments in RGCs, their intrinsic photosensitivity and the effect of light and dopamine (DA) on AA-‐NAT activity; in this context, DA may mimic the effect of light. When we examined the expression of clock genes and AA-‐NAT in the rat RGC line RGC-‐5, we found that dividing cells express the RGC marker Thy-‐1 together with Per1, Clock and Bmal1 and AA-‐NAT. RGC-‐5 also express a number of photopigments (Opn3, Opn5 and RGR) while chicken RGCs express melanopsin (Opn4) genes. Moreover, both chicken RGCs and RGC-‐5 cells respond to light stimulation causing significant changes in intracellular Ca2+ mobilization. By contrast, a light pulse of 30-‐60 min during the subjective day (CT 3) or subjective night (CT 17.5) caused a significant decrease in AA-‐NAT activity of PRCs while has no effect on RGC enzyme activity. DA treatment in the absence of light significantly inhibited AA-‐NAT activity in PRCs but not in RGCs. Overall the results suggest that chicken RGCs and the mammalian RGC-‐5 expressing non-‐canonical photopigments may act as non-‐visual photoreceptors while in the chicken retina, circadian rhythms observed in PRCs and RGCs are differentially regulated by light, DA and the circadian clock. The convergence of oscillatory and photoreceptive capacities in retinal cells and particularly in vertebrate RGCs could deeply impact on the functioning of the circadian system to temporally regulate physiology and behavior. Supported by ANPCyT-‐FONCyT,(PICT 04 967/PICT06 898), CONICET, SeCyT-‐UNC, and MinCyT-‐Cba.

86

Keywords: retina ganglion cell, melatonin, intrinsic photosensitivity

S10.4 FISH, AMPHIBIAN AND AVIAN CELLS AS PERIPHERAL CLOCKS: A COMPARATIVE APPROACH TO STUDY MELANOPSIN SIGNALING AND REGULATION OF CLOCK GENES Leonardo Graciani de Lima, Maria Nathália Moraes, Bruno Ribeiro Ramos, Maristela Poletini, Ana Maria Castrucci. University of São Paulo, Brazil. In the last two decades very important discoveries such as the existence of clock genes and of the new photopigment melanopsin helped chronobiologists to better understand the functioning of the molecular clock located in the suprachiasmatic nucleus (SCN) of mammals and its photoentrainment. Today it is well accepted that the molecular mechanism responsible for endogenous rhythmicity is present not only in the mammalian SCN but throughout the body of most vertebrate species. Therefore many cells are capable of functioning as independently phased circadian oscillators that are self-‐sustained peripheral clocks. Those findings have provided new possibilities to study the endogenous cues involved with entrainment and coupling of peripheral clocks. In the last few years we have been studying the circadian behavior of different cell lines within a comparative perspective in an attempt to better understand the signaling pathways of afferent and efferent signals involved with the molecular clock. We have previously shown that embryonic cells from the teleost Danio rerio (ZEM-‐2S) express melanopsins and that they are able to entrain clock gene expression under photoperiodic conditions; Xenopus melanophores express melanopsins with daily variations when kept in light-‐dark cycles and can be synchronized by a medium change; chicken embryonic retinal cells also are able to synchronize the melanopsin Opn4m, Clock and Tyrosine Hydroxylase gene expression under 12L:12D condition. We will discuss the signaling pathways evoked by blue light (450-‐475nm) and their ultimate effect on the peripheral clock machinery of vertebrate models. Work partially supported by FAPESP. Keywords: Light, Melanopsin, Peripheral clock

87

S11.1 TANYCYTES AND RFAMIDES, THE NEW PLAYERS IN SEASONAL REPRODUCTION 1Paul Klosen, 2Matei Bolborea, 3Marie-‐Pierre Laran-‐Chich, 3Kamontip Rasri, 4Stephan Steinlechner, 1Paul Pévet, 1Valérie Simonneaux. 1Institute of Cellular and Integrative Neurosciences, Neurobiology of Rhythms, CNRS UPR 3212, Strasbourg, France. 2Rowett Institute of Nutrition and Health, University of Abderdeen, United Kingdom. 3Department of Pre-‐Clinical Science, Faculty of Medicine, Thammasart University, Rangsit, Thailand. 4Veterinary School, Institute for Zoology, Hannover, Germany. The seasonal control of reproduction of mammals appears central among their various adaptations to the seasonal variations of the climate. The central role of melatonin secretion in seasonal adaptations has been established for several decades now, but the exact sites of action and neuroendocrine networks involved in this control remain elusive. In recent years, new actors in the neuroendocrine control of reproduction have emerged and have now also been linked to the seasonal control of reproduction. In 2003, the discovery of the role of Kisspeptin and its receptor Kiss1R (GPR54) in the control of puberty generated considerable interest in its potential role in the seasonal control of reproduction. We, as well as other laboratories, have since shown that Kisspeptin expression is subject to seasonal variations. These variations are controlled either directly by melatonin or indirectly through melatonin-‐controlled seasonal changes in gonadal steroids. There appear to be considerable species differences in the relative contributions of melatonin and gonadal steroid controls on Kisspeptin expression, which also seems to be affected by other influences such as metabolic signals. Also, Kisspeptin neurons do not appear to have melatonin receptors. More recently another member of the RFamide family, RFRP, the mammalian homologue of avian GnIH, has also been shown to be controlled by photoperiodic melatonin secretion. Most interestingly, RFRP expression does not seem to be influenced by seasonal changes in gonadal steroids. Furthermore, RFRP neurons are located in a brain area known to contain melatonin receptors. The pars tuberalis of the adenohypophysis contains the highest levels of melatonin receptors in a mammalian organism. Its involvement in the control of seasonal physiology has long been suspected, but the signals secreted by the pars tuberalis and their mode of action again remained elusive. We showed that the melatonin receptor expressing cells of the pars tuberalis produce TSH, however the target of this TSH was not known. In 2008, two research groups identified tanycytes as the target of pars tuberalis secreted TSH. Tanycytes are specialised glial cells located in the ependymal lining of the third ventricle that send their processes either to the meninges or to the blood vessels of the medio-‐basal hypothalamus. TSH controls the expression of the thyroid hormone activating Deiodinase 2 and thyroid hormone inactivating Deiodinase 3 enzymes in tanycytes. This discovery finally uncovered the link between melatonin secretion and the long suspected involvement of thyroid hormones in the seasonal control of physiology. Tanycytes are also known to locally modulate GnRH release in the median eminence and to display seasonal structural changes. We observed a reduction of vimentin immunostaining in tanycyte cell bodies and processes in short days which could not be reversed by testosterone supplementation. Similarly, tanycytes and their processes contain lower amounts of the neuronal cell adhesion molecule NCAM in short days. This discovery highlights the central role of tanycytes and neuroglial plasticity in the photoperiodic control of reproduction and probably also other seasonal adaptations.

88

Keywords: Plasticity, TSH, Deiodinases

S11.2 CNS SITES OF MELATONIN ACTION FOR REPRODUCTIVE AND BODY FAT RESPONSES IN SIBERIAN HAMSTERS 1Timothy J. Bartness, 1C. KaySong, 2Claudia Leitner, 1Brett J. Teubner. 1Dept of Biology, Georgia State University, USA. 2Zurich, Institute of Food, Nutrition and Health Physiology and Behavior, Switzerland. Seasonal responses often are triggered by daylength changes transduced into neuroendocrine signals by the pineal gland through the nocturnal duration of melatonin (MEL) secretion. Thus, long,”winter-‐like” MEL durations trigger short day (SD) responses by stimulating MEL receptors (MEL1a-‐Rs). The precise central sites necessary to receive, transduce and relay SD MEL signals into seasonal responses are not clear. Siberian hamsters exposed to SDs decrease body and lipid mass, and regress their testes. We previously demonstrated that MEL1a-‐R mRNA is colocalized on CNS sympathetic nervous system (SNS) outflow neurons that ultimately innervate white adipose tissue (WAT). SDs trigger an increase in WAT SNS drive thereby decreasing body and lipid mass, an effect blocked by WAT SNS denervation. The CNS components of this circuit include the subzona incerta (subZI), dorsomedial hypothalamic nucleus (DMH), thalamic reuniens nucleus (ReN) and the suprachiasmatic nucleus (SCN), the latter shown by us to be critical for SD MEL signal reception. We attempted to block SD-‐induced decreases in WAT mass distally by making electrolytic or sham lesions of the subZI, ReN or DMH in LD-‐housed hamsters. SD-‐like MEL signals were generated by injecting MEL 3 h before lights-‐out. ReN and subZI lesions did not block SD-‐like MEL signal-‐induced decreases in body, lipid or testes masses; by contrast, DMH lesions blocked these responses. This non-‐responsiveness was not due to lesion-‐induced inappropriate nocturnal LD MEL secretion that would have altered our creation of SD-‐like signals. Therefore, the DMH participates in the control of SD energy and reproductive responses joining the SCN as sites necessary for SD responses in this species. Others also have proposed the dorsomedial posterior arcuate nucleus (dmpARC) as a site critical for relaying SD information via modulation of several neurochemicals/receptors important for energy balance control, despite the lack of MEL1a-‐Rs there. We tested the necessity of an intact dmpARC for SD responses by making electrolytic lesions of the dmpARC and exposing hamsters to LDs or SDs for 12 wk. SDs triggered normal decreases in body and WAT mass, food intake, testicular volume, serum testosterone, pelage color change and increased UCP-‐1 protein expression in brown adipose tissue despite a non-‐intact dmpARC. These data demonstrate that an intact dmpARC is not necessary for SD responses. Conversely, we selectively stimulated the above sites (not dmpARC) by giving long duration, SD-‐like MEL signals applied site-‐specifically for 5 wk. Whereas SD MEL signals delivered to each of these sites induced testicular regression, all but the PVT also triggered

89

SD-‐like decreases in body mass. Collectively, these data suggest a distributed system of MEL-‐sensitive brain sites mediating SD responses in Siberian hamsters, the redundancy of which suggests its importance for appropriate seasonal responses critical for overwintering. Keywords: photoperiod, adipose tissue, hamsters

S11.3 ACUTE INDUCTION OF EYA3 BY LATE-‐NIGHT LIGHT STIMULATION TRIGGERS TSHβ EXPRESSION IN PHOTOPERIODISM Hiroki R. Ueda. Laboratory for Systems Biology and Functional Genomics Unit, RIKEN CDB Laboratory for Synthetic Biology, RIKEN Q-‐Bic, Japan. Living organisms detect seasonal change of day length (photoperiod) to alter their physiological functions according to seasonal environmental changes. The understanding of the photoperiodic system is also important for human health because this system also underlies season-‐associated diseases and symptoms such as seasonal affective disorders winter depressions), as well as season-‐associated symptoms observed in bipolar diseases and schizophrenia. Previously, we identified the induction of thyroid stimulating hormone beta subunit (Tshβ) in the pars tuberalis (PT) as a key pathway in animal photoperiodism. However, the upstream molecular mechanism for Tshβ expression remains to be elucidated. In this study, we show that late-‐night light stimulation acutely trigger Eya3-‐ Six1 pathway, which directly induces Tshβ expression. In details, we used melatonin-‐proficient CBA/N mice that preserves photoperiodic response of Tshb expression, and first performed genome-‐wide expression analysis of PTs in chronic short-‐day and long-‐day conditions. These genome-‐wide expression data comprehensively identify long-‐day and short-‐day genes, and also imply that late-‐night light stimulation induces long-‐day genes. We verified this hypothesis by extending light period in advance, which acutely induces Tshβ expression within one day. We thus performed second set of genome-‐wide expression analysis in this condition to search for direct upstream candidates, whose expression proceed the induction of Tshβ, leading to the identification of Eya3 gene. We demonstrated that Eya3 and its partner Six1 synergistically activate Tshβ expression through Six consensus sequence in Tshβ promoter, and that this activation is further enhanced by Tef and Hlf through D-‐box close to Six consensus sequence. [References] 1. Nature 452, 317-‐322 (2008). 2. Current Biology, 20(24):2199-‐206 (2010) Keywords: photoperiodism, Tshβ, Eya3

90

S11.4 PHOTOPERIOD AND THE MALE EFFECT CAN BE USED TO CONTROL THE REPRODUCTIVE ACTIVITY IN SUBTROPICAL GOATS José Alberto Delgadillo, José Alfredo Flores, Marie Bedos, Jesús Vielma, Gerardo Duarte, Gonzalo Fitz-‐Rodríguez, Horacio Hernández, Ilda Graciela Fernández. Centro de investigación en reproducción caprina, Universidad Autónoma Agraria Antonio Narro, México. Some goats originating from or adapted to subtropical latitudes display large seasonal variations in their reproductive activity. In some breeds such as local goats from subtropical Mexico, the reproductive activity is driven by an endogenous annual rhythm that is synchronized by photoperiod. This seasonal reproductive activity causes seasonal availability of milk, cheese and meat. To extend the availability of these products all year round, some animals must breed during the anestrous period. The male effect is a biostimulation technique that induces and synchronizes the sexual activity in female goats during the anestrous period. However, the sexual response of females is weak or absent when the male effect is performed during the mid-‐seasonal anestrous, probably because at this time males are also in the non-‐breeding season and display low sexual behavior. The stimulation of the male sexual activity during the non-‐breeding season by exposure to 2.5 months of long days (16 h of light by day) from November 1st, improves the quality of the male signals (odor and sexual behavior), increasing the proportion of females responding to the male effect. In addition, when these sexually active males are used, four hours of daily contact between sexes are sufficient to induce the sexual activity in anestrous goats. Thus, one sexually active male can stimulate several groups of female per day, increasing the efficiency of the male effect. Under subtropical conditions, the photoperiodic treatment applied to males and the male effects using these photoperiodic-‐treated males constitute an original manner to control the reproductive activity of goats and consequently extend the availability of goat products all year round. Keywords: Caprine, Reproductive seasonality, Socio-‐sexual relationships

91

S12.1 DESYNCHRONY AS A TOOL TO INVESTIGATE THE ROLE OF THE HUMAN CIRCADIAN SYSTEM IN PHYSIOLOGY AND PATHOPHYSIOLOGY Frank A. J. L. Scheer. Division of Sleep Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA. The use of desynchrony protocols in the study of physiology and pathophysiology has received increasing attention over the last few years. In animals, desynchrony caused by shifting the feeding/fasting cycle leads to metabolic disturbances, while desynchrony caused by shifting the light/dark cycle usually has profound adverse effects, up to and including decreased survival. In human experimental work, desynchrony caused by shifting the behavioral cycle while in constant dim light has been used for many years as a tool to study underlying circadian physiology while controlling for or balancing out behavioral effects. Also, in recent years such desynchrony experiments have been expanded to examine the interaction between the behavioral and circadian influences on physiology and neurocognitive function, and most recently, to study of the role of the circadian system in disease severity and risk. For example, recent studies utilizing the forced desynchrony protocol demonstrate that the human circadian system is important in regulating hemodynamic, autonomic, and hemostatic function and that there are significant circadian rhythms in the reactivity of the cardiovascular system to behavioral stressors such as changes in body posture and exercise. The high level of consistency in circadian rhythm amplitude and phase for cardiovascular measures in humans between different circadian protocols (20-‐hour forced desynchrony, 28-‐hour forced desynchrony, and constant routine protocol), demonstrate these are robust and reproducible circadian rhythms in cardiovascular control. Overall, these studies indicate a possible contribution of the circadian system in the morning peak in adverse cardiovascular events. While the cardiovascular system doesn’t seem affected much by the specific circadian protocol, circadian misalignment during forced desynchrony leads to impaired glucose tolerance and decreased leptin levels. Future studies are required to identify the critical differences in circadian control systems between cardiovascular function and metabolic function that may make metabolic function more sensitive to circadian disruption. Desynchrony protocols can thus be versatile tools to assess effects of both the circadian system and of circadian disruption in physiology and pathophysiology. Support: This work was supported by National Institutes of Health Grants R01-‐HL76409, P30-‐HL101299, K24-‐HL076446, and M01-‐RR02635. Keywords: Cardiovascular, Desynchrony, Metabolism

92

S12.2 DESYNCHRONIZED CIRCADIAN RHYTHMS: BOW TO THE MASTER Horacio O. de la Iglesia. Department of Biology, University of Washington, Seattle, USA. In the last years, our laboratory has developed an animal model of circadian forced desynchrony. Exposure of rats to a 22-‐h light-‐dark (LD) cycle leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral suprachiasmatic nucleus SCN (vlSCN) and the dorsomedial SCN (dmSCN). This desynchronization of neuronal oscillators within the master circadian pacemaker leads to the stable desynchronization of circadian outputs that shows all signature properties of human circadian desynchronization. Here we present evidence that the circadian desynchronization of sleep stages, core body temperature, and the release of melatonin, corticosterone and luteinizing hormone can be all accounted for by the forced desynchrony of vl-‐ and dmSCN oscillators. Internal desynchronization of circadian rhythms represents the most salient physiological outcome in humans exposed to temporal challenges such as shift-‐work and jetlag. Our data in the rat suggests that the neural bases for internal desynchronization may reside within the SCN neuronal network itself. Keywords: SCN, Forced desynchrony

S12.3 FOOD AS CHRONOTHERAPY TO AMELIORATE THE ADAPTATION TO A NEW TIME ZONE 1Manuel Angeles Castellanos, 1Jorge Miguel Amaya, 2 Roberto Salgado Delgado, 2Ruud Marinus Buijs, 1Carolina Escobar. 1Fac. Medicina, Universidad Nacional Autónoma de México, México. 2Instituto de Investigaciones Biomédicas, UNAM, México. Circadian desynchrony occurs when individuals are exposed to fast phase shifts of the light-‐dark cycle, as in “Jet-‐lag”. For reducing symptoms and for speeding up resynchronization, several strategies have been suggested including scheduled exercise, exposure to bright light, drugs and especially melatonin administration. Restricted feeding schedules have shown to be powerful entraining signals for metabolic and hormonal daily cycles, as well as for clock genes in tissues and organs of the periphery. This study explored in a rat model of jet-‐lag the contribution of melatonin or scheduled feeding on the re-‐entrainment speed of spontaneous general activity and core temperature after a 6 h phase advance of the light dark cycle. In a first phase the treatment was scheduled for 5 days prior to the phase shift, while in a second stage the treatment was presented combined and simultaneous to the phase advance of the light-‐dark cycle. Melatonin and especially scheduled feeding

93

simultaneous to the phase shift improved significantly the re-‐entrainment speed. The evaluation of the free running activity and temperature following the 5 day treatment proved that both, but mainly scheduled feeding, accelerated re-‐entrainment of the SCN driven general activity and core temperature. Present results show the relevance of feeding schedules as entraining signals for the circadian system and highlight the importance of using feeding schedules as a strategy for preventing internal desynchrony. supported by grants DGAPA PAPIIT IN-‐205809 and CONACyT 82462 Keywords: Food entrainment, Jet-‐Lag, Melatonin

S12.4 NIGHTWORK LEADS TO OBESITY AND DIABETES: A RAT MODEL OF NIGHTWORK UNCOVERS INTERNAL DESYNCHRONY AT THE LEVEL OF THE HYPOTHALAMUS AND WITHIN THE LIVER. 1Roberto Carlos Salgado-‐Delgado, 1Nadia Saderi, 1Maricarmen Basualdo, 2 Carolina Escobar, 1Rudolf M. Buijs. 1Departamento de Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, UNAM, MÉXICO, D.F. 2Departamento de Anatomía, Facultad de Medicina, UNAM, MÉXICO, D.F. Synchrony of internal oscillators with external cycles allows the adaptation of physiological functions to cyclic changes in energy demand. Night work and shift work lead to a disrupted phase relationship between external time cues and internal rhythms, associated with a loss of internal coherence among oscillations. This process results in internal desynchrony (ID) in which behavioral, hormonal, and metabolic variables cycle out of phase. Shift work or night work is associated with hypertension, metabolic syndrome, cancer, and other diseases. We investigated the mechanisms promoting metabolic desynchrony in a model for night work in rats, based on daily 8-‐h activity schedules during the resting phase .We demonstrate that major alterations leading to internal desynchrony induced by this working protocol, flattened glucose and locomotor rhythms and the development of abdominal obesity, were caused by food intake during the rest phase. Also we provide evidence that wakefulness and activity during the normal rest phase leads to ID at the level of the first output projections from the SCN, relaying nuclei that transmit temporal signals to other brain areas and to the periphery. Since these nights working animals also develop characteristics of the metabolic syndrome we investigated the impact of this desynchronization on the rhythmicity of metabolic and clock genes in the liver. Bmal1 and Clock completely inverted their rhythm as compared to ad libitum animals. Their rhythms become dissociated from Per2 and the metabolic genes Nampt, Pgc1 and the Ppars, which loose their rhythm and appear completely flattened. These results demonstrate that night work not only desynchronizes the rhythm between the SCN and the liver but also within the liver itself.

94

This may be the basis of the association of metabolic disorders in night-‐shift-‐workers such as diabetes and obesity. Keywords: Metabolic syndrome, Clock genes, Disease

S13.1 DAILY RHYTHMS IN OLFACTORY DISCRIMINATION DEPEND ON CLOCK GENES, BUT NOT THE SUPRACHIASMATIC NUCLEUS 1Daniel Granados Fuentes, 1Gal-‐Ben Josef, 1Gavin Perry, 2Donald A. Wilson, 3 Alexander Sullivan-‐Wilson, 1Erik D. Herzog. 1Dept. of Biology, Washington University, St. Louis, MO 63130-‐4899, U.S.A. 2New York Univ. Sch. of Medicine, U.S.A. 3Grinnell College, U.S.A. Mammals have circadian clocks in multiple peripheral tissues. Some tissue-‐specific roles for clock genes have been described, but there is no direct evidence for their physiological relevance. We have shown that the main olfactory bulb in rodents fulfills the criteria of a circadian pacemaker which regulates daily rhythms in firing rate and Period 1 gene activity in the absence of time cues from the environment or from the suprachiasmatic. We hypothesized that these daily changes mediate circadian oscillations in olfaction. We developed an automated assay to test olfactory sensitivity in mice at different times of the day. Using this automated assay, we found olfactory discrimination in mice increased approximately 10-‐fold from a minimum during the day to a peak in the early night. This rhythm was maintained in SCN-‐lesioned mice and mice deficient for the clock gene Npas2, but was lost in mice deficient for Bmal1 or both Per1 and Per2 genes. We conclude that an extra-‐SCN clock controls circadian rhythmicity in olfactory behavior.This work was supported by NIH grant MH63104. Keywords: Olfaction, Circadian rhythms, Clock

S13.2 THE CEREBELLUM; A CIRCADIAN OSCILLATOR SYNCHRONIZED BY FOOD Jorge Mendoza. Institute of Cellular and Integrative Neurosciences, Centre National de la Recherché Scientifique UPR3212, University of Strasbourg, France. The mammalian brain contains circadian clocks organized hierarchically in a network of oscillatory structures with the hypothalamic suprachiasmatic nucleus (SCN) at the top. The SCN coordinates extra-‐SCN oscillators so that they maintain proper phase relationships with each other. The cerebellum (CB) is thought to be a brain region regulating motor learning

95

and coordination. Recently, its role in circadian timing has been highlighted. The CB has competent local molecular circadian machinery (rhythmic clock genes expression) that permits it to continue to oscillate for several days when held in isolation. Whether the cerebellum is synchronized and sustained by the SCN or if it is a self-‐sustained clock, however, is still unknown. Nevertheless, in vivo, the circadian expression of clock genes in the cerebellar oscillator can be synchronized by feeding time, indicating that the CB can either directly sense metabolic signals or receive them indirectly via multiple neural projections. Therefore, current data indicate a role for the CB in the circadian timing network implicated in food synchronization and, additionally, suggest a potential role to regulate motor learning, coordination and other cognitive neural functions in a circadian manner. Supported by CNRS, University of Strasbourg and NEUREX Keywords: cerebellum, brain clocks, feeding

S13.3 DOPAMINERGIC MODULATION OF CLOCK MECHANISMS IN THE RAT LIMBIC FOREBRAIN Suzanne Hood, Shimon Amir, Jane Stewart. Concordia University, Canada. Many brain regions outside of the suprachiasmatic nucleus (SCN), the master circadian clock, rhythmically express clock genes and proteins, and these rhythms are presumed to control, in a tissue-‐specific manner, daily fluctuations in cellular and metabolic activity and functional output. Although signals from the SCN entrain the timing of clock gene expression in these regions, behavioral and physiological events can also influence their rhythmic activity independently of the master clock. One example of this form of regulation is the effect of motivationally salient stimuli on clock gene expression in regions of the limbic forebrain. Both appetitive (e.g., drugs of abuse) and aversive (e.g., stressors) stimuli have been found to shift daily patterns of expression of clock genes in regions such as the striatum, the oval nucleus of the bed nucleus of the stria terminalis, and the central nucleus of the amygdala, regions which are themselves involved in the regulation of motivational states. The mechanisms by which these stimuli influence clock gene expression in these regions remain unclear; however, a number of findings suggest that activity of midbrain dopamine (DA) pathways, long associated with the regulation of motivated behavior, might play an important role. In this presentation, I will review evidence demonstrating that midbrain DA release is a regulator of clock gene expression in the limbic forebrain and present recent findings from our laboratory that indicate that timed, daily stimulation of D2 DA receptors is necessary to maintain the daily expression pattern of the clock protein, PERIOD2, in the dorsal striatum. Together, this evidence suggests a mechanism by which motivational events can influence selected circadian oscillators normally downstream of SCN control.

96

Keywords: PERIOD2, dopamine, striatum

S13.4 PUTATIVE CIRCADIAN OSCILLATORS IN THE EPITHALAMUS AND HYPOTHALAMUS Hugh David Piggins, Alun Thomas Hughes, Clare Guilding. Faculty of Life Sciences, University of Manchester, UK. Since the determination that the mammalian suprachiasmatic nuclei (SCN) of the hypothalamus contain a circadian pacemaker, important progress has been made in identifying the molecular and cellular basis of biological timekeeping in this structure. We now know that SCN neurons function as cell autonomous pacemakers, generating daily rhythms in electrical excitability (spontaneous action potentials and resting membrane potential) and clock gene/protein (per1-‐2/PER1-‐2, cry1-‐2/CRY1-‐2, etc) expression. Further, action potential production and intercellular communication are now known to be necessary for these cellular clocks to maintain synchrony and to drive robust rhythms in behavior. With the development of rodent models containing fluorescent/bioluminescent reporters of clock gene/protein expression and the visualization and measurement of these signals in cultured explants, we now know that the SCN is not the only mammalian circadian timekeeper. Indeed many peripheral tissues and extra-‐SCN brain structures manifest oscillatory capabilities. However, what is not known is the extent to which individual cells in these tissues/brain structures share the properties of SCN neurons. To investigate, this, we have prepared and cultured coronal epithalamic and hypothalamic brain slices from adult PER2::LUC mice and used 1) a sensitive EM-‐CCD camera-‐equipped microscopy system as well as photomultiplier tube assemblies to visualize and measure bioluminescence and 2) extracellular recordings of multiunit activity (MUA) targeted at sites rhythmically expressing PER2::LUC. In epithalamus, we visualized rhythmic PER2::LUC expression in a small number of cells along the medial border of the lateral habenula as well as the glial ependymal cell layer of the dorsal third ventricle. Unlike the SCN, these oscillations in PER2::LUC were reset by culture, were of a low amplitude and damped within 3 days of culturing. Complementary recordings of MUA in lateral habenula revealed that robust circadian oscillations in electrical activity were absent in most neurons sampled. Therefore, the lateral habenula contains a very weak circadian oscillator. In the mediobasal hypothalamus, rhythmic PER2::LUC expression was readily visualized and localized to the arcuate and dorsomedial nuclei, and to the median eminence/pars tuberalis as well as the ependymal cell layer of the third ventricle. In the lateral and dorsal arcuate as well as the dorsomedial nuclei, single bioluminescing cells could be tracked over several days. Unlike the SCN, in which single cell oscillators remain tightly synchronized, these single cell oscillators damped and desynchronized within a week culturing. Complementary MUA recordings indicated that weak electrical rhythms were present in the dorsal part of the arcuate only. Collectively, these investigations reveal that the extra-‐SCN structures examined have very weak (lateral

97

habenula) to moderate oscillatory capabilities and that the cells in these areas do not possess the full complement of properties observed in SCN neurons. Keywords: extra SCN, bioluminescence, electrophysiology

S14.1 THE CONSEQUENCES OF DYS-‐ENTRAINMENT

1Till Roenneberg, 1Céline Vetter, 1Karla Allebrandt, and 2Martha Merrow. 1Ludwig-‐Maximilians-‐University of Munich, Department of Medical Psychology, Germany. 2Department of Molecular Chronobiology, University of Groningen, The Netherlands.

Since the circadian clock controls sleep timing, sleeping at the ‘wrong’ time has consequences. In a large-‐scale, on-‐going epidemiological study on sleep behaviour, we find that the disparity between biological and social timing (social jetlag) affects health. The higher an individual’s social jetlag, the more likely he/she is a smoker and the higher his/her alcohol and caffeine consumption. But social jetlag also affects body mass index (BMI), which has been shown to correlate with sleep duration. The conflict between an individual’s biological clock and social constraints can result in a chronic sleep debt on workdays, which people compensate for on work-‐free days. The association between BMI and sleep duration is twice as strong on workdays than on work-‐free days, indicating social jetlag, rather than individual sleep need as the basis for increased BMI. Obesity has reached crisis proportions in industrialised societies, and social jetlag is among the many factors that converge to yield increased BMI. This finding is of key importance in pending discussions on artificial manipulations of clock time, such as Daylight Saving Time, over-‐proportioned time zones, or inappropriately early work and school times. We additionally show a trend towards increased social jetlag over the last decade, stressing the timeliness of these discussions. Improving the correspondence between biological and social clocks will lead to improved health on a population-‐wide level.

98

S14.2 ON THE EVOLUTION OF Drosophila BLUE LIGHT PHOTORECEPTOR CRYPTOCHROME AND ITS RELATION TO THE VISUAL SYSTEM

1Gabriella M. Mazzotta, 1,2Alessandro Rossi, 1Emanuela Leonardi, 1Moyra Mason, 3Cristiano Bertolucci, 1Silvio C.E. Tosatto and 1Rodolfo Costa. 1Dept. of Biology, University of Padova, Italy. 2Dept. of Chemistry, University of Padova, Italy. 3Dept. of Biology and Evolution, University of Ferrara, Italy.

A fundamental aspect of the circadian clock is the ability to synchronize with 24h environmental cycles. In Drosophila melanogaster, the resetting of the clock relies mostly on the blue light photoreceptor CRYPTOCHROME (CRY). In presence of light, CRY associates with TIM and promotes its proteasome-‐mediated degradation. Besides the brain, in Drosophila CRY is expressed also in other body cells, like eyes, wings, legs, antennae, where “peripheral” clocks probably control the local physiology of these organs. In peripheral clocks, in addition to its role as photoreceptor, CRY has been shown to act as transcriptional repressor, similarly to its mammalian homologs mCRY1 and mCRY2. Although the light dependent CRY activation mechanism is still not fully understood, a regulatory role for the C-‐terminus has been proven.

We had previously reported that CRY contains a number of putative interaction motifs in its C-‐terminal, suggesting that CRY may be regulated in a two-‐step process modulated by light. The search for new partners of CRY has led to the identification of several elements of the phototransduction cascade, that are assembled in a multiprotein signalling complex organized by INAD, a PDZ containing scaffold protein. We have demonstrated that CRY interacts with the visual system through INAD and identified the domains responsible for this interaction. These experimental data are in accordance with the results of a novel in silico search for evolutionarily conserved CRY interaction motifs, that suggest the existence of an evolutionary pressure for CRY to maintain these binding sites all over the organisms with eyes.

Taken together, our data suggest for the first time a connection between the circadian clock and the vision.

99

S14.3 THE EARLY WORM CATCHES THE LIGHT (AND THE HEAT): CIRCADIAN ENTRAINMENT IN C. Elegans 1,2Diego A Golombek, 1,2Andrés Romanowski, 1,María Laura Migliori, 1,2María Eugenia Goya, 2,3Sergio H Simonetta. 1Universidad Nacional de Quilmes Argentina. 2CONICET, Argentina, 3Fundación Instituto Leloir, Argentina. C. elegans is a soil dwelling nematode widely used in various fields of research but not very well characterized in chronobiological studies. We have recently described rhythms in various physiological variables such as stress resistance, oxygen consumption, defecation and feeding. In addition, we have characterized locomotor activity rhythms in individual nematodes and found the main circadian features conserved: free-‐running rhythms, entrainment and temperature compensation. Such rhythms are entrained by both light and temperature cycles, although the mechanism for synchronization is not well understood. Since C. elegans lacks a specific light-‐sensing organ we are trying to determine if the nematodes sense light. Our data shows a positive phototaxis index towards the green wavelength of light. While searching for this elusive entrainment pathway, we tested lite-‐1 (strain KG1180) and the double mutant lite-‐3 / tax-‐2 (strain PR691) (both members of a novel photoreceptor family sensitive to UV/blue light in C. elegans) and found that both strains are capable of light entrainment (LD 12 h : 12 h). Recent work has hinted a possible role for tax-‐2, a cyclic-‐nucleotide gated channel involved in thermotaxis and chemosensation, as a doorway for light/temperature signals. We tested tax-‐2 mutants (strains PR691, PR694 and PR671) and found that although masking to light is conserved, photic entrainment is deficient in these strains. When tested for temperature entrainment, we found that while a 4°C fluctuation masks circadian rhythms, a cycle with a 1°C amplitude is able to entrain such endogenous rhythmicity. In addition, we found that C. elegans exhibits a diurnal/circadian rhythm in aaNAT activity and melatonin production. Interestingly, the nocturnal aaNAT peak was inhibited by white a blue light, indicating that this assay can be used as light-‐sensing probe in this species. Indeed, a question arises regarding the adaptive value of circadian rhythmicity in a mostly subterranean animal. A cyclic surrounding (in terms of potentially stressful challenges) might have selected rhythmic behaviors, which do need to be entrained even by the subtle signals provided by the environment which might include light, temperature and chemical cues. Keywords: Caenorhabditis elegans, entrainment, zeitgeber

100

S14.4 PROTEIN PHOSPHATASE 1 (PP1) REGULATES PERIOD LENGTH AND PHASE RESETTING OF THE MAMMALIAN CIRCADIAN CLOCK 1Isabelle Schmutz, 2Sabrina Wendt, 2Achim Kramer, 3Isabelle M. Mansuy, 1Urs Albrecht. 1University of Fribourg, Switzerland. 2Charité Berlin, Germany. 3University and ETH Zürich, Switzerland. Circadian clocks coordinate the timing of important biological processes. Interconnected transcriptional and post-‐translational feedback loops based on a set of clock genes generate and maintain these rhythms with a period of about 24 hours. Many clock proteins undergo circadian cycles of post-‐translational modifications. Among these modifications, protein phosphorylation plays an important role in regulating activity, stability and intracellular localization of clock components. Several protein kinases were characterized as regulators of the circadian clock. However, the function of protein phosphatases, which balance phosphorylation events, in the mammalian clock mechanism is less well understood. Here, we identify protein phosphatase 1 (PP1) as regulator of the period length of the mammalian circadian clock and of light-‐induced resetting. Down-‐regulation of PP1 activity in cells by RNA interference and in vivo by expression of a specific inhibitor in the brain of mice provoked a lengthening of the circadian period. Moreover, reduction of PP1 activity in the brain altered light-‐mediated clock resetting behavior in mice, enhancing the phase shifts in either direction. At the molecular level, diminished PP1 activity affected the nuclear accumulation of the clock component PER2 in neurons and modulated the subcellular localization of this protein. Hence, PER2 may be a molecular target of PP1, which affects its location by influencing its phosphorylation status. By consequence, this may lead to period lengthening and alteration of phase shifting properties of the mammalian circadian clock. Keywords: resetting, Per2, protein

S15 INTRODUCTION TO THE SESSION “THE MAMMALIAN CIRCADIAN TIMING SYSTEM: HORMONAL KEY MECHANISMS INVOLVED IN ORGANIZATION AND COORDINATION OF CENTRAL AND PERIPHERAL CLOCKS” Paul Pevet. INCI-‐CNRS and University of Strasbourg, France. Disruptions of rhythmicity in humans are characteristic of, and may underlie, a variety of troubles. Sleep and circadian rhythms are often disrupted in neurological disorders and evidence indicates that alterations in the sleep/wake cycle accompany (or may be

101

responsible for) many types of neurological disorders. Epidemiological data demonstrate also the consequences of circadian rhythm disruption in terms of public health, particularly in metabolic, neurobiological and cardiovascular diseases and cancer. To develop strategies to treat, prevent or delay such disturbances is a new challenge for science and medicine. A network comprised of circadian clocks, synchronizing inputs, various clock outputs and multiple peripheral self-‐sustained oscillators is responsible for daily rhythmicity. In mammals, the focal point is a master clock within the suprachiasmatic nucleus (SCN). Self-‐sustained circadian oscillators are also present in numerous tissues. Peripheral oscillators share similar molecular mechanisms to generate rhythms but they are distinct at the functional level. Even if peripheral oscillators can be affected independently, in intact animals all the identified synchronisers of peripheral oscillators (glucocorticoids, feeding cues…) depend on the SCN for their temporal expression. Therefore the SCN clock has not only the capacity to build a circadian message, but can also distribute this signal to other structures. It is thus the complex interactions of neural, hormonal and behavioural outputs from the SCN that drive the circadian expression of events within the body. In terms of health issues, problems may occur at various levels in the circadian network and drugs may be directed toward input pathways, the clock itself, output pathways, peripheral oscillators or ultimately the organ(s) expressing a particular rhythm. Even if mechanisms of action at the genetic level are the main topic we must not forget that general integrative approaches have also proven to be successful and opened important perspectives for therapeutic innovations, One absolutely essential step in this session will be a clear understanding of the role of the hormonal outputs of the clocks (Melatonin, glucocorticoids) Keywords: Melatonin, corticosterone, 5-‐HT

S15.1 HSD3B1: A NEW ENZYME LINKING CIRCADIAN CLOCK AND HYPERTENSION Masao Doi, Hitoshi Okamura. Kyoto University Graduate School of Pharmaceutical Sciences, Japan. Physiologically relevant circadian time is generated at the level of transcription-‐(post)translation feedback loop of clock genes in most cells throughout the body. Of course we know that the regulation of this system is known to be ultimately regulated by the hypothalamic suprachiasmatic nucleus (SCN), it seems that the importance of cell-‐specific clock is unrelenting. The consequences of a compromised circadian clock in model animals are clear: increased mortality of aged mice arising from chronic jet-‐lag, and metabolic syndrome in Clock mutant mice. It was also demonstrated that Per2 knockout mice have a higher ratio of radiation-‐induced lymphoma. Similarly in humans, rotating shift workers have a higher risk of developing cancers. Supporting these observations, the cell cycle is under clock control via the regulation of key genes such as wee1, a G2-‐M transition

102

inhibiting kinase. Dimerization of CHK1 protein kinase with CRY1/CRY2, and CHK2 with PER1, suggest that cell cycle arrest and apoptosis are also clock-‐controlled phenomena. Interestingly, when we surveyed the pathologies of genetically engineered mice, deficient for both Cry1 and Cry2 genes (Cry-‐null) and as a result showing arrhythmic behavior, physiology and metabolism, we observed that these mice, while having normal blood pressure under normal conditions, exhibited dietary salt-‐sensitive hypertension, with remarkably high secretion of aldosterone. These mice suffered from hyperaldosteronism, likely originating from a disorder of the adrenal. Then, by analyzing clock-‐controlled gene expression in the adrenal gland using DNA microarray, we discovered a novel link between the circadian clock and this pathological condition relevant to human health. Hyperaldosteronism in these animals is caused by a dramatic increase in the expression of a previously unappreciated enzyme, HSD3b6, involved in steroid synthesis. This enzyme is specifically expressed in the aldosterone-‐producing zona glomerulosa cells of the adrenal gland. Hsd3b6 in these cells is normally a clock controlled gene, but in arrhythmic Cry-‐null mice, Hsd3b6 mRNA and protein levels are both constitutively upregulated, leading to a drastic increase in the HSD3b enzymatic activities, leading to an over-‐production of aldosterone. These data place Hsd3b6 in a pivotal position through which circadian clock malfunction is coupled to the development of hypertension. Since human HSD3B1 gene is functionally similar to mouse Hsd3b6, extension of these results to humans will be fruitful to understand the pathophysiology of human hypertension. Recently we examined its regulation by humoral factors in human adrenocortical H295R cells, and found that HSD3B1 is regulated by angiotensin II by de novo protein synthesis for HSD3B1. Thus, clock controlled HSD3B1 is also an enzyme in rennin-‐angiotensin-‐aldosterone system, which has a crucial role for maintaining the blood pressure. Keywords: hypertension, suprachiasmatic nucleus, shift workers

S15.2 PHYSIOLOGY OF THE ADRENAL CIRCADIAN CLOCK Henrik Oster, Silke Kiessling, Gregor Eichele. Max Planck Institute for Biophysical Chemistry Göttingen, Germany. Under non-‐stressed conditions adrenal glucocorticoid (GC) secretion shows strong circadian rhythms with blood levels peaking around wake-‐up time (i.e. in the morning in humans and at the beginning of the night in nocturnal rodents). These diurnal variations are primarily regulated by rhythmic activation of the hypothalamic-‐pituitary-‐adrenal (hpa) axis and the secretion of adrenocorticotropin (ACTH) from the pituitary. While circadian hpa axis activity is ultimately controlled by the circadian pacemaker residing in the suprachiasmatic nuclei (SCN) of the hypothalamus, we found that clock gene activity at the level of the adrenal cortex regulates the sensitivity of the steroidogenic machinery to ACTH stimulation and,

103

hence, the GC responsiveness during the course of the day. Using a combination of molecular, genetic and surgical approaches we describe the mechanisms of SCN-‐to-‐adrenal clock interaction in the regulation of GC rhythms in the mouse. Using a rapid light/dark cycle shifting paradigm we discovered a role for the adrenal clock and adrenal GC rhythms in the re-‐synchronization of rest-‐activity cycles during jet lag and devised a novel pharmacological strategy to accelerate jet lag adaptation based on "pre-‐flight" manipulation of circadian GC secretion. Keywords: adrenal, circadian, glucocorticoids

S15.3 GLUCOCORTICOID REGULATION OF CLOCK GENE EXPRESSION IN THE MAMMALIAN BRAIN Lauren Arielle Segall, Shimon Amir. Concordia University, Canada. Glucocorticoids regulate a wide variety of functions, including synaptic plasticity, hypothalamic-‐pituitary-‐adrenal axis activation, conditional fear learning, metabolism, and sensitization to drugs of abuse. The diurnal secretion of glucocorticoids, driven by the mammalian master clock located in the suprachiasmatic nucleus of the hypothalamus, has been shown to induce and entrain clock gene expression in peripheral tissues. However, little attention has been given to the form and function of centrally located subordinate oscillators, and the synchronizing factors that influence them. Recent findings implicate glucocorticoids in the circadian regulation of clock genes in mammalian brain and propose mechanisms whereby glucocorticoids can feed back on rhythms downstream from the master clock and possibly alter the functional output of these nuclei.

S15.4 MELATONIN, AN ENDOCRINE OUTPUT OF THE CENTRAL CLOCK INVOLVED IN THE REGULATION OF HUMAN CIRCADIAN RHYTHMS Bruno Claustrat. Hospices Civils de Lyon, France. Melatonin is synthesized from tryptophan via serotonin in the pineal gland. It is also synthesized in various tissues and cells (retina, gut, platelets, immunocompetent cells….) where it may exhibit autocrine or paracrine properties. It acts via G protein-‐coupled receptors expressed in various areas of the central nervous system and in peripheral tissues. The blood melatonin profile faithfully reflects the pineal secretion which is under the control of the suprachiasmatic nuclei (SCN). Melatonin is released at night in the blood-‐stream and gains access to various tissues. It modulates the brain activity after passage

104

across the blood-‐brain barrier. Melatonin secretion displays a robust rhythm, stable, and very reproducible from one day to another. About 5 % of normal subjects show low or undetectable plasma melatonin levels, whose meaning and delayed consequences remain unknown. Artificial light, as low as the domestic one, can display a masking effect on melatonin secretion. Also, the women’s melatonin secretion is more sensitive to light than the men’s one. Melatonin metabolism is regulated to favour a clear-‐cut hormone message with a peak at night and low or undetectable blood levels during the day. Physiological melatonin effects are difficult to study in humans, especially in the circadian area. Studies with exogenous melatonin administration should provide blood profiles superimposed upon the endogenous ones. The model of melatonin deficiency, corrected by exogenous supplementation, which is classical in endocrinology, is not available in human pathology. Patients whose melatonin rhythm is abolished after removal and/or radiotherapy of pinealoma are not a pure model of hormone defect, due to after-‐effects, especially in adjacent nervous structures. The study of the effects of melatonin suppression with light, especially on sleep is not sound, since light administration needs to maintain the subjects waken up and â-‐blockers display a specific activity on sleep. Finally, specific antagonists of different types of melatonin receptors are not available for clinical studies. A general opinion on the role of melatonin is that its rhythm is an endogenous synchronizer, able to entrain rhythms, to stabilize or to reinforce them. Data are available in humans mainly for the sleep-‐wake-‐cycle and temperature rhythm. Also, melatonin stabilizes the circadian system since receptors are present in the SCN. The synchronizing role of melatonin extends to other physiological functions or cellular mechanisms which display a circadian organization: immunity, blood pressure, hemostasis, bone metabolism, cell multiplication, anti-‐oxydative activity etc…We will present some data on the synchronizing role of melatonin obtained following alteration of melatonin secretion with light and in clinical situations (pinealoma, migraine, Smith-‐Magenis syndrome…) Keywords: melatonin, humans, synchronizer

S16.1 CIRCADIAN REGULATION OF BEHAVIORS IN MAMMALS. INTRODUCTION. 1Ken-‐ichi Honma and Horacio de la Iglesia. 1Department of Physiology, Hokaido University Graduate School of Medicine Sapporo, 060-‐8036, Japan. The circadian system in mammals is a hierarchical multi-‐oscillator system, consisting of the central clock in the suprachiasmatic nucleus (SCN) and the peripheral clocks in a variety of tissues and organs. Although behaviors are the most prominent and probably important output of the circadian system, we are still ignorant in the regulatory mechanism of circadian behavior rhythms on molecular or cellular levels. Several parameters associated with circadian behavior rhythms such as a phase-‐angle difference to the ambient light

105

conditions and an interval between the onset and end of activity (activity time) are the subjects of circadian regulation. In addition, we know behavior rhythms in the circadian domain which are independent of the SCN. Furthermore, a short cycle of 3 to 6 hours is evident in behaviors of some mammal species which used to be called as the basic res-‐activity cycle (BRAC). Recently, the circadian pacemakers are suggested in the mouse SCN which respectively regulate the onset and end of activity time (Inagaki et al., PNAS, 2007). Changes of activity time in respond to photoperiod are explained by changes in the coupling of these circadian pacemakers. The peripheral clocks for behavior rhythms are suggested to be involved in methamphetamine-‐induced circadian behavior rhythm (Honma & Honma, Eur J Neurosci, 2009). And a pacemaker for the BRAC could be in the SCN (Nakamura et al., Curr Biol, 2008). In this symposium, the overall picture for the circadian regulation of behavior rhythm in mammals is discussed.

S16.2 THE MASTER AND SLAVE OSCILLATORS FOR BEHAVIORAL RHYTHMS IN MICE 1Yujiro Yamanaka, 1Sato Honma, 2Ken-‐ichi Honma. 1Dept. Physiology, Hokkaido University Graduate School of Medicine, Japan. 2Dept. Chronomedicine, Hokkaido University Graduate School of Medicine, Japan. The hierarchical system in mammalian circadian oscillators shows several features after an abrupt shift of environmental light cycles, such as jet-‐lag travel. The first is circadian behavioral rhythms re-‐entrain to a new light cycle with transient period. The second is that the period of transient relates to the direction of phase-‐shifts (advance or delay). At now, one hypothesis has been addressed that this transient shown in circadian behavioral rhythms reflect a gradual phase-‐shift of slave oscillator to reestablishment a steady state phase-‐relationship with the master pacemaker in the SCN. Also, it has been reported that circadian rhythm in the SCN rapidly finish the re-‐entrainment to a new light cycle, although the rhythms in behavior and peripheral organs gradually phase-‐shifted and require several transient days. However, it remains unknown that different transient rate in two behavioral component (onset and end of activity) relates to the instantaneous shift in the SCN. Recently, using transgenic Per1-‐luc mice, we examined whether or not the phase-‐shift of two behavioral components relates to the phase-‐shift of mPer1 expression rhythm in the SCN. We found different transient period for re-‐entrainment to an 8-‐h advanced light cycle not only between onset and end of activity but also among the Per1 expression rhythms in different SCN areas. We will discuss the possibility that specific region of the SCN regulates different components of behavioral rhythms in response to an abrupt shift of LD cycle.

106

S16.3 A FUNCTIONAL PROPERTY OF CIRCADIAN PACEMAKERS WHICH CONTROL BEHAVIORAL RHYTHMS Wataru Nakamura. Laboratory of Oral Chronobiology, Osaka University Graduate School of Dentistry, Japan. Behavioral circadian rhythms are the final output of the internal biological clock, the master pacemaker of which is located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. By detailed analysis in behavior, it has been predicted basic properties of the circadian pacemaker such as self-‐sustained oscillation (free-‐run) and entrainment (phase adjustment). The predictions have been verified in vivo and in vitro and further study has been made into molecular levels. Recent discovery of molecular machineries of circadian oscillation enables us to examine the genetically manipulated, molecular-‐clock-‐deficient mice and use of these animal models has enhanced our understanding regarding the biological clocks. Although basic behavioral assay using molecular-‐clock-‐deficient mice is really informative, moreover we have been applied a couple of classic techniques on it. (i) In vivo multi-‐unit neural activity recording (MUA) in freely moving mice enables us to monitor the circadian output of the pacemaker itself that reflect environmental input for entrainment. (ii) Specific lesion of SCN is the indispensable technique for studies of extra-‐SCN oscillators which might be able to control behavioral rhythms without the master pacemaker. By applying modern and classics on behavioral analysis, we present here the functional property of circadian pacemakers. Keywords: SCN, in vivo MUA, Lesion

S16.4 REDUCED FOOD INTAKE HAS PRONOUNCED EFFECTS ON BEHAVIOURAL ENTRAINMENT AND INTERNAL SYNCHRONISATION Roelof A. Hut, Violetta Pilorz, Sjaak Riede, Vincent van der Vinne, Serge Daan. Center for Behavior and Neurosciences, Chronobiology Unit, University of Groningen, the Netherlands. Food restriction modifies behavioural entrainment and internal circadian organisation. Food restriction involves both a time component when food is delivered as well as a reduced food intake component. Here we evaluate the effect of reduced food intake per se, on behavioural entrainment and internal circadian organisation. To this purpose we simulate natural food shortage in mice that work for food. We show that reduced food intake alone

107

has severe impact on circadian organisation and shifts the activity phase from the night into the day. Relative to the light-‐dark cycle, we found various phase angle changes for different organs, but we found no evidence for phase angle change within the SCN. This flexibility in timing of behaviour and organ function may be highly adaptive since it allows mice to exploit the diurnal temporal niche while minimizing energy expenditure under poor feeding conditions in nature. This study reveals an intimate link between metabolism and mammalian circadian organization.

S16.5 A ROLE FOR THE HABENULA IN THE REGULATION OF LOCOMOTOR ACTIVITY CYCLES Matthew J. Paul, Premananda Indic, William J. Schwartz. University of Massachusetts Medical School, Worcester, USA. While much is known about the regulation of the circadian rest-‐activity cycle by the hypothalamic suprachiasmatic nucleus in nocturnal rodents, little is known about the neural substrates that regulate the temporal organization of nocturnal activity within the active phase. In this talk, data are presented in Syrian hamsters to implicate the habenula “believed to be involved in motivation, reward, and motor control“ as a candidate site for such a role. First, by examining hamsters during the day and night and by introducing a "novel" running wheel in order to induce daytime motor activity, we show that immunoreactive c-‐Fos expression in the lateral and medial habenula is related to motor activity / arousal. Second, by transecting the major efferent pathway of the habenula (fasciculus retroflexus), we show that the interruption of habenula neural output alters the daily amount of motor activity; lengthens the period of the circadian rest-‐activity rhythm; and disrupts the species-‐typical pattern of nocturnal motor activity, measured as either wheel running behavior or general locomotor activity. Instead of the usual pattern of nighttime locomotion, characterized by a prolonged bout of elevated activity in the early night followed by shorter sporadic bouts or the cessation of activity altogether, lesioned animals exhibited a more homogeneous, undifferentiated temporal profile extending across the night. These data suggest a previously unrecognized function of the habenula whereby it regulates the temporal pattern of activity occurring within a circadian rest-‐activity window set by the suprachiasmatic nucleus. Keywords: Syrian hamster, circadian rest-‐activity cycle, fasciculus retroflexus

108

S17.1 DMH-‐SCN INTERACTION PERMITS FOOD ANTICIPATORY BEHAVIOR DURING THE REST PHASE 1Ruud Buijs, 1Guadalupe Acosta-‐Galván, 2Jack Jhamandas, 3Manuel Ángeles Castellanos, 1María del Carmen Basualdo, 3Carolina Escobar. 1Instituto de Investigaciones Biomédicas, UNAM, México. 2Division of Neurology, Department of Medicine, University of Alberta, Canada. 3Fac. Medicina, Universidad Nacional Autónoma de México, México. Food anticipatory behavior (FAA) is induced by limiting access to food for a few hours daily. Animals anticipate this scheduled meal event even without the suprachiasmatic nucleus (SCN), the biological clock. Consequently a food entrained oscillator (FEO) has been proposed to be responsible for meal time estimation. Recent studies suggested the dorsomedial hypothalamus (DMH) as the site for this FEO which has led to considerable controversy in the literature. Herein we demonstrate by means of c-‐Fos immunohistochemistry that the neuronal activity of the suprachiasmatic nucleus (SCN), which signals the rest phase in nocturnal animals, is reduced when animals anticipate the scheduled food and simultaneously neuronal activity within the DMH increases. Using retrograde tracing and confocal analysis, we show that inhibition of SCN neuronal activity is the consequence of activation of GABA containing neurons in the DMH that project to the SCN. Next we show that DMH lesions result in a loss or diminution of FAA simultaneous with increased activity in the SCN. A subsequent lesion of the SCN restored FAA. We conclude that in intact animals, FAA may only occur when the DMH inhibits the activity of the SCN thus permitting locomotor activity. As a result, FAA originates from a neuronal network comprising an interaction between the DMH and SCN. Moreover, this study shows that the DMH-‐SCN interaction may serve as an intra-‐hypothalamic system to gate activity instead of rest overriding circadian predetermined temporal patterns. Acknowledgments: This study was supported by grants from CONACyT 79797, DGAPA PAPIIT IN215308-‐3 UNAM, Mexico Keywords: Food anticipatory behavior, DMH, FEO

109

S17.2 ROLE OF FOOD COMPONENTS IN ENTRAINMENT OF MOUSE LIVER CLOCK Shigenobu Shibata. Department of Physiology & Pharmacology, School of Advanced Science and Engineering, Waseda University, Japan. Since the discovery of clock gene in 1997, molecular mechanism of circadian oscillation and daily entrainment has been elucidated. When the expression profile of clock genes such as Per1 and Per2 is examined in the whole body, almost all organs such as kidney liver, lung, and skeletal muscle clearly show the circadian rhythms, suggesting that peripheral clock may operate the local clock function. Daily restricted feeding entrains the circadian rhythm of mouse clock gene expression in the central nervous system excluding the suprachiasmatic nucleus (SCN), as well as in the peripheral tissues such as the liver, lungs, and heart. Although the circadian liver clock is entrained by the feeding cycle, the factors such as food volume and starvation interval are poorly understood. Per2:luciferase knock-‐in mice were given two meals per day with different food volume sizes and/or with different intervals of starvation between two mealtimes under the total food volume per day fixed at 3.6g (80 food pellets, approximately 75% of free-‐feeding) per mouse. The bioluminescence rhythm in the liver produced an unimodal peak but not bimodal peak under the regimen of two meals per day over 14-‐15 days. Peak Per2 expression occurred concurrently with the mealtime of the larger food volume, when the first and second meal was given as different food volume ratios under a 12 h feeding interval. When an equal volume of food was given under different starvation interval (8h:16h), the peak of the Per2 rhythm was close to peak by mealtime after long starvation (16h). When food volumes for each mealtime were changed under (8h:16h), the peak rhythm was influenced by combined factors of food volume and starvation interval. Food intake after the 16 h starvation caused a significant increase in liver Per2, Dec1, and Bmal1gene expression in comparison with food intake after the 8 h starvation under (8h:16h) feeding intervals. In conclusion, the present results clearly demonstrate that food-‐induced entrainment of the liver clock is dependent on both food volume and the starvation interval between two meals. We recently examined the effect of carbohydrates, including sugar and starch, on the entrainment of the circadian liver clock using Per2::luciferase knock-‐in mice. Humans generally consume an appropriately balanced diet containing starch, protein, and oil, but not simple nutrients including glucose. In order to elucidate the role of nutrition in inducing phase shifts, components of the AIN-‐93M diet were partially or completely substituted. In carbon hydrates, corn, rice, and wheat starch posses a stronger entrainable signal rather than green beans and potato starch, suggesting that easily digestible starch and sugar are important component for food entrainment. When casein was substituted by amino acids or polypeptides, and casein percentage (14%) in diets was decreased to 8% or 6%, the magnitude of phase-‐advance was unaffected by

110

these treatments. These data suggest that increase of glucose and/or increase of insulin secretion may be involved in process of food-‐induced entrainment of peripheral clock. Keywords: feeding, liver, nutrients

S17.3 “SWEET TRANSITION” OF NEURONAL ACTIVITY INDUCED BY ENTRAINMENT ON PALATABLE FOOD Elena Timofeeva. Laval University, Canada. Feeding activity is synchronized with light-‐dark cycles by the brain light-‐entrainable oscillator (LEO). However, when food availability is restricted to a few hours scheduled at fixed time of the day, the light-‐dark-‐related activity is compromised, and rodents develop food-‐entrained circadian rhythms with pronounced food-‐anticipatory activity (FAA). The FAA, expressed by increased exploration and foraging locomotion, is driven by the food-‐entrainable oscillators (FEOs). The nutritive and hedonic values of food seem to have strong entraining effects on the FAA. The metabolic fluctuation created by time-‐limited caloric intakes and following negative metabolic states is a powerful Zeitgeber. On the other hand, the limited access to palatable food in free-‐feeding animals also entrains the FAA. In the brain, restricted feeding schedules and limited access to palatable food seem to activate distinct neuronal circuitries. During FAA, the restricted feeding schedules produces neuronal activation within the hypothalamic strictures while the limited access to palatable food mainly activates the limbic structures. It was not clear whether this palatable-‐food-‐induced pattern of neuronal activation during FAA will persist for combination of negative energy balance and scheduled access to palatable food. To investigate this question we compared the pattern of early induction of expression of mRNA of immediate early gene c-‐fos, a marker of neuronal activation, during FAA in rats maintained on restricted feeding schedules (2-‐h daily food access between ZT6-‐8) on regular chow (chow-‐FA group) or chow and sucrose (sucrose-‐FA group). The chow-‐FA rats gradually increased daily intake of chow so that at the end of the three-‐week treatment they consumed during their 2-‐h access to food the amount of chow comparable to 24-‐h intake of freely-‐fed rats. The sucrose-‐FA rats dramatically escalated intake of sucrose but did not demonstrate increase in chow intake. The chow-‐FA rats showed early (at ZT3) activation of the dorsomedial hypothalamic nucleus (DMH), septohippocampal nucleus, and the paraventricular thalamic nucleus. As time of feeding approached (at ZT5-‐6), the activation also extended to the prefrontal cortex (PFC) and the paraventricular hypothalamic nucleus (PVN). The sucrose-‐FA rats early (at ZT3) activated the PFC, lateral septum and the anterior lateral hypothalamus. As the time of access to food approached (at ZT5-‐6), the accumbens shell and core, but not the DMH and PVN, were also involved in the neuronal activation in the sucrose-‐FA rats. Therefore, adding

111

sucrose to feeding schedules reorganizes brain activity by activating limbic-‐related regions but not medial hypothalamic nuclei. These data also provide evidence that brain may contain different oscillatory systems which are specifically entrained according to food palatability and metabolic state, and food palatability has a particularly strong entraining power. Keywords: food-‐entrainable oscillator, palatable food, c-‐fos mRNA

S17.4 GUT PEPTIDES ARE NOT NECESSARY FOR FOOD ANTICIPATORY ACTIVITY INDUCED BY A HIGHLY PALATABLE MEAL Megan J Dailey, Timothy H Moran. Johns Hopkins University, USA. Animals learn to anticipate a meal as evidenced by increased activity prior to a scheduled mealtime. This learned response appears to be independent of nutrient status because food anticipatory activity (FAA) can be seen after entrainment by a highly palatable food when rats remain ad lib on chow. Mealtime feeding not only induces an increase in activity, but also appears to entrain the secretion of various peptides prior to a meal including insulin, ghrelin and glp-‐1 when rats are conditioned to a 4 h meal each day. It is not clear if these meal anticipatory increases in gut peptides are also associated with FAA or those induced by a highly palatable food. In order to assess if these preprandial peptide changes are associated with the FAA that occurs with palatable meal entrainment, rats were conditioned to receive a 2 h access of chocolate in the middle of the light cycle while remaining ad lib on chow. FAA was measured for 4 h prior to the chocolate meal. Rats were then sacrificed at 90, 60, 30min prior to the chocolate mealtime and plasma was collected. Although the chocolate-‐entrained rats showed FAA compared with the non-‐chocolate entrained animals, they did not show anticipatory increases in the ghrelin or glp-‐1. In fact, chocolate entrained animals had significant decreases in insulin prior to mealtime. Thus, FAA can occur without increases in insulin, ghrelin or glp-‐1. This suggests that separate mechanisms may underlie the meal entrainment to chow versus entrainment to a palatable food when freely feeding. Keywords: food anticipatory activity, gut peptides, palatable meal

112

S18.1 CIRCADIAN RHYTHMS IN GLIAL CALCIUM SIGNALING MEDIATE RHYTHMIC EXTRACELLULAR ATP ACCUMULATION IN THE SUPRACHIASMATIC NUCLEUS Mark J. Zoran. Department of Biology, Texas A&M University, USA. Extracellular ATP accumulates rhythmically in the suprachiasmatic nucleus (SCN) of the rat with peak ATP levels occurring in late night and persisting in constant darkness. Furthermore, ATP gliotransmitter release in an immortalized rat SCN2.2 cell line peaks every 24 hours (Womac et al., 2009; Eur J Neurosci). However, the intracellular signaling pathways that link the SCN clockworks to this circadian rhythm in extracellular ATP accumulation remain unclear. Since the rat SCN contains a high density of astrocytes and stimulus-‐evoked ATP release from astrocytes is a calcium-‐dependent process, we used ratiometric imaging to determine if calcium levels oscillate in SCN glial cells. In rat SCN2.2 astrocytes in vitro, intracellular Ca2+ levels fluctuated in an antiphase relationship with rhythmic extracellular ATP accumulation. Subsequently, we found that mitochondrial Ca2+ levels were also rhythmic in SCN2.2 astrocytes and that peak mitochondrial Ca2+ occurred during maximal clock-‐controlled ATP release and was in precise antiphase with the peak in cytosolic Ca2+. Furthermore, inhibition of the mitochondrial Ca2+ uniporter disrupted rhythmic production and extracellular accumulation of ATP. Taken together, these results demonstrate that mitochondrial Ca2+ signaling links the circadian clockworks to gliotransmission in SCN astrocytes. Sensitivity to ATP, as determined by intracellular calcium responses of glial cells to exogenously applied ATP, differs over circadian time, with highest sensitivity to ATP and most expansive ATP-‐dependent calcium waves present when circadian-‐regulated ATP release is lowest. Interestingly, this trough time in the circadian astrocytic ATP cycle is also when melatonin has its greatest modulatory effects on glial ATP and calcium signaling. To investigate the physiological consequences of clock-‐controlled ATP release in SCN glial cells, we blocked specific P2X and P2Y purinergic receptors. The P2X7 receptor antagonist, BBG, abolished rhythms in ATP release and these ATP rhythms were dampened by 72 hours of treatment with several other P2XR antagonists. In addition, BBG treatment of mouse SCN cell cultures produced a dampening of PER2::LUC reporter luminescence, indicating that alterations in purinergic signaling can influence the synchrony of ensemble SCN clock gene expression. Taken together, these studies suggest that ATP and calcium signaling are both important mechanistic regulators of clock-‐controlled ATP release rhythms in mammalian glial cells. Keywords: Adenosine triphosphate, Astrocyte, suprachiasmatic nucleus

113

S18.2 ASTROCYTE-‐ASTROCYTE COMMUNICATION AND CONNEXIN 43 REGULATE MAMMALIAN CIRCADIAN RHYTHMS Luciano Marpegan, Connie Tsai, Tatiana Simon, Erik Herzog. Washington University, USA. Evidence from in vivo and in vitro studies indicates that intercellular communication modulates the rhythmic physiology in mammals. Glial cell activity has been implicated, but understudied, in the circadian system. We showed previously that astroglia are competent circadian oscillators which synchronize to diffusible signals from the SCN and that vasoactive intestinal polypeptide (VIP) can coordinate rhythms in astrocytes. Here, we studied the effects of cell-‐cell interactions on circadian timing in cultured astrocytes. Measuring PER2::LUC bioluminescence rhythms from cultured, mouse cortical astrocytes we found that astrocytes plated at high density (1200 cells/mm2) displayed longer periods and faster damping rates than low density cultures (100 cells/mm2; 24.7±0.1 vs. 25.9±0.1 h, p< 0.01). These period and damping rate differences were preserved when astrocytes at high density were cultured next to astrocytes at low density, leading us to hypothesize that glia can modulate circadian timing in neighboring glia. Since gap junctions can mediate short-‐range interactions between astrocytes, we tested the effects of gap junction blockers in astrocyte cultures and SCN explants. Meclofenamic acid (MEC, 150µM) increased the damping rate in both high and low density astroglia and decreased the period in high density cultures (25.4±0.1h to 23.6±0.3h, p<0.05). Addition of 50µM MEC to SCN explants also shortened the period and damped PER2::LUC rhythms. Similar results were observed with Carbenoxolone (200microM), another potent gap junction blocker. Using immunocytochemistry and Western blots, we found connexin 43, the major component of gap junctions in astrocytes, was circadian in cultured cortical astrocytes and in vivo. Taken together these results suggest that intercellular communication among astrocytes via gap junctions modulates the period and sustainability of the circadian clock. Keywords: glia, astrocytes, gap junctions

S18.3 ADULT GLIAL CELLS MODULATE THE CIRCADIAN NEURONAL CIRCUIT F. Rob Jackson, Fanny S. Ng. Tufts University School of Medicine, USA. Contemporary neuroscience investigations have the goal of defining the neural circuits and synaptic interactions that mediate behavioral plasticity. In both mammals and Drosophila,

114

the neuronal circuitry underlying circadian behavior has been studied extensively, but roles for glial cells in circadian networks have only begun to be defined. We have shown in recent studies that conditional, cell-‐specific genetic manipulations of glia lead to arrhythmic circadian behavior in adult behaving Drosophila. Correlated effects on behavior and Peptide Dispersing Factor (PDF) “a known clock neuron peptide transmitter” demonstrate the capacity for glia-‐to-‐neuron signaling in the circadian circuitry. Results from these studies also reveal the importance of glial internal calcium stores and a single type of fly glial cell “the astrocyte” in the regulation of circadian rhythms. Our results indicate that adult glial cells can physiologically modulate the circadian neuronal circuitry and behavior. Keywords: glia, Drosophila, locomotor activity

S18.4 THE INVOLVEMENT OF GLIA IN THE CIRCADIAN REGULATION OF NEURONAL MORPHOLOGY Elzbieta Malgorzta Pyza, Milena Damulewicz, Jolanta Gorska-‐Andrzejak. Jagiellonian University, Institute of Zoology, Department of Cytology and Histology, Poland. In the first optic neuropil (lamina) of the fly’s visual system six types of glial cells have been distinguished. This includes the fenestrated, pseudocartridge and satellite glia located between the retina and lamina and in the lamina cortex, and the epithelial glia distributed in the lamina synaptic neuropil. The epithelial glial cells surround the lamina modules called cartridges which consist of lamina interneurons, photoreceptor terminals and processes of other neurons. In each cartridge two the largest lamina interneurons, L1 and L2 monopolar cells, which receive visual and photic information, show circadian morphological plasticity. They change their size and shape during the day and night and the pattern of these changes is correlated with the circadian rhythm of locomotor activity of flies. Moreover, these changes are offset by the epithelial glial cells which swell when neurons are shrank. In Drosophila L1 and L2 increase girth of their axons in the morning and in the evening. These changes seem to be driven by cyclical physiological and morphological changes in the epithelial glial cells since disruption of glia metabolism affect sizes of L1 and L2 axons. Moreover, these glial cells show cyclical expression of clock genes and changes in levels of the Na+/K+-‐ATPase and the V-‐ATPase subunits. The level of the alpha subunit of the Na+/K+ATPase increases in the morning and in the evening and this pattern is correlated with the bimodal pattern of L1 and L2 swelling and shrinking. In turn the V-‐ATPase in glia is active during the night and together with Ebony seem to take part in metabolism and transport of histamine, a neurotransmitter in tetrad synapses formed between the photoreceptor terminals and L1 and L2 monopolar cells. Although clock genes are expressed in the lamina glia, circadian inputs from the retina photoreceptors and from clock neurons in the brain are also important. The lack of the clock gene expression in glia

115

changes the pattern of circadian rhythms in neurons. They also depend on a circadian input from the clock lateral neurons that invade the lamina as a fine projection terminating in glia above the lamina cortex. This projection is immunoreactive to ion transport peptide (ITP) but not to pigment-‐dispersing hormone (PDF), a neurotransmitter of the ventral lateral neurons. ITP may facilitate ions and fluids shifts responsible for morphological changes of the epithelial glial cells and L1 and L2 interneurons. Keywords: neuronal plasticity, clock genes, ion pumps

S19.1 CIRCADIAN REGULATION OF SENSORY PROCESSING Erik Herzog. Washington University, St. Louis, USA.

S19.2 CIRCADIAN REGULATION OF OLFACTORY SENSITIVITY AND OLFACTORY BEHAVIOR IN THE COCKROACH, LEUCOPHAEA MADERAE Terry L. Page, Madeleine V. Garren. Department of Biological Sciences, Vanderbilt University, USA. Olfaction is a dominant sensory cue in both vertebrates and invertebrates and, particularly in insects, is often the critical signal for a variety of well-‐studied behaviors including reproduction, feeding, host-‐identification, and intra-‐specific competition. There have been several studies on circadian regulation of olfactory receptors and olfactory behaviors in the cockroach Leucophaea maderae. Cockroaches exhibit a robust circadian rhythm in the olfactory response of the antenna to both food-‐related odors and sex pheromone components. The results indicate there is a global regulation of olfactory sensitivity that extends to the responses of single olfactory receptors. Interestingly, peak olfactory sensitivity occurs in the early subjective day, a time when the animals are inactive. These data raise the question of what impact the rhythms in olfactory sensitivity might have on olfactory behavior, and we have examined two aspects of behavior that are strongly dependent on olfactory input. First, we have found that mating behavior, which is believed to be regulated by sex pheromone reception, is controlled by the circadian system, with mating behavior being restricted to the late subjective day/ early subjective night. We have also found that there is robust rhythm in olfactory learning and memory that appears to be regulated by the circadian system. Following training by classical conditioning, cockroaches trained in the early subjective night showed excellent learning and retained the memory for at least two days. In contrast, animals trained and tested at other circadian phases showed significant deficits in performance for both short-‐ and long-‐term memory. Interestingly,

116

with an operant conditioning paradigm, learning and memory appear to be unaffected by circadian phase, and animals perform equally well when trained in the early subjective night or early subjective day. Thus far we have no evidence that the circadian rhythms in olfactory behaviors are dependent on the circadian rhythms in olfactory sensitivity. The results indicate that the impact of the circadian modulation of olfactory input may be subtle or perhaps will only be evident near stimulus threshold. In contrast, the data clearly support the view that the rhythms in olfactory behavior arise from circadian modulation of the central processing of sensory input. Keywords: insect, olfactory behavior, learning and memory

S19.3 CIRCADIAN RHYTHM IN OLFACTION IN A SUBTERRANEAN ORGANISM

Martha Merrow and Maria Olmedo. Department of Molecular Chronobiology, University of Groningen, The Netherlands.

Experiments with simple microbes clearly establish that circadian clocks confer fitness to organisms that are subject to environments with daily light/dark cycles. However, many organisms experience only muted daily cycles due to insular spatial niches (e.g. caves or soil) leading to speculation that they may dispense of circadian behaviour. Indeed, regulation of behaviour by the circadian clock in the model organism C. elegans is generally so subtle as to limit e.g. genetic dissection of the molecular clock. We show that the circadian system in the nematode has far-‐reaching and ecologically important functions via the regulation of chemotaxis in response to an olfactory stimulus. We furthermore have a handle on molecular clock-‐regulated processes: nematodes that express LUCIFERASE show rhythms in its activity and oscillations metabolites are described. This work describes the circadian system in the nematode from molecules to behaviour, revealing a potentially novel molecular mechanism of a biological clock.

S19.4 CIRCADIAN GATING OF PHOTIC INPUTS IN PLANTS Andrew Millar. University of Edinburgh, UK.

117

S19.5 CIRCADIAN MODULATION OF SIMPLE AND SELECTIVE ATTENTION IN HUMANS Kenneth P. Wright Jr., Ph.D. Sleep and Chronobiology Laboratory, Department of Integrative Physiology, University of Colorado Boulder, Boulder CO 80309 USA For over one hundred years scientists have used reaction time measures to assess sensory-‐motor performance. We utilized reaction time to simple and selective attention tasks to examine the circadian contribution to these cognitive functions in humans tested during 28-‐40 h long constant routine and two week long T=28h forced desynchrony circadian protocols. Findings show a robust circadian modulation to simple and to some, but not all, selective attention tasks (p<0.05). The amplitude of the circadian modulation of simple and selective attention was dependent upon the level of sleep inertia (grogginess upon awakening from sleep) and upon the level of sleep homeostasis (hours awake). From a circadian perspective, simple and selective attention was worst in the early morning hours during sleep inertia or when sleep homeostasis was high. These findings have important implications for humans required to work at an adverse circadian phase following long work hours or immediately upon awakening from sleep.

S20.1 TRACKING PHASE IN A NETWORK OF HETEROGENEOUS SCN CELLS Stephanie Taylor. Colby College, USA. I plan to discuss the interplay between modeling and measurement. We are using wavelet analysis on bioluminescence data, wavelet analysis on complex model simulations, and (simple) phase-‐only model simulation.

118

S20.2 APPLYING WAVELET TRANSFORMS TO CIRCADIAN DATA Tanya L Leise. Amherst College, USA. Circadian rhythms are characterized by ~24 hour cycles of some observable variables like locomotor activity or clock gene expression. The period of some rhythms can be quite precise, but circadian oscillations can also exhibit considerable variability in period and amplitude over time. Wavelet transforms provide a means of quantifying this variability in oscillatory time series. Wavelet methods can also be used to quite effectively remove trend and noise from time series, as well as assessing the strength of rhythms in different frequency bands, for example, ultradian versus circadian components in an activity record. I will describe how to apply discrete and continuous wavelet transforms to circadian data, illustrated with examples. Keywords: time series analysis, wavelet, period variability

S20.3 NEW METHODS OF ANALYSIS FOR CHRONOBIOLOGY: COMPLEXITY, FRACTALS, DIMENSIONS, RECURRENCE PLOTS... Antoni Diez-‐Noguera. Dept. Physiology. Fac. Pharmacy, Univ. Barcelona, Spain. The analysis of large data sets obtained through time (time series = TS) is a fundamental activity in Chronobiology research. Traditionally, the analysis has been focused on the detection and quantification of periodicities using periodograms, spectral analysis, circular statistics and conventional regression methods. Given that a TS reflects the activity of many other physiological processes, in addition to rhythms, and considering the advent of new techniques of mathematical analysis, it is of great interest to extract more information about the real nature of these processes. In most cases this information is related with the functional structure and variability of the process, and is embedded in the TS but usually remains hidden or unseen by traditional methods. One can distinguish two levels or scales of analysis: one is the overall structure of the rhythm, in which the interest is on the level of complexity of the cycles, its repetitiveness, its variability and its predictability. The other level of analysis is the fine structure of the data beyond their rhythmicity. Also at this level one can study the complexity of the series, the degree of coherence, its fractal structure, the memory of the process, or the number of dimensions (variables) governing the process.

119

Here, we review and propose the use of various techniques and discuss their characteristics and possible applications in Chronobiology. The methods to be discussed include: fractal analysis, analysis of complexity, entropy estimation, the calculation of the Hurst index, phase space reconstruction, correlation dimension, several exploratory graphical techniques and the novel analysis of recurrence plots. Keywords: Analysis, time-‐series, complexity

S20.4 MODELING ENTRAINMENT OF THE MAMMALIAN CIRCADIAN CLOCK Adrian E. Granada, Hanspeter Herzel. Institute for Theoretical Biology, Humboldt University Berlin, Germany. This talk will discuss two problems related to entrainment of circadian rhythms: 1) How intercellular coupling controls the entrainment region? 2) Can locomotor patterns outside entrainment unveil SCN subpopulations? Regarding the first question, we perform a systematic theoretical and experimental study and find that intercellular coupling governs the range of entrainment (Abraham et al. MSB 6:438, 2010). In the second case, we study irregular behavioural rhythms using signal analysis and nonlinear dynamics. We show that a periodically driven oscillator can generate the observed complex locomotor patterns ("beating", "side-‐bands"). We developed a framework to study desynchronization patterns and discuss the interpretations and limitations of a reverse engineering approach on this data (Granada et al. J. Royal Society Interface Focus 1:153-‐166, 2011).

S21.1 EFFECTS OF PERIOD3 POLYMORPHISM ON CIRCADIAN RHYTHMICITY AND SLEEP HOMEOSTASIS IN HEALTHY OLDER INDIVIDUALS 1Antoine U Viola, 1Sarah L Chellappa, 2Simon N Archer, 2Derk-‐Jan Dijk, 1Christian Cajochen. 1Centre for Chronobiology, University of Basel, Switzerland. 2Surrey Sleep Research Centre, University of Surrey, United Kingdom. Aging is associated with a decrease in non-‐rapid eye movement (non-‐REM) sleep consolidation and circadian phase advance, which can reflect changes in the sleep homeostatic and/or circadian drive. In young subjects, a polymorphism of the clock gene PERIOD3 (PER3) can predict inter-‐individual sleep differences, such as slow EEG oscillations during NREM sleep, REM sleep and wakefulness, with no changes in circadian rhythmicity. Predictors of these inter-‐individual differences in sleep in older people are still unknown. Here we investigated circadian rhythms and sleep EEG characteristics in older participants

120

homozygous for the longer (PER35/5) and for the shorter (PER34/4) allele of the clock gene PER3. Healthy older volunteers were selected exclusively on the basis of their PER3 genotype, andPER3 polymorphism was determined in 133 participants (55-‐75 years). Twenty-‐one PER35/5 and 16 PER34/4 participants completed the 3-‐week field segment of the study, which comprised actigraphy monitoring and sleep diaries to characterize habitual sleep and wake times. Wake-‐up times from sleep diaries indicated a tendency for earlier timing for PER35/5 participants. Similarly, actiwatch analysis revealed significant earlier timing of the rest-‐activity cycle in PER35/5 participants. For the laboratory study, 13 PER35/5 (5 men, 8 women, 62.23 ± 1.01 years) and 13 PER34/4 (5 men, 8 women, 62.38 ± 1.39 years) participants were selected and matched by age, gender, body mass index and ethnicity. Following a baseline night, all volunteers underwent approximately 40 hours of extended wakefulness under constant routine conditions (CR), to assess endogenous circadian phase and amplitude in the absence of the confounding effects of light-‐dark and behavioural cycles. The CR was followed by a recovery sleep. Circadian rhythms of core body temperature and cortisol did not differ between genotypes. Interestingly, melatonin profile across extended wakefulness revealed that PER35/5 subjects had a phase-‐advance of fitted melatonin maximum compared to PER34/4 subjects. Sleep structure and consolidation differed between genotypes: Homozygosity for the longer allele (PER35/5) had a significant effect on baseline sleep structure, with lower total sleep time, sleep efficiency, shorter non-‐REM sleep stage-‐2 duration, and more wakefulness. Spectral analysis of baseline and recovery sleep EEG activity further indicated differences between the genotypes: EEG delta activity (0.75-‐1.75Hz) in non-‐REM sleep was significantly higher (increase of 39.7% for the entire night) and spindle activity (11-‐13.5Hz) was significantly lower (decrease of 31.9% for all night) in PER35/5 compared to PER34/4 individuals (p<0.05). Within the framework of the circadian and homeostatic regulation of sleep, our data imply for the first time that the interaction of aging and the PER3 VNTR polymorphism affects both the circadian and the homeostatic aspects of sleep regulation. These data have implications for our understanding of the basis of inter-‐individual differences in age-‐related changes in circadian rhythmicity and sleep. Keywords: clock gene, age, sleep

S21.2 INVOLVEMENT OF CLOCK GENES IN SLEEP HOMEOSTASIS IN MICE 1,2Valerie Mongrain, 2Francesco La Spada, 2Thomas Curie, 2Paul Franken. 1Center for Advanced Research in Sleep Medicine, HSCM; Department of Psychiatry, Université de Montréal; 2Center for Integrative Genomics, University of Lausanne, Switzerland Sleep is regulated by a circadian and a homeostatic process. The molecular wiring of the circadian timing system is known to depend on complex molecular feedback loops involving

121

clock genes and their protein products. However, clock genes have been reported to play a clock-‐independent role in the regulation of sleep homeostasis. This notion was based mostly on the observation that clock gene expression changes in parallel with sleep pressure (e.g., after sleep deprivation), and by the altered dynamics of sleep homeostasis in animals lacking different clock genes (i.e., Cry1/Cry2, Clock, Npas2, and Bmal1). Strain comparisons revealed that the sleep deprivation-‐dependent changes in clock gene expression varied with genetic background such that the strain with the smallest increase in sleep pressure, as measured by the electrophysiological marker of homeostatic sleep pressure EEG delta power, displayed the largest increase in the expression of Per1 and Per2. Our most recent work indicates that two distinct molecular pathways appear to importantly contribute to the sleep-‐wake dependent changes in clock gene expression: a glucocorticoid-‐dependent pathway and the core clock transcriptional machinery itself. The strain comparison mentioned above equally revealed differences in stress-‐susceptibility with increases in plasma corticosterone after sleep deprivation being proportional to the increase of Per1 and Per2 expression. Using adrenalectomy, we could causally link the surge in corticosterone associated with the sleep deprivation with the increase in Per1, -‐2, and -‐3 expression. Moreover, sleep pressure directly impinges on the core molecular clock. We observed specific changes in the DNA binding activity of the three core clock transcription factors (i.e., CLOCK, NPAS2, and BMAL1) to the E-‐box elements in the promoter of Per2 and of the clock controlled gene Dbp in the cerebral cortex. Overall, our results shed light on the molecular mechanisms linking clock genes to sleep homeostasis and on the considerable variability associated with the molecular response to increased sleep pressure. Keywords: Sleep regulation, gene expression, rodents

S21.3 FUNCTIONAL GENETIC POLYMORPHISMS OF BDNF AND ADA MODULATE SLEEP AND NEUROBEHAVIORAL PERFORMANCE DURING PROLONGED WAKEFULNESS 1Valérie Bachmann, 2Peter Brugger, 3Wolfgang Berger, 1Hans-‐Peter Landolt. 1Institute of Pharmacology and Toxicology, University of Zürich, Switzerland. 2Department of Neurology, University Hospital Zürich, Switzerland. 3Institute of Medical Molecular Genetics, University of Zürich, Schwerzenbach, Switzerland. A sleep deficit impairs waking functions and is reliably compensated for by increased sleep duration and intensity in recovery sleep. This facet of sleep-‐wake regulation has been referred to sleep homeostasis, and may reflect plastic synaptic changes across the wake-‐sleep continuum (Tononi and Cirelli, 2006). The molecular mechanisms of sleep homeostasis are poorly understood. We hypothesized that functional single nucleotide polymorphisms (SNP) in genes encoding established molecular components of neuronal plasticity such as brain-‐derived neurotrophic factor (BDNF) and the adenosine

122

neuromodulator/receptor system contribute to inter-‐individual differences in sleep-‐dependent neurobehavioral functions and sleep-‐wake homeostasis in humans. The alleles and genotypes of the Val66Met polymorphism of BDNF (Egan et al., 2003) and the Asn8Asp polymorphism of the adenosine metabolizing enzyme adenosine deaminase (ADA) (Rétey et al., 2008) were determined in 127 men and 118 women (all healthy; age: 18-‐40 years). Memory, executive functioning, attention and self-‐reported habitual sleep duration were systematically quantified in all individuals. To investigate the impact of functional allelic variation of BDNF and ADA on homeostatic sleep regulation, two groups of 11 subjects carrying the variant allele were carefully matched according to a case-‐control design with 11 Val (BDNF) and 11 Asn (ADA) homozygotes in two separate studies in the sleep laboratory. Sleep and sleep EEG were recorded in baseline and recovery nights after 40 hours of prolonged wakefulness. Psychomotor and working memory performance, subjective state, and the waking EEG were intermittently quantified throughout sleep loss. Consistent with the literature (Egan et al., 2003), Val/Met allele carriers of BDNF showed reduced working memory (2-‐back task) when compared to Val/Val homozygotes. By contrast, subjective sleepiness (Stanford Sleepiness Scale) and sustained attention (psychomotor vigilance task) were similar in both groups. However, the Met allele carriers showed reduced deep stage 4 sleep and EEG slow-‐wave activity (SWA; 0.5-‐4.5 Hz) in nonREM sleep in both baseline and recovery nights. This difference was restricted to the first nonREM sleep episode when homeostatic sleep pressure is highest, and reflected an attenuated build-‐up of SWA in the initial 30 minutes after sleep onset. The genotype-‐dependent changes differed from those in REM sleep and wakefulness, suggesting that BDNF plays a specific role for nonREM sleep homeostasis. In vitro data indicate that the facilitatory action of BDNF on long term potentiation requires endogenous adenosine (Fontinha et al., 2008). We, thus, investigated whether carriers of the non-‐functional Asp-‐allele of ADA differ from Asn homozygotes. Working memory was not affected by genotype. Nevertheless, individuals with the variant allele performed consistently worse on tasks of vigilant attention (d2, PVT). Vigor was reduced, whereas EEG alpha activity (8.5-‐12 Hz), sleepiness, and fatigue were enhanced throughout prolonged wakefulness. Suggesting elevated homeostatic sleep pressure, slow wave sleep and EEG 0.75-‐1.5 Hz oscillations in nonREM sleep were more prevalent in G/A than in G/G genotype. In conclusion, the data show that genetic variation in candidate genes can profoundly alter waking performance and sleep phenotypes in healthy humans. The affected signaling pathways provide insights into molecular mechanisms underlying sleep-‐wake regulation. Research was supported by the Zurich Centre for Integrative Human Physiology and the Swiss National Science Foundation grant # 310000-‐120377. Keywords: adenosine, homeostasis, cognition

123

S21.4 Patrice Bourgin. Institute for Cellular & Integrative Neurosciences, CNRS UPR 3212 and Sleep Clinic, University of Strasbourg, France. The circadian (C) and homeostatic (S) controls of sleep continuously interact resulting in the large diversity of sleep/wake patterns observed in humans. Light also affects sleep and waking, either indirectly through the phase adjustment of circadian rhythms, or directly through non-‐circadian mechanisms. These latter non-‐circadian effects of light were poorly understood and their significance and possible interactions with C and S remain to be clarified. In addition to rod and cones, a third component, melanopsin, is crucial for light detection. Melanopsin is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) that convey light information to the brain. We and others have characterized the sleep response to light in melanopsin-‐deficient or melanopsin-‐ipRGC-‐ablated mice providing significant advances towards the understanding of the direct effects of light on sleep. Furthermore, we have performed sleep deprivation experiments and analyzed the power spectrum of the EEG for further qualitative analysis. After a brief presentation of recent data on the interaction between C and S in mice, we will show a set of findings providing evidence that melanopsin-‐ipRGCs, yet also rod and cones, mediate the acute sleep-‐promoting effect of light. Analysis of the EEG reveals that the alerting effect (as evaluated by EEG correlates of alertness and exploratory behavior) of a 1-‐hour dark pulse also depends on melanopsin-‐based pathways. In addition, our analysis of an ultradian LD1h:1h cycle in mice lacking melanopsin revealed that light and dark non-‐circadian, direct effects greatly depend on circadian time. Analysis of EEG delta activity, a marker of sleep need, under various conditions including a 6h sleep deprivation, indicates that this lack of melanopsin also alters sleep homeostasis. Our results suggest that melanopsin-‐mediated direct effects of light, the circadian drive and sleep homeostasis, interact together to determine the timing and quality of sleep and waking. These findings in the murine model suggest that a revaluation is needed as to how the input of light or its absence shapes the inter-‐individual temporal organization of human behavior and performance via continuous modulatory effects on brain function and its interaction with the homeostatic and circadian timing systems.

124

125

ABSTRACTS

Posters

126

127

P001 WINTER LIGHTING CONDITIONS ACCELERATE RE-‐ENTRAINMENT TO SIMULATED TIME ZONE TRAVEL IN HAMSTERS Elizabeth M. Harrison, Michael R. Gorman. Department of Psychology and Center for Chronobiology, University of California, San Diego, United States of America. Dysrhythmia as seen in jet lag and other circadian disorders has negative effects on health and performance. Photoperiodic history has been shown to modulate phase-‐shifting responses and even more recently, sensitivity, to light. In short days that mimic winter conditions, the amplitude of the phase response curve to resetting by light is increased, and phase shifting via light pulses occurs more rapidly and requires less light relative to long summer days. In this experiment we used a simulated jet lag paradigm to examine whether or not this photoperiodically-‐dependent change in entrainment state facilitates adjustment to a wider range of phase shifts than previously explored. Photoperiodic history had a significant effect on the ability of the animals to entrain to a new photoperiod. More specifically, when measuring the absolute phase angle of entrainment averaged over the first 3 days after the light shift, animals entrained to a short day had effectively shifted their activity rhythms more than twice as far as the animals entrained to a long day photoperiod (hours from readjustment: SD mean = 2.66 ± 0.45; LD mean = 5.66 ± 0.40). This confirms and extends previous findings from our lab wherein photoperiod mediates plasticity in the circadian system, and future studies will be directed towards uncovering the mechanism for this enhanced entrainment. These results may inform decisions regarding treatment for individuals with dysrhythmia. Keywords: photoperiod, phase-‐shift, jet lag

P002 AFTEREFFECTS OF ENTRAINMENT IN THE FIELD PROVIDE NEW INSIGHTS INTO THE SWITCH FROM DIURNALITY TO NOCTURNALITY IN THE SUBTERRANEAN RODENT TUCO-‐TUCO (Ctenomys Cf. Knight) 1Barbara Mizumo Tomotani, 1Danilo Eugênio de França Laurindo Flôres, 1Patricia Tachinardi, 2José Demetrio Paliza, 1Gisele Akemi Oda, 2Verónica Sandra Valentinuzzi. 1Instituto de Biociências, Universidade de São Paulo, Brazil. 2Centro Regional de Investigaciones Científicas y Transferencia Tecnológica, La Rioja, Argentina. Ctenomys cf. knighti, a subterranean rodent from La Rioja, Argentina, is clearly nocturnal when kept in the laboratory under LD 12:12 (L: 200 lux) in cages with running-‐wheels. However, in a semi-‐natural enclosure (10x5 meters) continuous observation during the light

128

phase of the day of one individual during summer and another in winter surprisingly revealed intense aboveground activity throughout the day, outside their burrows. This indicates that C. cf. knighti could be another species that switches from diurnality to nocturnality when transferred from the field to the laboratory, as has been reported in some other wild diurnal rodent species (Smale et al., 2003). The next step was to verify whether the observed diurnal aboveground activity is a component of their total daily diurnal activity (above and underground) or, alternatively, tuco-‐tucos are not entrained in the field and thus the observed aboveground activity is just part of randomly timed activity bouts during the 24 hours of the day. In order to approach this question, and based on the fact that circadian oscillators often display aftereffects of entrainment, after the finalization of each observation experiment we immediately transferred the animals from the field enclosures to constant laboratory conditions. In this way, we could register the aftereffects of the previous field synchronization and access the phase of the synchronized oscillator in the field. The result was surprising: the activity phase of both animals coincided with the external night. In order to verify the generality of these results, we brought 11 more animals now directly from the field to the laboratory under constant conditions: five in running wheels and six with infrared motion sensors. Independently of the measuring device, aftereffects indicate nocturnality in the field. We conclude that the activity phase of tuco-‐tuco is nocturnal, as dictated by the LD entrained oscillator but its expression is diurnal in the field, due to masking by some environmental cycle other than the light-‐dark. Some hypothesis about the masking factors are temperature, oxygen content cycles in the underground environment or even predation risk in the surface. Support: CONICET, CNPq, FAPESP, CAPES. Keywords: diurnal, nocturnal, subterranean

P003 OVERWEIGHT REDUCES ENTRAIMENT EFICIENCY OF LOCOMOTOR ACTIVITY IN THE VOLCANO MOUSE NEOTOMODON ALSTONI Vania Patricia Carmona Alcocer, Manuel Miranda Anaya. Departamento de Biología Celular, Facultad de Ciencias, UNAM, México. Overweight (OW) and obesity becomes an increasing problem in industrialized countries. Recent studies have related this problem with changes in the circadian regulation. The volcano mouse Neotomodon alstoni, endemic of central Mexico, develop OW in most organisms in captivity, when fed regular rodent diet, and develop symptoms related with the metabolic syndrome, which makes this species interesting to study circadian behavior and obesity. The aim of the present work was to elucidate whether OW affects the entraining of locomotor activity in the volcano mouse, explored by a phase response curve (PRC, 200 lux, 1hr) and jet lag protocols. Control (49.57± 0.81 g g) and OW (75.81± 2.06g) male adult mice were used. Freely moving locomotor activity was recorded by means of

129

infrared light beams. Two experiments were set; first, mice were exposed to LD 12:12 photoperiod and 6 h advances and 6 h delays were set every 10 days. Correspondent results indicate that OW animals require larger number of transient days to fully entrain to either advances or delays. A second experiment consisted on animals entrained to LD (12:12), then maintained in DD during at least 10 days. A light pulse was given at different circadian times (CT) and phase shifts were estimated. Results indicated that in OW animals phase delayed zone is reduced compared with controls and the phase advance zones in OW is not evident. Preliminary observations in C-‐Fos expression at the suprachiasmatic nucleus indicate differences between normal and OW animals. Our results indicate that the photic entrainment in mice N alstoni is negatively affected by the overweight condition, and that this species could be of particular interest to study the effect of obesity upon circadian physiology. Supported by PAPIIT IN225311 Keywords: entrainment, locomor activity,

P004 WAVELENGTH DEPENDENCE FOR EFFECTS OF DIM SCOTOPHASE ILLUMINATION ON CIRCADIAN WAVEFORM AND REENTRAINMENT IN SIBERIAN HAMSTERS 1Michael R. Gorman, 2Jeffry A. Elliott. University of California, San Diego, USA. Compared to totally dark nights, dim scotophase illumination (DSI), promotes rapid expansion of activity time (Î±) in winter-‐like short days (10L:14D) and an increased incidence of rhythm splitting in 7L:5D, a light cycle offering two photo-‐ and two scoto-‐phases per 24 h (LDLD). In our working hypothesis, the DSI of D promotes increased speed and flexibility in circadian entrainment responses to the bright light of L through unspecified modification of the coupling interactions among the multiple cellular oscillators comprising the SCN pacemaker. In effect DSI somehow lubricates the circadian clock, strongly affecting the kinetics of re-‐entrainment and the achievement of altered circadian waveforms, whether seasonally appropriate (expanded subjective nights in 14D) or exotic (split subjective nights in LDLD). While there is expanding evidence that bright light effects on circadian phase-‐shifting and LD entrainment involve an interplay of photoreception by rods, cones and melanopsin containing retinal ganglion cells, no comparable information is available for the potent effects of DSI. Here we report the results of experiments undertaken to characterize the influences of DSI irradiance and wavelength on the above two entrainment responses using a specifically designed 28 night assay protocol. Previously reported dose response curves for Î± expansion under green or red DSI gave estimated EC50s of 2.388 x 109 and 1.313 x 1011 photons/cm2/s for 560 nm and 695 nm DSI, respectively, showing responsiveness to 560 nm DSI at irradiances several orders of magnitude lower than others have reported for phase-‐shifting. In continuation, this poster will update the above with on-‐going analysis of more recent data on shorter wavelength (< 560 nm) DSI and on responses

130

to DSI provided by broad spectrum white LEDs. [Supported by NSF-‐IBN-‐034639, NICHD-‐36460]. Keywords: action spectrum, coupling, plasticity

P005 SHORT-‐DAY RESPONSE IN DJUNGARIAN HAMSTERS DISPLAYING DIFFERENT CIRCADIAN PHENOTYPES Dietmar Weinert, Konrad Schöttner, Maren Schmidt. Martin-‐Luther-‐University Halle, Germany. In Djungarian hamsters (Phodopus sungorus) bred at our institute, a certain amount of animals show activity patterns that seem incompatible with proper entrainment of the endogenous circadian pacemaker to the environmental light:dark cycle. The activity offset in those animals is stably coupled to “lights on”. The activity onset, however, is increasingly delayed, leading to a compression of the activity time (alpha). When alpha falls below a critical value (~ 3h), the rhythm in those, so called DAO-‐hamsters, starts to free-‐run and finally breaks down. Hamsters show arrhythmic activity patterns only. Previous studies revealed that the mechanisms of photic entrainment are deteriorated. This mainly concerns the nonparametric effects of light. The phenomenon may also have consequences for the photoperiodic time measurement. Therefore, the impact of changes of the photoperiod was investigated. 6 adult males each of DAO, arrhythmic (AR) and wild type (WT) hamsters were kept under standard housing conditions (light:dark = 14:10h, long day [LD]) at the beginning of the experiment for two weeks. Thereafter, the light regime was changed to short day conditions (light:dark = 8:16h, [SD]) for 8 weeks. Beside the locomotor activity rhythm, body mass, testis size and fur coloration were measured to quantify the photoperiodic reaction. The results clearly indicate that only hamsters of the wild type were able to adapt to the changed photoperiod properly. The activity time decompressed, body mass and testis size decreased significantly, and fur coloration changed from summer to winter pelt. By contrast, DAO and AR hamsters displayed no reaction. In subsequent experiments, DAO and AR hamsters were kept under constant darkness for 8 and 13 weeks, respectively. Interestingly, after 8 weeks of constant darkness DAO hamsters showed a similar photoperiodic reaction as observed in wild type hamsters that have been kept for 8 weeks under SD conditions. However, since the DAO hamsters did not display the same level of adaptation compared to the WT, we repeated the experiment but kept animals in constant darkness for 13 weeks. At the end of the experiment, these hamsters were fully adapted to short day conditions. Arrhythmic animals by contrast displayed no photoperiodic reaction, either after 8 or 13 weeks in constant darkness. The results show that the physiological mechanisms necessary for seasonal adaptation are working in DAO hamsters and led us to the conclusion, that the light:dark cycle prevents rather than mediates the photoperiodic reaction. The findings of the study confirm our hypothesis, that

131

the phenomenon observed in DAO hamsters is the result of a disturbed interaction of the endogenous circadian clock with the environmental light:dark cycle. In AR hamsters, however, the entire disruption of circadian rhythmicity doesn’t enable photoperiodic time measurement. Keywords: Djungarian hamster, impaired photic entrainment, photoperiodic time measurement

P006 REORGANIZATION OF THE MAMMALIAN PACEMAKER UNDER ULTRA LONG DAY LENGTHS PROVIDES AN ASSAY FOR OSCILLATOR COUPLING 1Jennifer A. Evans, 2Tanya L. Leise, 1Oscar Castanon-‐Cervantes, 1Alec J. Davidson. 1Morehouse School of Medicine, United States. 2Amherst, United States. In mammals, the suprachiasmatic nucleus (SCN) acts as a central pacemaker to control daily rhythms in behavior and physiology. Circadian regulation is maintained at the cellular level via ~24h rhythms in clock gene and protein activity (e.g., per2/PER2). At the population level, neural oscillators within the SCN adopt specific phase relationships that are modulated by light, and oscillator interactions across this network are critical for pacemaker function. To investigate SCN network properties, we employed a photoperiodic manipulation that dissociates clock gene rhythms within SCN shell and core regions and exploited this reorganized state to investigate regional coupling mechanisms. In a preliminary study, PER2::LUC knockin mice were entrained to either a standard 12h light: 12h dark cycle (LD12:12) or a light:dark cycle with an ultra long day length (LD20:4). After three months under these respective photoperiods, clock gene expression within the SCN was monitored in vitro via PER2::LUC imaging. Consistent with our previous work, after exposure to LD12:12, SCN regions displayed 1-‐3h phase differences in peak PER2::LUC expression on the first cycle in vitro and these regional phase differences were relatively stable over subsequent cycles in vitro. After LD20:4, in contrast, shell and core SCN adopted an initial phase difference greater than 7h, which increased by more than 3h over subsequent cycles. To investigate the basis of these photoperiod-‐dependent dynamics, the magnitude and stability of SCN phase relations over time in vitro were assessed after exposure to one of five photoperiod conditions (LD12:12, LD16:8, LD18:6, LD20:4, LD22:2). Consistent with predictions based on previous research, the phase relation between shell and core reflects the entraining day length. Moreover, dynamic changes in the phase relation between SCN shell and core displayed over time in vitro depend on the initial phase relation between SCN shell and core in a systematic and non-‐linear manner. The pattern of results obtained thus far is consistent with the hypothesis that these dynamic changes in regional phase relations reflect the actions of oscillator coupling. By altering temporal coordination in the SCN, photoperiodic reorganization of the central pacemaker may provide a robust tool to investigate mechanisms underlying oscillator coupling.

132

Keywords: photoperiod, SCN reorganization, coupling

P007 LOCOMOTOR ACTIVITY AND SLEEP RHYTHMS IN THE MONGOLIAN GERBIL MERIONES UNGUICULATUS ARE DIFFERENTIALLY AFFECTED BY SHORT AND LONG PHOTOPERIOD 1Cinthia Rocío Juárez-‐Tapia, 1,2Pilar Duran, 1Manuel Miranda-‐Anaya. 1Facultad de Ciencias, UNAM., México. 2INB, UNAM, México. Changes in the photo-‐phase length in a 24-‐hour cycle, influences the circadian system in several organisms, including mammals. This effect may be reflected in different physiological functions and behavior such as locomotor activity, hormones release and the sleep-‐wake cycle. Mongolian gerbil Meriones unguiculatus, presents changes in the activity profile regarding photoperiod and have been considered as a semidiurnal or crepuscular mammal. It also has been established that seasonal photoperiod induce physiological changes in the reproductive system of this species. The aim of this study was to evaluate the effects of three different photoperiods (LD 12:12, long photoperiod 16:08-‐LP-‐ and short photoperiod 08:16-‐SP-‐) on the architecture of locomotor activity and sleep-‐wake cycle in adult male Mongolian gerbils. Our results indicate that the locomotor activity profile is organized according to the photoperiod tested. In LD 12:12, animals showed a bimodal pattern with main components associated to lights on and off respectively. When exposed to LP, gerbils increased their total activity and maintain their bimodal profile associated with light transitions. When exposed to SP, total activity gets reduced and the component related to lights on disappears. By other hand, results of temporal distribution of the vigilance states indicate that, in LD 12:12, gerbils spent about 65% of the recording time in wakefulness, mainly diurnal; and near 5% of REMs. When exposed either to SP or LP, wakefulness increases in both light and darkness, while sleep gets reduced. Also, during SP the main wake state becomes nocturnal. We proposed that the changes observed in both locomotor activity and sleep-‐wake patterns, could be analyzed considering the morning and evening oscillators theory, and become Mongolian gerbils as a good animal model for studying the effects of seasonal photoperiod changes in both circadian and homeostatic processes. Suported by CONACyT fellowship to CRJT. Keywords: gerbil, photoperiod, activity

133

P008 PHOTOPERIODIC WAVEFORM AND AMPLITUDE REGULATION OF MAMMALIAN PHASE RESPONSE CURVES Jeffrey A Elliott, Michael R Gorman. UCSD, USA. Mammalian circadian rhythms are synchronized to the day-‐night cycle via retinal light exposure mediating daily phase shifts that adjust phase and period to match the entraining light-‐dark (LD) cycle. Entrainment is mediated by a circadian rhythm in the resetting response to light that is described by a circadian phase response curve (PRC) traditionally measured by administering relatively brief bright light stimuli to rhythms otherwise free-‐running in continuous darkness (DD). The magnitude and direction of phase-‐resetting varies systematically with the timing of light relative to the free-‐running rhythm with phase delay shifts elicited earlier, and phase advances later in a subjective night (CT12 – CT24) that in rodents begins with the onset of wheel running activity at CT12. Recent studies from our laboratory have confirmed that LD entrainment history (photoperiod) influences the magnitude of phase shifts elicited at specific CTs in both the delay and advance regions of the PRC independent of pineal or gonadal secretions. Additionally, we have described a 40-‐fold increase in photosensitivity (dose response ED50) following entrainment to long (14 h) as compared to short nights (e.g. 10L:14D vs. 14L:10D; Glickman et. al. in preparation). These photoperiodic effects on the PRC entrainment mechanism may be based in seasonal modulation of retinal and/or SCN neuronal physiology, including interactions key to the synchrony, phase-‐relationships, and coupling among multiple cellular oscillators. Here we present studies in male Syrian hamsters testing the prediction that LD entrainment history regulates the circadian phase-‐shift response by broadly altering both the amplitude and the waveform (shape) of the PRC. Measured after entrainment to LD cycles with different photoperiods (T=24 h), different period lengths (T<>24 h), and after different durations of continuous darkness (1-‐20 days), the functional duration and amplitude of the PRC varies markedly and correlates closely with the waveform of the activity rhythm quantified in the duration of activity time (α). Specifically, the length of the responsive region of the PRC (D+A) and the area of the PRC curve (log (D+A)) are each linearly correlated with wheel-‐running α (r=0.91, p<.001 and r=0.78, p<0.01), but not with free-‐running period (). Thus, photoperiodic history strongly influences the shape and amplitude of the Syrian hamster PRC thereby contributing to seasonal modulation of entrainment. As recent studies have shown that human phase resetting is likewise modulated by photoperiod, further studies into the neural mechanisms of this PRC modulation will be important in the search for practical methods to ameliorate adverse effects of shift-‐work and jet-‐lag through enhancement of the phase resetting capabilities of the human circadian system. [Supported by NIH grants GM 14516 and NICHD 36460]

134

Keywords: waveform, amplitude, PRC

P009 EFFECT OF DIFFERENT PHOTOPERIODS ON THE RHYTHMIC INDUCTION OF HYPOXIA-‐INDUCIBLE FACTOR-‐1 AND HEAT SHOCK PATHWAY Rosa María Velázquez-‐Amado, Elsa G. Escamilla-‐Chimal, Ana Gabriela García-‐Tecpan, María Luisa Fanjul-‐Moles. Neurofisiología Comparada, Facultad de Ciencias, Universidad Nacional Autónoma de México, México.

Crayfish use different day lengths for phasing photoperiodic functions as molting and reproduction. This critical value seems to vary with the latitude of origin and depends on the abilty of the circadian system to entrain to the seasonally changing light-‐dark (LD) daily ratio. Procambarus clarkii, an invasive species introduced into Mexico from northern latitudes shows great tolerance to environmental stressors. Previous works our laboratory have shown that both irradiance and photoperiod length of the environmental daily cycles are stressors that strain Procambarus clarkii circadian rhythms, and as a consequence changes in this species metabolic, oxidative, and behavioral rhythms occur. Extreme luminous situations may drive this species to a state of behavioral and metabolic depression that allows it to cope with the adverse luminous environment. This adaptive response should involve signaling pathways able to translate the environmental changes into metabolic and behavioral response by means of genes expression mechanisms. The response to stress is controlled by specific transcription factors as hypoxia-‐induced factor (HIF-‐1) which interacts with chaperones such as heat shock proteins (HPS) stabilizing the stress response. Although in crayfish this response has not been studied the oxidation produced by extreme light cycles is coupled to cellular fluctuations in the production of free radicals and reactive oxygen species (ROS). ROS are regulatory mediators in signaling processes and antioxidant responses. Many of the ROS-‐mediated responses, as GSH system protect the cells against oxidative stress and reestablish redox homeostasis. Then it is plausible to propose HIF-‐1 as a transcription factor related to ROS, to heat shock pathway activity and participating in the daily adaptive response to extreme light cycles in crayfish. Here we investigated two structures proposed as circadian pacemakers of crayfish: the retina and the brain-‐optic lobe complex (BOL), determining whether equatorial (LD 12:12) and extreme (20:4) daily cycles, activate the expression of HIF-‐1, HSP 70 and 4-‐hydroxy-‐2-‐nonenal (HNE), a marker of lipid oxidation, and whether these markers expression levels depend on the LD cycle and the time of day. Adult crayfish P. clarkii acclimated to the laboratory in intermoult stage, were divided into three groups: 1) This group was subjected to light-‐dark cycles (LD) 12:12 (lights on at 07:00) for 15 days, 2) animals were subjected to the same conditions of photoperiod as group 1 and subsequently to 72 h of darkness (DD), 3) this group of crayfish underwent a cycle LD 20:4 (lights on at 0700, off 03:00). After each experimental condition the animals were killed at two times of day. Retinas and BOL complex were dissected and processed by western blotting using polyclonal anti-‐HIF-‐1 (C-‐

135

19), anti-‐HSP 70 and anti-‐HNE. The immunoreactivity was visualized by chemiluminescence These results revealed an effect of time on HIF-‐1 and HSP-‐70 relative abundance both in retina and BOL. In BOL both proteins increased at 08:00 decreasing at 20:00 in LD 20:4 and DD. Although we did not find any effect of time on lipid oxidation levels paradoxically retina decreased the levels of lipid oxidation in 20:04 with respect to DD conditions, suggesting an effect of ROS produced by light on the activation of antioxidant systems. This study suggests a rhythmic HIF-‐1-‐dependent increase in HSP expression that should be highly up-‐regulated during extreme photoperiodic conditions. Supported by PAPIIT IN218811. Keywords: Crayfish, Photoperiodic-‐response, Hypoxia-‐inducible Factor

P010 PERIOD 1 GENE EXPRESSION IN THE BRAIN OF A DUAL PHASING RODENT, THE OCTODON DEGUS 1Beatriz B Otalora, 2Megan H Hagenauer, 1Maria Angeles Rol, 1Juan Antonio Madrid, 2Theresa M Lee. 1Department of Physiology, Faculty of Biology, University of Murcia, Spain. 2Department of Psychology and Neuroscience Program, University of Michigan, United States. Chronotype in mammals is defined as diurnal or nocturnal depending upon whether animal activity occurs during the day or at night. While most species are either diurnal or nocturnal, under certain conditions, the Octodon degus can shift from day to night-‐time activity. Recent studies show that some nocturnal degus are entrained to the scotophase while others become nocturnal by negative masking. It is well established that in mammals circadian rhythms in behaviour and physiology are generated by daily oscillations in clock genes in the suprachiasmatic nucleus (SCN) of the hypothalamus. In the SCN the period 1 (Per1) gene is an integral component of the molecular clock. Its levels are high during the day and low at night. Daily rhythm in Per1 expression has been reported elsewhere in the brain, and recent studies show that although the time of peak expression of SCN Per1 levels does not differ greatly with chronotype, its expression in other brain regions is elevated at the time of activity and are therefore out of phase in diurnal and nocturnal species. This suggests that activity phase preference in mammals is located downstream from the SCN. Our objective was to study Per1 day-‐night levels in the SCN and brain areas outside the SCN in diurnal and nocturnal degus, in order to establish the relationship between brain clock gene activity and chronotype. Further, we compared for the first time Per1 expression between entrained and masked nocturnal degus. A total of 30 male degus were individually housed in cages with wheels. Their activity patterns were recorded for at least 15 days. They were classified as diurnal, entrained or masked nocturnal using criteria previously described. Brains were collected at two time-‐points, ZT4 and ZT16 (ZT0 corresponds to lights on). Per1 mRNA levels were analyzed by in situ hybridization. In the SCN, Per1 signal was higher at ZT4 than at ZT16 irrespective of chronotype. However, in areas outside the

136

SCN, such as the CA1 region of the hippocampus and the dentate gyrus, Per1 expression in entrained nocturnal degus was out of phase (higher values at ZT16) when compared to SCN values. Interestingly, in masked nocturnal animals this inversion in Per1 expression was not significant. These results support growing studies involving extra SCN brain areas in activity phase preferences, and suggest that there are fundamental differences between entrained and masked nocturnal degus. Supported by RD06/0013/0019, BFU2010-‐21945-‐C02-‐01 and 12005/PI/09. Keywords: Octodon degus, Per1, chronotype

P011 LIGTH/DARK CYCLE AND CLOCK GENES Mario Pedrazzoli. Universidade de São Paulo/EACH, Brazil. The light/dark cycle given by the rotation of earth around its own axis and around the sun has been shown to be the strongest zeitgeber that entrain circadian rhythms, inclusive to humans. Due to tilt in earth north/south axis in relation to the sun plan, the sunlight reaches earth differentially depending mainly on the latitude, resulting in an enormous variability of isolation levels and day length along the latitudinal cline. Practically all physiological processes are regulated by the signal coming from light/dark. The suprachiasmatic nuclei, in the hypothalamus, is the first step in temporal processing of light/dark signaling and this processed signal is widespread through a net of nuclei inside hypothalamus giving rise to circadian control of endocrine, autonomic and muscular systems and consequently behavior. Part of the signal processing is related to gene expression, a group of genes, called clock genes, are directly involved in the regulation and maintenance of circadian rhythms in the body. Variations in these genes, as polymorphisms and mutations, are associated with aberrant or differential expression of circadian behavior. The strong link between these clock genes and environmental conditions derived from the position of the earth in the solar system make them evident candidates to natural selection associated to light/dark cycle. The study of these genes and their interaction with environment results in a better comprehension of the human evolution related to the adaptation to annual variations of light/dark cycle through the latitudinal cline and its consequences to human health and social organization. Keywords: clock genes, sunlight, latitude

137

P012 EFFECT OF EXPERIMENTAL GLAUCOMA ON THE NON-‐IMAGE FORMING VISUAL SYSTEM 1Nuria M. A. de Zavalía, 2Santiago A Plano, 1Diego C Fernandez, 1María Florencia Lanzani, 1Ezequiel Salido, 1Nicolás A Belforte, 1María Inés Keller Sarmiento, 2Diego A Golombek, 1Ruth E Rosenstein. 1Department of Human Biochemistry, School of Medicine, University of Buenos Aires, Argentina, 2Department of Science And Technology, University of Quilmes, Argentina. Purpose: Glaucoma is a leading cause of blindness worldwide, characterized by specific visual field defects due to the loss of retinal ganglion cells and damage to the optic nerve head. Medications and surgery can help to slow the progression of some forms of the disease, but at present, there is no cure. Recent evidences indicate that a population of retinal ganglion cells is intrinsically photosensitive (through the expression of a specific photopigment, melanopsin), and transmits light information to the suprachiasmatic nuclei (SCN), where the principal pacemaker center for circadian rhythm is located, regulating several non-‐visual processes, like synchronization of the biological clock, photic suppression of pineal melatonin synthesis, and pupillary light responses. The aim of this work was to study the effect of glaucoma induced by weekly injections of chondroitin sulphate (CS) on the levels of rhodopsin and melanopsin, pupillary light reflex, photic suppression of pineal melatonin synthesis, expression of cfos in the suprachiasmatic nuclei (SCN) and locomotor activity rhythms. Methods: CS was injected in one eye and vehicle in the contralateral eye, once a week, during 10 weeks. In a group of animals, the injections of CS or vehicle were performed bilaterally. For the assessment of pupillary light reflex, animals were weekly injected in one eye with vehicle or CS, while the contralateral eye remained intact. The levels of rhodopsin and melanopsin were measured by western blot and immunohistochemistry, the melatonin content was determinated by RIA, after a light pulse of 20 min (white or blue light) and the expression of cfos induced by a light pulse of 10 min was analyzed by immunohistochemistry. Locomotor activity rhythms were determinated with infrared detectors of motion. Results: Melanopsin levels and pupil contraction was significantly lower in eyes injected with CS with respect to controls (p<0.01). White and blue light significantly decreased pineal melatonin content in vehicle-‐treated eyes, whereas in animals bilaterally injected with CS blue light was ineffective in reducing pineal melatonin content. Midnight pineal melatonin content was significantly higher in control than in glaucomatous animals. The immunohistochemical study indicated that light-‐induced expression of cfos in the SCN significantly decreased in CS-‐injected eyes. Glaucomatous animals exhibited a delayed phase angle with respect to lights off and a significant increase in the percentage of diurnal activity. Conclusions: These results indicate that chronic ocular hypertension induces a significant functional deficit of retinal ganglion cells and provoked significant alterations in the non-‐image-‐forming visual pathway. Keywords: glaucoma, melanopsin, rhythms

138

P013 PINEAL CHRONOBIOLOGY, SUNSPOTS AND GEOMAGNETIC STORMS 1José Luis Bardasano Rubio, 1José Luis Aldeanueva Aguirre, 2José Luis Arranz, 3Juan Álvarez-‐U de la Torre, 4Miguel Raposo Sánchez. 1Department of Medical Specialities. University of Alcalá, Spain, 2International Society for Proton Dynamics, Spain, 3Bioelectromagnetism European Foundation, Spain, 4Department of Physics. University of Alcalá, Spain. Based on the magnetoreceptor hypothesis of the pineal gland (pg) in mammals, we propose an helioterrestrial method of observation (using an refractor telescope and magnetograms) that links solar magnetic activiry (through the relative number of sunspots or “Wolf number”) to geomagnetism (“Zeitgeber” or additional external photoperiod synchronizer) for the general study (morphological, physiological, etc.)) of the pg chronobiological activity. We assess the relation between the numerical variation of some organelles of the pinealocytes (morphological markers of the pg rhythmic activity) and one of the most peculiar aspects of geomagnetism: the universal phenomenon of magnetic storms more frequently seen in the eleventh year acrophase cycle of sunspots. Using an electron microscope, we establish the number of myeloid bodies (MBs) associated with lipid droplets in the pg of 40 Wistar rats (20 males an 20 females) submitted to continuous darkness, during the calm days (10 sacrificed males and 10 sacrificed females) and during those with magnetic storms (6 sacrificed males and 14 sacrificed females) By using “Student t” and ANOVA we infer that during days of magnetic calm, no significante differences are observed in the number of MBs between the sexes. During the day of geomagnetic storms, the number of MBs for the females is 5±2.9 and for the males 2±1 with p<0.05 (Chi Square Test) for both sexes. The mathematical model suggest that the magnetic storms are involved in the drastic numerical decrease in the MBs, according to our experimental design. Keywords: pineal gland, sunspots, magnetic storms

139

P014 POSTEMBRYONIC MATURATION OF THE DIURNAL RHYTHM OF PINEAL MELATONIN BIOSYNTHESIS PATHWAY IS SEASON-‐DEPENDENT IN CHICKEN KEPT IN CONTROLLED LABORATORY CONDITIONS 1Aneta Piesiewicz, 1Urszula Kedzierska, 2Krystyna Zuzewicz, 1Krystyna Skwarlo-‐Sonta, 1Pawel Marek Majewski. 1Department of Animal Physiology, Faculty of Biology, University of Warsaw, Poland. 2Central Institute for Labour Protection, Warsaw, Poland. Previously, we have found that in chicken kept from the day of hatch in laboratory photoperiod L:D 12:12, activity of the pineal serotonin N-‐acetyltransferase (AANAT), a key enzyme in melatonin biosynthetic pathway exhibited age-‐ and season of hatch (winter vs summer)-‐ dependent diurnal changes. Namely, in chicks hatched in winter AANAT activity was lowest in the youngest birds (2-‐day-‐old) and increased with age (up to 16-‐day-‐old), while in summer magnitude of its nocturnal peak was age-‐independent and even in 2-‐day-‐old was similar to that found in adult birds reared under natural photoperiod. This suggested the existence of a photoperiodic memory in the pineal gland of newly-‐hatched chicken. The aim of present study was, therefore, to check whether in the same experimental conditions this photoperiodic memory operates on the transcriptional level of enzymes involved in the melatonin biosynthesis. Pineal glands from 2-‐, 9-‐ and 16-‐ day-‐old birds were isolated under dim red light every 2 hs over a 24 hs period, immediately frozen and subsequently used for analysis of the level of mRNA (real-‐time PCR) of genes encoding all enzymes involved in the melatonin biosynthetic pathway: tryptophan hydroxylase 1 (Tph1), aromatic L-‐amino acid decarboxylase (Ddc), Aanat and Hydroxyindole-‐O-‐methyl transferase (Asmt). It has been found that in 2-‐day-‐old birds the influence of season was the best visible and consisted in the lack of the diurnal rhythm in winter (Tph1 mRNA) or in summer (Asmt mRNA), low amplitude in the rhythmical changes in Aanat transcript and highest amplitude in those of Ddc mRNA. Above mentioned season-‐related differences were less expressed in 9-‐day-‐old chickens however still present in the pineal gland of winter chickens while in the oldest birds examined the effect of season on the diurnal changes in mRNA of all 4 enzymes was almost invisible. These results clearly indicate that the diurnal rhythm of melatonin biosynthetic machinery in the chicken pineal gland evaluated at the transcriptional level undergoes the season-‐related postembryonic modifications. Genes encoding particular enzymes are transcribed rhythmically already in 2-‐day-‐old chickens in both seasons but the amplitude of rhythm reaches the value comparable with that in adult birds much slower in winter birds, suggesting a kind of maturation of the rhythm, the best seen in the case of Aanat and Asmt mRNA. Supported by the Polish MSHE grants N N303 3177 33 and NN 303 5957 39. Keywords: seasonality, chicken pineal gland, melatonin biosynthesis

140

P015 THE DAILY MELATONIN PATTERN IN DJUNGARIAN HAMSTERS IS DEPENDING ON THE CIRCADIAN PHENOTYPE 1Dietmar Weinert, 1Konrad Schöttner, 2Valérie Simonneaux, 2Patrick Vuillez, 3Stephan Steinlechner, 2Paul Pévet. 1Martin-‐Luther-‐University, Halle, Germany. 2University of Strasbourg, France. 3University of Veterinary Medicine, Hannover, Germany, Djungarian hamsters (Phodopus sungorus) bred at the institute in Halle reveal three different circadian phenotypes. One of them shows the classical activity pattern with respect to the light:dark cycle and is therefore designated as wild type (WT). In the DAO phenotype, activity onset is increasingly delayed from the beginning of the dark phase whereas the activity offset is stably coupled to “lights on”, and this leads to a compression of the activity time (á). When á falls below a critical value, the rhythm starts to free-‐run and finally breaks down; animals show then an arrhythmic activity pattern and were identified as the AR-‐phenotype. Previous studies showed that a disturbed interaction of the circadian system with the light:dark cycle contributes to the phenomenon observed in DAO hamsters. To get a better insight into the underlying mechanisms, we investigated the daily melatonin rhythm as a second, independent marker of the circadian clock (SCN). Hamsters of all three phenotypes were kept individually under a light:dark cycle of 14:10h (light on 04:00h CET). DAO hamsters with a 5h delay of the activity onset were used for both experiments. Pineal melatonin content was determined at 3 times points (4h after “light off” [D+4], 1h before “light on” [L-‐1], 1h after “light on” [L+1]) using a melatonin RIA. To compile a 24h profile of urinary melatonin (6-‐sulfatoxymelatonin, aMT6s), hamsters were transferred to metabolic cages for 27h. Urine was collected every 3h and the amount of aMT6s was measured using an ELISA kit. WT hamsters showed high pineal melatonin content during the dark time (D+4, L-‐1) which significantly decreased at the beginning of the light period (L+1). DAO hamsters on the contrary displayed low melatonin levels during the part of the dark period when animals were still resting (D+4). At the end of the dark period (L-‐1), melatonin content increased significantly and dropped again when light was switched on (L+1). AR hamsters showed low, comparable to daytime values melatonin levels at all 3 time points. The results were confirmed by the aMT6s data. WT hamsters showed a marked circadian pattern of aMT6s excretion. The concentration started to increase 3h after “light off” and reached daytime values again 5h after “light on”. In DAO hamsters by contrast, aMT6s excretion started about 6h later and reached significantly lower levels compared to WT hamsters. In AR animals, aMT6s excretion was low at all times. The results clearly indicate that the rhythm of melatonin secretion in DAO hamsters is delayed according to the delayed activity onset, whereas AR hamsters displayed no melatonin rhythm at all. Since the regulatory pathways for the rhythm of locomotor activity and melatonin synthesis downstream from the SCN are different but obviously convey the same signal, we conclude that the origin of

141

the phenomenon observed in DAO hamsters must be located upstream of the SCN or in the SCN itself. Keywords: Djungarian hamster, daily melatonin rhythm, disturbed photic entrainment

P016 ENVIRONMENTAL FACTORS AFFECTING THE ROOSTING BEHAVIOR OF INDIAN CLIFF SWALLOW HIRUNDO FLUVICOLA Arati Singh, Shrutika Kankariya, Arti Parganiha, A. K. Pati. School of Life Sciences, Pt. Ravishankar Shukla University, Raipur 492010, India. We investigated the influence of environmental factors on roosting behavior of Indian cliff swallow, Hirundo fluvicola in different seasons. We monitored the exit time of the first bird in the morning and entry time of the last bird in the evening for eight consecutive days every month over a period of 15 months. The sunrise & sunset times, temperature, humidity, and light intensity of the study site were recorded simultaneously. Morning and evening phase angles for exit and entry times were computed with reference to sunrise & sunset times, temperature, humidity, and light intensity. Results depict that exit time was significantly positively correlated with sunrise time, humidity, and light intensity, whereas negatively correlated with the morning temperature. The entry time exhibited significant positive association with sunset time and evening temperature, and negative relationship with humidity. The phase angle for exit time showed negative relationship with the evening light intensity only. However, phase angle computed for entry time exhibited significant positive correlation with humidity and light intensity, and negative correlation with evening temperature. The birds exited from their roosts before sunrise and entered after sunset time, irrespective of season. The factor ‘season’ produces significant effect on exit and entry times of the bird. Hirundo fluvicola, exited later and entered earlier during winter as compared to summer and rainy season. We can conclude that exit and entry timings that represent roosting behavior of Hirundo fluvicola are modulated by environmental factors. Keywords: Exit time, Entry time, Phase angle difference

142

P017 ROOSTING BEHAVIOR OF INDIAN HOUSE SWIFT, APUS AFFINIS WITH REFERENCE TO ENVIRONMENTAL FACTORS Shrutika Kankariya, Arati Singh, A. K. Pati, Arti Parganiha. School of Life Science, Pt. Ravishankar Shukla University, Raipur, India. We discovered a roosting site of a colony of Indin house swift, Apus affinis on the busiest commercial area of Raipur city and studied roosting behavior of the birds for eight consecutive days, every month over a period of one and half year. The exit time of the first bird in the morning and entry time of the last bird in the evening and corresponding phase angles were recorded with reference to sunrise and sunset, respectively. The ambient temperature, humidity, light intensity of the study site was recorded simultaneously. The exit time and entry time showed positive relationship with the time of sunrise and sunset, respectively. The bird exited from their roosts before sunrise and entered to their roosts after the sunset time, irrespective of the season. The factor ‘season’ produced statistically significant effect on exit and entry time of the bird. Apus affinis, exited later and entered earlier during winter as compared to summer rainy season. The corresponding evening phase angle differences are insignificant with season. The exit time showed negative, but the entry time showed positive relationship with temperature. The corresponding phase angle differences are insignificant with the temperature. The exit time showed positive, but the entry time showed negative relationship with humidity of the study site. The corresponding phase angle differences are negatively significant with humidity. The exit time, entry time and corresponding phase angle differences are positive correlated with light intensity of the study site. On the basis of the findings we conclude that roosting behavior of Apus affinis is modulated by the environmental factors. Keywords: Roosting behavior, Seasonal variations, Phase angle difference

P018 THE CIRCADIAN BODY TEMPERATURE RHYTHMS OF DJUNGARIAN HAMSTERS REVEAL DIFFERENT CIRCADIAN PHENOTYPES 1Dietmar Weinert, 1Konrad Schöttner, 2Jim Waterhouse, 1Martin-‐Luther-‐University Halle, Germany. 2Liverpool John Moores University, UK. Djungarian hamsters (Phodopus sungorus) of our breeding stock show three different rhythmic phenotypes. In wild type (WT) animals, activity onset starts shortly after “lights-‐off” and the hamsters are active until the lights are switched on. In delayed activity onset (DAO) hamsters by contrast, the activity onset is reliably delayed after “lights-‐off”, but the

143

activity offset is still coupled to “lights-‐on”, leading to compression of the activity time (á). On falling below a critical á, animals start to free-‐run and the rhythm finally breaks down; hamsters then become arrhythmic (AR, the third phenotype) and are episodically active throughout the 24-‐h day. The delayed activity onset in DAO hamsters indicates an attenuated ability to synchronize with the periodic environment and may result from an aberration in the endogenous circadian system. To better understand the underlying mechanism, the main aim of the study was to investigate whether the observed phenotypic differences are caused by an altered output from the suprachiasmatic nuclei (SCN). As a marker of the circadian clock, the body temperature rhythm, purified from masking effects due to motor activity, was used. Hamsters were kept individually under standardized laboratory conditions (L:D = 14:10h; T = 22°C ± 2°C; food and water provided ad libitum). Body temperature and motor activity were monitored by means of implanted G2-‐E-‐Mitters and the VitalView® System (MiniMitter). Each phenotype showed distinctive rhythms of overt activity and body temperature, these two rhythms being very similar to one another and characteristic of each phenotype. Correcting body temperatures for the effects of activity produced purified temperature rhythms which retained profiles that were distinctive for each phenotype. These results show that the body temperature rhythm is not simply a consequence of the activity pattern but is caused by the endogenous circadian system. The purification method also allowed estimation of the thermoregulatory efficiency using the gradients as a measure of the sensitivity of body temperature to activity changes. In WT and DAO hamsters, the gradients were low during activity period and showed two peaks. The first peak occurred after “lights-‐on”, and the second preceded activity onset. In AR hamsters, the gradients did not reveal circadian changes. The results provide good evidence that the different phenotypes result from differences in the circadian clock rather than from the different patterns of activity. In AR hamsters, the SCN do not produce an obvious circadian signal. Since both activity and body temperature rhythms show the same phenomena independently and also have different regulatory pathways downstream from the SCN, we assume that the circadian signals coding for the specific phenotypes arise as direct outputs of the SCN. With regard to DAO hamsters, it remains to be investigated whether the clockwork itself or the afferent entraining pathways are abnormal compared to WT hamsters. Keywords: Djungarian hamster, circadian system, unmasked body temperature

144

P019 MOTOR ACTIVITY OF MANATEE (Trichechus manatus manatus) IN SEMI-‐CAPTIVITY CONDITIONS: PRELIMINARY RESULTS 1Victoria Eugenia Holguín, 1Victor Manuel Alcaraz, 2Jairo Ignacio Muñoz. 1Universidad Veracruzana, México, 2Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, México. Motor activity is being registered of 7 manatees in exterior captivity at the Aquarium of Veracruz, Mexico, with AW4 actiwatch actimeters for a year. The actimeters are programmed to collect data continiously, with a recording interval of 5 minutes, 24 hours a day during 12 months. Activity data has been collected from all individuals, obtaining Actograms and Periodogram through the day, along with behavioral data records during the photoperiod for posterior correlation analyses. This observation records are divided in morning and evening periods. Based on data from the U.S. Navy website (http://aa.usno.navy.mil/data/docs/RS_OneDay.php) with the location coordinates of Veracruz Aquarium (19º 11’14.8’’ N, 96º 07’ 19.8’’ W), average photoperiod data, sunrise and sunset (hr), as well as average of morning and evening twilight start (hr) was obtained. As preliminary data, the average intensity record of each individual was obtained; plus the feeding schedules of observation days for each individual were recorded. Our preliminary results show 4 important activity peaks, possibly related to the feeding schedules, suggesting an anticipatory behavior at feeding hours. However, more records are needed to corroborate this finding. This study is contributing in the chrono-‐ecological aspects that could be relevant for the species adaptation to diverse temporal cycles, which are determined by geophysics phenomena and are very difficult to study under natural conditions. Actimetry can be a robust technique to obtain long term records of activity-‐rest rhythm, allowing the investigation of relationships of environmental factors that could affect the manatee activity cycle. Keywords: activity-‐rest rhythm, manatee, photoperiod

P020 YAWNING AND PENILE ERECTION OSCILLATES ALONG THE CIRCADIAN CYCLE IN THE HY SUBLINE 1,2José R. Eguíbar, 1Ma. Del Carmen Cortés, 1Araceli Ugarte. 1Instituto de Fisiología, B. Univ. Autónoma de Puebla, México. 2Secretaría General, BUAP, México.

145

At the Institute of Physiology we have two sublines of Sprague-‐Dawley rats which differ in their spontaneous yawning behavior. The high-‐yawning (HY) have around 20 yawns/h and the low-‐yawning (LY) less than 3 yawns/h. In HY rats dopaminergic-‐induced penile erection is tightly associated with the expression of yawning. The high incidence of this behavior allows us to analyze the circadian distribution of yawning and penile erection. The rats were maintained under standard conditions with free access to rodent pellets and tap water. The observations were made in Plexiglas cages (32x47x20 cm) continuously 48 h in a sonoamortiguated room by two trained researchers. Yawning and penile erection had an acrophase before the lights off; importantly this circadian oscillation of penile erection is not associated with any sexual stimulus suggesting an endogenous mechanism. In the case of yawning, constant light conditions disorganized circadian rhythm, so it is not an endogenous rhythm. Feeding restriction paradigm to just 2 to 3 h/day produced an anticipatory peak of yawning, suggesting that the expectancy which is associated with an increase in the glucocorticoid levels in the bloodstream, particularly corticosterone could be responsible of this increase. In conclusion, yawning is associated with the expectancy produce by the change of light conditions and/or food disposition. In fact, adrenalectomy prevents the dopaminergic-‐ and/or cholinergic-‐ induced yawning suggesting that glucocorticoids had a facilitatory role in this innate motor pattern. This research was supported by Dr. E. Agüera-‐Ibañez Rector-‐BUAP, VIEP-‐BUAP SAL/G/2010, CONACyT 106694 grants to JRE. Keywords: CIRCADIAN, YAWNING, STRESS

P021 CRONOECOLOGY OF REST-‐ACTIVITY RHYTHM AND THE BEHAVIORS OF THE SPIDER MONKEY Ateles geoffroyi IN SEMI-‐LIBERTY: A ELECTRIFIED ENCLOSURE 1José Carlos Sánchez-‐Ferrer, 2Domingo Canales-‐Espinoza, 3Ana María Santillán-‐Doherty, 3Jairo Muñoz-‐Delgado. 1Estudiante de Maestría en Nueroetología, Instituto de Neuroetología, Universidad Veracruzana, México. 2Dirección del Área Académica Biológico Agropecuarias, Universidad Veracruzana, México. 3Laboratorio de Cronoecología y Etología Humana, Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz”, México. All animals, including primates, show regular variations in their behavior and physiology. The temporary system that synchronizes the internal processes of the organism with the daily events of the environment is the circadian rhythm, while the environmental factors that impact these rhythms are called Zeitgeber. Such is the effect of stronger environmental Zeitgeber, the natural light-‐dark cycle. But there are other physical and/or social variables that reduce or increase the rest-‐activity rhythms; this is the case of geophysical phenomena. The present study analyzed the relationship between astronomical and

146

meteorological parameters and its impact on rest-‐activity rhythm and behavior of the spider monkey (A. geoffroyi) in semi-‐liberty in an electrified enclosure. We studied the behavior of four individuals, two adult females, one adult male and a juvenile male, located in an electrified enclosure in a natural environment. Motor activity was recorded in three of these individuals through actimetry. Recordings were analyzed using the program ACTIWARE. The data were averaged for every 32 days as monthly factor, 64 days for dry-‐rain seasonal information, and 128 days for the total recording information. The information of the astronomical parameters, such as the rising and setting sun, solar day length, and duration of astronomical twilight, was obtained from the U.S. Naval Observatory. The meteorological parameters considered: environmental temperature, sky and time condition, were obtained by the information provided by the Comisión Federal de Electricidad de México. The behavior was recorded with direct observations during two daily sessions (morning and afternoon) in four periods throughout the year. We calculated the average frequency and duration of the behaviors of the group to determine the differences between the peaks of activity. The results showed no effects of the astronomical variables on the parameters of rest-‐activity rhythm of individuals. However it did show an effect on the pattern of the activity variables. The temperature and precipitation showed strong masking effects on the rest-‐activity rhythm. We observed a clear biphasic pattern of activity, with a peak in the morning and a wider peak in the afternoon, similar in duration and amount of activity. We conclude that the morning peak is regulated by human interaction (food provisioning), in which we observed a longer duration of the feeding behavior, and also due to the environmental temperature. The evening peak is regulated by precipitation associated with social behavior and foraging. Seasonal differences (dry-‐rain) indicated increased activity during the dry season, in contrast to that reported for the species in external captivity, which indicates an increase in activity during the rainy season. It is confirmed that A. geoffroyi is a diurnal species with an adaptive capacity which allows it to respond to changes in their environment, observed in the degree of plasticity of their rest-‐activity rhythm. Keywords: rest-‐activity rhythms, Behavior, Ateles geoffroyi

P022 CIRCADIAN VARIATION IN SHORT -‐TIME ESTIMATION AND THYROID HORMONES LEVELS IN YOUNG HUMANS DURING 30 H CONSTANT ROUTINE 1Babita Pande, 2Atanu Kumar Pati, 1Arti Parganiha, 2Pradeep Kumar Patra. 1School of Life Sciences, Pt. Ravishankar Shukla University, Raipur 492010, India. 2Department of Biochemistry, Center for Genetic Diseases & Molecular Biology, India. We evaluated circadian variations in 60 s interval estimation and thyroid hormones (triiodothyronine; T3, thyroxine; T4 and thyroid stimulating hormone; TSH) levels in eight

147

apparently healthy young subjects (male and female, four each; median age, 23.5 y) during 30 h constant routine. All subjects were free from any neurological disorders. They were instructed to maintain their regular sleep-‐wake cycle, five days prior to beginning of the study. They were also administered a brief demo before the experiment. During the study subjects stayed awake, in semi recumbent position with minimum physical/ locomotor activity, continuously for 30 h in a hall under partial social isolation. Room temperature (32.3 °C +/-‐1.59) and light intensity (30 lux at subject’s eye level) were nearly constant. Subjects were served with light food (around 250-‐300 kcal) at 2-‐hourly intervals on completion of each session till termination of the study. Each subject prospectively produced 60 s at 2-‐hourly intervals using digital stopwatch. In addition, oral temperature (OT) was measured and blood (5 ml) was collected from each subject simultaneously. From serum samples the levels of T4 (£gg/dl), T3 (ng/ml) and TSH (IU/ml) and cortisol (£gg/dl) were measured. The results substantiated a statistically significant circadian variation in 60 s production in two males and one female only, and in total males in group. Prominent circadian rhythms also detected in OT and cortisol level in total subjects, females and males at group level. Furthermore, statistically significant circadian rhythms documented in serum T3 and TSH concentration in total subjects as well as in males and females at group level. The peak level of T3 and TSH level in serum was found to be during early morning hours. It was observed that the 60 s production was significantly positively associated to T4 level. Both T3 and TSH were significantly negatively correlated to OT. We conclude that interval timer could interact with the circadian clock for 60 s interval estimation. Circadian rhythm in TSH has enriched the already existing reports additively. In humans, variation in serum T3 level seems to be under circadian clock control. It could also be suggested that T4 have effect on cognitive attributes with reference to short-‐time estimation. Keywords: Circadian rhythm, constant routine, time estimation

P023 THE CIRCADIAN SYSTEM INLFUENCES SHORT-‐TIME PERCEPTION Patricia V. Agostino, Ivana L. Bussi, Micaela do Nascimento, Diego A. Golombek. Laboratorio de Cronobiología, Universidad Nacional de Quilmes, Buenos Aires, ARGENTINA Temporal perception is fundamental to environmental adaptation in humans and other animals. To deal with timing, organisms have developed multiple systems that are active over a wide range of magnitude, the most important being circadian timing, interval timing and millisecond timing [1]. Time estimation in the second-‐to-‐minutes range – known as interval timing – involves the interaction of the basal ganglia and the prefrontal cortex. We have previously reported that short-‐time perception in mice is not independent of the influence of the circadian pacemaker [2]. In this work we tested the hypothesis that dopamine signaling is involved in the interaction between circadian and interval timing.

148

Animals were trained following the peak-‐interval (PI) procedure [3]. Results show significant differences in the estimation of 24-‐second intervals at different times of day, being more accurate the group trained at night. Interval timing was also studied in animals under constant light (LL) conditions, which abolish circadian activity and temperature rhythms. Mice under LL conditions were unable to acquire temporal control in the peak interval procedure. However, daily injections of L-‐DOPA before the experiment improved timing performance in LL mice, suggesting that an increase of dopamine is necessary for the interval to be timed. We are currently studying the circadian regulation of dopamine levels in the striatum, as well as the expression of clock genes – such as Per2 – in pre-‐frontal cortex and basal ganglia. We also aim to develop theoretical models which tell about the mechanisms involved in interval timing. References: [1] Buhusi C.V. and Meck W.H. (2005). Nature Reviews 6:755-‐765. [2] Agostino et al. (2010). Brain Res. 1370:154-‐63. [3] Cheng R.K. and Meck W.H. (2007). Brain Res. 1186:242-‐254. Keywords: circadian rhythms, interval timing, cortico-‐striatal circuits

P024 DIURNAL VARIATIONS IN THE LEARNING OF A MOTOR SKILL 1Alejandra Galeana Garcia, 1Georgina Almeida Rosas, 1Maribel Delgado Herrera, 2Raúl Aguilar Roblero, 1Alejandra Ruiz Contreras. 1Laboratorio de Neurogenomica Cognitiva, depto. Psicofisiología, Fac. Psicología, UNAM, México, D.F. 2División de Neurociencias Cognitivas, Instituto De Fisiología Celular, UNAM, México, D.F. In our daily activities, we use a variety of motor skills that are learned and perfected through repetition. These skills are perfected with practice and their acquisition is determined by the reduction in the time of execution and the number of errors made. The acquisition of motor skills is a gradual process that consists of two stages: a rapid learning that occurs during initial trials; followed by a small learning process that takes place during subsequent attempts. There is evidence that cognitive (e.g., in attention, short-‐term memory) and visuomotor performance (e.g., serial reaction time tasks) is different as a function of circadian rhythms. These changes in efficiency may be associated with changes that occur in the nervous system throughout the day (eg., changes in the expression of ligands, mRNA and receptors). It is possible that these changes also trigger a difference in the efficiency of learning a motor skill. The purpose of this study was to detect whether training in a motor skill is more efficient when acquire in the morning (8 hrs) than at night (20 hrs). To this end, two groups of young volunteers (n=14 per group, 50% male) underwent training, one at 8 hrs and the other at 20 hrs. Participants had to trace a third inner star in a clockwise direction inside a five-‐pointed, double-‐margin star (5 mm between borders) on a letter-‐size sheet of paper. They could not look at the drawing directly, only its

149

reflection in a mirror. A total of 15 trials were conducted in one session. In each trial, the number of errors, execution time, index of errors per minute, and the mean time interval elapsed between the errors in each session were measured. Mixed Analyses of Variance, using the between groups factor: Group (training at 8 hrs vs. 20 hrs) and the within groups factor: Trial Number (15), were conducted. The group trained at 8 hrs showed a reduction in the mean number of errors, execution time, and the time interval elapsed between each error, in comparison to the group trained at 20 hrs. Also, for all of the dependent variables, an interaction between the factors Group and Trial Number was observed: the rapid-‐learning phase occurred in the first four trials which differed from the following ones; and the two groups differed in the rapid-‐learning phase but not in the slow-‐learning phase. These results indicate that the efficiency in learning motor skills varies during the day. One probable explanation is that the circadian rhythms of physiological variables (e.g., levels of melatonin or cortisol, expression of receptors and their ligands) in the brain may influence directly the learning of motor tasks. Keywords: DIURNAL VARIATIONS, MOTOR SKILL, CIRCADIAN RHYTHMS

P025 CIRCADIAN VARIATIONS IN COGNITIVE INHIBITION AND FLEXIBILITY Benito Martínez, Aída García, Javier Talamantes, Candelaria Ramírez, Pablo Valdez. Universidad Autónoma de Nuevo León, México

Errors and accidents at work increase during the night; this could be due to circadian variations in inhibition and flexibility, two components of executive functions. Inhibition is the capacity to restrain inappropriate responses, and flexibility is the capacity to change responses according to environmental demands. The objective of this study was to identify circadian variations in inhibition and flexibility, using a shifting criteria task. Participants were 8 undergraduate students, age: 17.75 ± 0.46 years (17-‐18 years), 1 male and 7 female. They were kept in a constant routine protocol for 28 h. Rectal temperature was recorded each minute and responses to a shifting criteria task were recorded every 100 min. In this task, numbers in color blue or red were displayed on a computer screen. Participants had to press keys according to the following conditions. First, they had to press a key that matched the number displayed. Second, they had to press the non-‐matching key to the number shown; these responses were taken as indices of inhibition. Third, they had to shift the criteria (match, non-‐match) every 4 to 6 numbers, depending on the color of the number; these responses were taken as indices of flexibility. Rectal temperature showed circadian rhythms (mean acrophase = 15:45 ± 1:23; cosinor fit %R = 80.25 ± 11.83, p < 0.001). During the night and early morning, there was a decrease of efficiency in inhibition (Friedman = 42.03, p < 0.001), an increase of latency in inhibition (Friedman = 44.61, p < 0.001) and a decrease of efficiency in flexibility (Friedman = 66.07, p < 0.001). In conclusion,

150

there were circadian variations in inhibition and flexibility. The decrease in these functions during night and early morning could be a risk factor for night shift workers. Keywords: circadian rhythms, executive functions, shifting criteria task

P026 CIRCADIAN VARIATIONS IN ALERTNESS Javier Talamantes López, Aida Garcia Garcia, Candelaria Ramírez Tule, Pablo Valdez Ramírez. Universidad Autónoma De Nuevo León, México

Alertness is a basic cognitive process that enables the person to produce general responses. Alertness supports other cognitive processes such as attention and memory. The objective of this study was to analyze circadian variations in alertness. 10 females and 2 male students, age: 18.31 years (± 2.39 SD), participated in this study. They were recorded in a constant routine protocol during 30 h. Rectal temperature was recorded each minute, and performance was assessed every 100 minutes using a continuous performance task (CPT) and a concurrent alarm task, with a duration of 12 min. CPT required using the right hand to press key 1 to any number (except “9”) appearing at the screen, key 2 to a “9”, and key 3 to a “4” after the “9”. The concurrent alarm task required to hold a key pressed with the left hand; if the key was released, an alarm sound indicated the participant to press the key again. Alertness was measured as sequences of omissions and sequences of commission errors in the CPT and frequency of alarms and latency to cancel the alarms in the alarm task. Rectal temperature and performance in the CPT showed circadian variations. There were circadian variations in frequency of alarms, latency to cancel the alarms and sequences of omissions. There were higher frequency of alarms (Friedman = 133.51, p< 0.001), longer latency to cancel the alarms (Friedman = 122.95, p< 0.001) and higher sequences of omissions (Friedman = 145.71, p< 0.001) during the night and early in the morning. Sequences of commission errors did not showed circadian variations (Friedman = 22.44, p = NS). In conclusion, there were circadian variations in frequency of alarms, in the latency to cancel the alarms as well as in the sequence of omissions, but not in the sequences of commission errors. These findings contribute to explain the time of day variations on daily activities such as driving a car as well as on the efficiency on many working activities. Keywords: Chronobiology, Human Performance, Alertness

151

P027 THE VALUE OF MONITORING AMBULATORY BLOOD PRESSURE IN OLD PEOPLE 1Zi Yan Zhao, 1Zhi Hui Yan, 2Yue Rong Fu. 1Anti-‐Senility Research of Shandong, Institute of Materia Medica, Shandong Academy of Medical Sciences, P.R China. 2Affiliated Hospital of Shandong Academy of Medical Sciences, P.R. China. We have monitored the ambulatory blood pressure (ABP) since 1999, mostly in the old Chinese population. Overview our results, we have seen the important value of monitoring ABP for both diagnosis and proper treatment of hypertension in old people. Our data showed that in old Chinese population, there were higher prevalence for both white coat hypertension and masked hypertension; in longevity population (i.e., centenarians), the health status and activities of daily living scores in were associated significantly with blood pressure, the higher BP, the better quality of life; the trace of ABP monitoring more than ten years in an old women (born in March, 1918, from normotensive to white coat hypertensive, age related hypertensive, then after anti-‐hypertensive treatment return normotensive and withdrawl of medication for more than 3 years, and now hypertensive after fracture of 8 ribs with anti-‐hypertensive treatment) showed that age-‐related hypertension need not take anti-‐hypertensive medication for lifelong. Keywords: ambulatory BP monitoring, old, hypertension

P028 ASSOCIATION OF CIRCADIAN ACTIVITIES, SLEEP, AND QUALITY OF LIFE IN CHINESE CENTENARIANS 1Zi Yan Zhao, 1Zhi Hui Yan, 1Yue Rong Fu, 2Feng Zhang. 1Antisenility Research Center of Shandong, Institute of Materia Medica, Shandong Academy of Medical Sciences, P. R. China, 2Affiliated Hospital of Shandong Academy of Medical Sciences, P. R. China. We have surveyed 109 centenarians (13 men and 96 women, aged 101.9 ±2.0 years old) lived in Jinan, China by home visits. Health survey was done by questionnaires including the Athens Insomnia Scale (AIS), the Activity of Daily Living Scale (ADLS), and measurements of blood pressure (BP), pulse rate (by digital BP monitors UA-‐787, A&D, Japan), and arterial oxygen saturation (SpO2, by JERRY-‐1, China on the third finger). Survey data were analyzed by SPSS-‐Windows. Results The centenarians were divided into three groups, A. 30 (30/109, 27.5%) were bedridden, they were fragile, lost circadian activities and suffered from severe chronic diseases, such as stroke, fracture, psychiatric disorders, or acute infections at visits,

152

their life quality was very poor with significant higher ADL scores (51.5¡À5.8) than other two groups (P<0.01); B. 15 (15/109, 13.8%) were suffered from insomnia to different degrees (AIS scores were 6.9¡À3.4) but lived with circadian activities, their ADL scores (39.0¡À12.5) were significant lower than group A, a little higher but with no significant difference than the group C (normal sleeper) of 64 centenarians (64/109, 56.7%) with AIS scores (1.0¡À0.9, significant lower than group B, P<0.01) and ADL scores (32.0¡À8.3). Conclusions Our survey results showed that 56.7% centenarians were normal sleepers with better life quality, 13.8% were insomniacs with better life quality than those bedridden centenarians (27.5%) who had lost circadian activities. Therefore, the circadian activities might influence on the quality of life in centenarians. Keywords: centenarian, circadian activity, quality of life

P029 SLEEP-‐WAKE CYCLE AND ACTIVITY-‐REST CYCLE IN ELDERLY PEOPLE Anahí Flores, Xóchitl Angélica Ortiz, Minerva Aída García, Candelaria Ramírez, Pablo Valdez. Universidad Autónoma de Nuevo León, México. In elderly people the nocturnal sleep period shortens, with an associated increase in diurnal sleepiness and frequency of naps. These modifications in sleep can be due to changes in the period and the phase of the sleep-‐wake and the activity-‐rest cycle as age advances. The objective of this study was to analyze the sleep-‐wake and activity-‐rest cycle in elderly people. It is expected that elderly people will present more awakenings, more naps and a fragmentation of the activity-‐rest cycle. Participants were 6 seniors (4 male, 2 female) mean age 70.33Â±7.87 (65-‐86) years, without neurological or degenerative diseases and without sleep disorders. All participants answered the mornigness-‐eveningness self-‐assesment questionnaire, then answered a sleep diary and used a wrist activity monitoring device in the non-‐dominant hand, for 18 consecutive days. One participant was definitely morning chronotype (age: 70 years), two were moderately morning type (ages: 65, 67 years) and three were intermediate type (ages: 66, 68, 86 years). The participants slept from 23:10±1:37 h to 07:37±1:27 h (midsleep: 03:23±1:37 h; total sleep time: 8:25±0:26 h). They woke up an average of 1.26±1.34 times per night, higher frequency of awakenings occurred in the oldest participant (3.81±0.81 times per night). Only two participants took naps during the day (ages: 66, 86 years). Using a spectral analysis, an activity cycle with a 24:16±0:00 h period was found in all participants. Five participants also showed a 12:00±0:00 h, only the youngest of the sample did not show this cycle (age: 65 years). In conclusion, it was found that in elderly people, nocturnal sleep period shortens, awakenings increase and the activity cycle is fragmented into two cycles: 24 h and 12 h. Keywords: sleep-‐wake cycle, activity-‐rest cycle, elderly people

153

P030 SERUM FACTORS CHANGE CELLULAR CLOCK PROPERTIES IN OLDER INDIVIDUALS 1Lucia Pagani, 1Karen Schmitt, 1Fides Meier, 1Jan Izakovic, 2Ermanno Morriggi, 1Konstanze Romer, 1Antoine Viola, 1Christian Cajochen, 1Anna Wirz-‐Justice, 1Anne Eckert, 2Steven A. Brown. 1Univesity of Basel, Switzerland. 2University of Zurich, Switzerland. Human ageing is accompanied by dramatic changes in daily sleep-‐wake behaviour: activity shifts to an earlier phase, and the consolidation of sleep and wake is disturbed. Although this daily circadian rhythm is brain-‐controlled, its mechanism is encoded by cell-‐autonomous circadian clocks functioning in nearly every cell of the body. In fact, human clock properties measured in peripheral cells like fibroblasts closely mimic those measured physiologically and behaviourally in the same subjects. To better understand molecular mechanisms by which human ageing affects circadian clocks, we therefore characterised the clock properties of fibroblasts cultivated from dermal biopsies of young and older subjects. Fibroblast period length, amplitude and phase were identical in the two groups even though behaviour was not, thereby suggesting that basic clock properties of peripheral cells do not change during ageing. Interestingly, measurement of the same cells in the presence of human serum from older donors shortened period length and advanced the phase of cellular circadian rhythms compared to treatment with serum from young subjects, implying that a circulating factor might alter human chronotype. Further experiments demonstrated that this effect is due to a thermolabile factor present in serum of older individuals. Thus, even though the molecular machinery of peripheral circadian clocks does not change with age, some age-‐related circadian dysfunction observed in vivo might be of hormonal origin and therefore pharmacologically remediable. Keywords: circadian, fibroblast, elderly

P031 CIRCADIAN RHYTHMS AND TIME ESTIMATION IN HEALTHY YOUNG AND ELDERLY HUMANS Mario Leocadio Miguel, Luiz Menna-‐Barreto. Universidade de São Paulo, Brazil. Introduction: Several aspects of the circadian timekeeping system change with age, including alterations in phase relationship of rhythms to the environmental zeitgebers, reduced sensitivity of the circadian pacemaker to time cues, decreased amplitude and stability of the circadian rhythms. Besides being sensitive to the aging processes, timing or time estimation capacities are also subject to diurnal variability, representing an interface

154

to the study of the relationship between these two endogenous timing systems. The aim of this present study is to understand the association between age-‐related chances in the circadian system and the capacity of estimation of short time intervals. Methods: Two groups of healthy volunteers were recruited: young adults (n=15, 20-‐30 years) and older adults (n=16, 50-‐70 years). Sleep/wake cycles, wrist temperature and rest/activity were monitored by sleep diary and actimetry for 10 days. Following their usual daily routines, the subjects were instructed to perform a time estimation procedure comprising the production of 10 seconds, in 5 different phases of day for 10 consecutive days. Results and Discussion: The onset of sleep of the young group was delayed only on weekends (01:00h vs. 24:00h, ANOVA F=11.29, p<0.05). Moreover, the total amount of sleep of both groups was different on weekdays (ANOVA, F=13.34, p<0.001). Cosinor analysis revealed that the acrophase of wrist temperature is delayed in the young group, whereas no phase difference was found concerning the rest/activity rhythm. A circadian phase-‐dependent modulation on time estimation was found for both groups (morning vs. afternoon vs. evening sessions), and the magnitude of deviation or accuracy from the proposed 10 seconds was similar between groups, showing no age dependent modulation. The age-‐related differences found in the circadian timekeeping organization were only related to phase and seem to be insufficient to impair the capacity of estimation of short intervals. Keywords: Time Estimation, Ageing, Cognition

P032 CIRCADIAN BLOOD PRESSURE VARIABILITY AMONG SOUTHEAST INDIAN POPULATION AS FUNCTION OF AGE, GENDER AND DIPPING PATTERN Nishtha Vaidya, Atanu Kumar Pati, Arti Parganiha. Pt. Ravishankar Shukla University, Raipur, 492010, India. Ambulatory blood pressure monitoring has become increasingly important for the evaluation of blood pressure variability in normotensive and hypertensive human subjects. Information on blood pressure variation among Southeast Indian population is altogether absent. Therefore, in the present study, we aimed to investigate the circadian blood pressure rhythm in a cohort of normotensive human subjects inhabiting the Southeastern region of India. In addition, variation in BP was examined as function of age gender nocturnal dipping pattern and work type in BP. One hundred fourteen normotensive human subjects consisting of 57 males and 57 females voluntarily participated in the present study. Blood pressure in each subject was monitored using Ambulatory Blood Pressure Monitor (ABPM, TM 2430) for 2-‐4 consecutive days. Data were analyzed using A&D, Cosinor, Spectre, Costat, and SPSS software. Result indicates that More than one fourth of the studied population was identified as non-‐dippers. A statistically significant circadian rhythm was validated in all studied variables, irrespective of age, gender, dipping pattern and work

155

type in BP. Rhythm detection ratio was higher in extreme dipper followed by the dipper and nondippers. Significant differences for the circadian Mesors of (SBP, DBP, HR, and MAP) were noticed between males and females. Age, dipping pattern and work type produced a significant effect on the rhythm parameters of studied variables. Significant interaction effect was validated between time and dipping patterns. Further, day-‐ and night-‐time averages of SBP, DBP and MAP exhibited positive correlation with age, height weight, body surface area (BSA) and body mass index (BMI). Findings indicate that factors, age, gender dipping pattern and work type are modulating the levels of BP during sleep and wake period. Further, in the present study 32% of the subjects are non-‐dippers of this region may have higher risk of cardiovascular diseases. Keywords: ABPM, circadian rhythm, nocturnal dipping pattern

P033 EFFECTS OF A 30-‐MINUTES ADVANCE IN SCHOOL SCHEDULE ON SUSTAINED ATTENTION IN CHILDREN Juventino Cortez, Diana Juárez, Jorge Borrani, Aida García Candelaria Ramírez, Pablo Valdez. Universidad Autónoma De Nuevo León, México. People are exposed to several types of changes in schedule, such as shiftwork and daylight saving time. An advance in the schedule produces changes in the sleep-‐wake cycle and sleep deprivation. These disturbances may affect sustained attention, which is the capacity to respond efficiently to the environment during prolonged periods. There are three indices of sustained attention: general stability of efficiency, time on task stability and short-‐term stability. The objective of this study was to determine the effects of a 30-‐minutes schedule advance on the indices of sustained attention in children. Participants were 21 elementary school students (13 males and 8 females; age: 10.94 ± 0.65 years); attending school in a morning shift, from Monday to Friday. Recordings were made at school settings in February, when some elementary schools of the State of Nuevo León advance their start time, from 08:00 (winter schedule) to 07:30 (spring schedule); the other schools did not change their start time (08:00 h). Nine participants attended a school that did not change its schedule and 12 participants attended a school that advanced 30 minutes its start time. All participants kept a sleep diary during three days before and one day after changing schedule. All participants responded a continuous performance task (CPT) at 08:00 h, one Monday before and one Monday after changing schedule. Participants attending the school that change the schedule advanced their waking time by 32:24±9:45 minutes, and showed a lower level of short term stability as measured by the median longest hit runs (before change = 24 ±16.47 correct responses, after change 16.33 ±6.86 correct responses, T=3, p<0.01). After the change in schedule, this measure was also lower compared to participants that did not change schedule (median longest hit runs = 28.22 ±14.77 correct

156

responses, U=26.50, p<0.05). In conclusion, a 30-‐minutes advance in schedule impairs short-‐term stability, an index of sustained attention of children, early in the morning. Keywords: SCHEDULE CHANGE, ATTENTION, CHILDREN

P034 CHRONOTYPE ASSESSMENT: VALIDATION OF THE MCTQ IN A SAMPLE OF UNIVERSITY STUDENTS FROM BRAZIL 1Marilene Farias Alam, 2Karla V. Allebrandt, 1Mabel Mascarenhas Wiegand, 1Giovana Dantas, 1Rosa Maria Levandovski, 3Rosana Mendonça de Souza, 1Maria Paz Hidalgo. 1Universidade Federal de Pelotas (UFPel), Departamento de Fisiologia e Farmacologia /Universidade Federal do Rio Grande do Sul (UFRGS), Faculdade de Medicina Laboratório de Cronobiologia, Brazil. 2Ludwig-‐Maximilians-‐University of Munich (LMU), Department of Medical Psychology, Germany, 3Fundação Universidade Federal do Rio Grande (FURG)Departamento de Medicina Interna-‐Faculdade de Medicina In the last years, the interest in the study of the individual difference known as circadian typology or chronotype is increasing. Several instruments have been developed for identification of circadian typology with proven reliability and validity. Objectives: The validation of the Munich ChronoType Questionnaire (MCTQ) in a sample of undergraduate students from Brazil. Methods: This cross-‐sectional study analyzed data from 246 subjects, university students from Brazil, aged 17-‐35 years who filled the MCTQ and MEQ in a Portuguese version. Preferences in sleep schedules were assessed taking into account workdays (study hours) and free days and the amount of exposure to ambient light. The mid-‐sleep on free days was corrected for sleep debt and the validation was made using the ROC curve. Statistical analyses (SPSS 18.0) included the Pearson’s correlation coefficient (r). Also discriminant analyses was used to define which variables of MCTQ presented the highest discriminant coefficient between evening-‐type and the other chronotypes. Results: The mid-‐sleep phase distribution in this sample was around 5:00 to 6:00 am (local time). MCTQ and MEQ showed normal distribution. The ROC curve showed AUC = 0.76 (CI95%: 0.70; 0.83), Sensitivity of 74% and Specificity of 68% for a cut-‐off = 5.5. MEQ presented negative correlation with mid-‐sleep phase corrected to sleep debt (Pearson correlation: r = -‐ 0.48; p<0.001). The variables that presented the highest discriminant coefficient were MSF (0.89) and sleep onset in free days (0.86). The total discriminant coefficient was 70%. Conclusion: This study showed a good sensitivity and specificity. Also a good correlation between the MEQ and MCTQ questions related to sleep times on free days was observed. But in discriminat analysis only MSF and sleep onset presented higher levels of discrimination between evening-‐type and the other chronotypes. This means that MCTQ

157

and MEQ measure different dimensions of human behavior. This study is part of a project which is intended to continue evaluating an extended number of chronotypes, in Brazil. Keywords: Chronotypes, MCTQ, Validation

P035 CHRONOTYPES: A CORRELATION WITH EATING BEHAVIOR 1Ana Beatriz Harb, 2Wolnei Calmo, 1Rosa Maria Levandovski, 3Ceres Oliveira, 4Kelly Allison, 4Albert Stunkard, 1Maria Paz Hidalgo. 1Human Chronobiology Program of Hospital de Clínicas de Porto Alegre, Brazil. 2Anesthesia Service and Perioperative Medicine at Hospital de Clínicas de Porto Alegre, Brazil. 3Guest Professor of the Post-‐Graduation Program of Hospital Moinhos de Vento, Brazil. 4University of Pennsylvania School of Medicine, Department of Psychiatry, Center for Weight and Eating Disorders, EUA. Objective: This study examined the association between the morningness/eveningness dimension and eating patterns. Method: A cross-‐sectional study with a sample of 100 subjects who were screened at a nutrition clinic. Results: Mean age was 39.5 years (± 11.7), 77% were women and 66% were overweight. Bivariate analyses showed significant relationships between the morningness/eveningness Questionnaire (MEQ) and binge eating behaviors measured by the Binge Eating Scale (BES) and night eating behaviors measured by the Night Eating Questionnaire (NEQ). In the multivariate analyses, only binge eating was significantly associated with the MEQ (p=0.027). Binge eating and night eating behaviors were significantly correlated (p<0.001). Discussion: Binge eating was significantly related to the eveningness dimension. BED and NES are associated with the development of overweight. These disorders represent an important public health concern. Moreover, the study of chronotypes is related to mechanisms that may be identified through chronobiological methods and may be an important tool to be used in the comprehension of abnormal eating behavior. Keywords: Chronotype, Eating disorders, Night eating syndrome

158

P036 LIGHTING AND HEALTH: CASE STUDY IN RETAIL STORES 1Betina Tschiedel Martau, 2Maria Paz Hidalgo. 1Faculdade de Arquitectura-‐UFRGS, Brazil. 2Laboratório de Cronobiología -‐ HCPA -‐ UFRGS, Brazil. Despite the decades of research, it was only after the discovered of the connection between light and a third type of photoreceptor in the retina that the description of the mechanism of biological effects controlled by the light and dark cycle has advanced. This work addresses the issue of non-‐visual impacts of human exposure to light, in an attempt to relate the quality of lighting design to health, comfort, and well-‐being of female retail store employees. The sample for the cross-‐sectional study was randomly established with three groups of ten female volunteers each. Assessment of lighting considered glare, color appearance of light, flexibility, and possibility of lighting control by employees. The tools to assess well-‐being and health were psychometric scales internationally validated by the psychiatric field to measure depression, anxiety, and stress symptoms. Assessment of sleep conditions and analysis of the activity/rest rhythm was carried by a wrist monitor with attached luximeter and the analysis of the body temperature rhythm was made by a temperature sensor, to which each participant was submitted for five consecutive days. The lighting pattern’s influence on the circadian system was verified by measuring saliva melatonin and cortisol levels. The degree of satisfaction of employees and their preferences regarding work environment lighting were surveyed by applying questionnaires. Data were analyzed using Pearson correlations, ANOVA, and stepwise regression. In street retail store group, results indicate that crossing the assessment of satisfaction and emotional aspects with biological ones indicated that the higher the employees’ general satisfaction with lighting, the higher their melatonin level at 12 p.m. and the lower their depression scores. Possibility of outside visual contact in that group leads to better physiological conditions, especially sleep conditions, than the other groups. In shopping mall groups, the reverse correlation found between the store’s average general illuminance and general satisfaction with lighting conditions in the work environment is worth pointing out, since scores were highest in that category (worse emotional conditions) in all scales applied and there are changes both in cortisol rhythm (tendency to lower rhythm in the morning and afternoon mall group) and in melatonin (tendency to phase delay in the afternoon and evening mall group) as well as differentiation in activity rhythm and temperature in the afternoon and evening mall group. Shopping mall employees’ miss visual contact with the outside, being able to vary lighting during the workday, and consider lighting as excessive. The study concludes that poor lighting design can actually have negative consequences for health and well being and that it is necessary to review the stores’ lighting design strategies, to seek

159

new guidelines able to solve the possible conflicts between light oriented to sell products and to light people in stores. Keywords: lighting design, health, retail stores

P037 SLEEP PATTERNS AND RISK FACTORS FOR METABOLIC SYNDROME AMONG PROFESSIONAL PUBLIC TRANSPORT DRIVERS Ma. Elena Durán Lizárraga, Juan Manuel Ponce, Miguel Ángel Palomino Garibay. Academia de Biología Humana, Colegio de Ciencia y Tecnología, Universidad Autónoma de la Ciudad de México, México. Sleepiness and sleep complaints are common among professional drivers. Sleepiness is a problem that affects the driver’s well-‐being, and has consequences in their performance and safety. Assessment of prevalence and research into the risk factors are an important health issue and as public transport drivers have a high prevalence of sedentary habits, diabetes and obesity. Our aim is to describe the distribution of chronotypes and explore the association between sleep patterns and risk factors for metabolic syndrome (MS) in this population with the collaboration of the Centro para el Fomento de la Educación y la Salud de los Operarios del Transporte Público de la Ciudad de México (CENFES, AC). The Horne-‐Östberg questionnaire, the Sleep Hygene Index (SHI), the fatigue severity scale (FSS), the obstructive sleep apnea syndrome (OSAS) questionnarie and a medical examination and laboratory analisys, (both of these were made in the medical unit of CENFES, AC) were completed by 200 professional public transport drivers during september and august 2010. The mean (SD) age of the population was 39.4 (11) yrs. The 83% of them have a matutine chronotype, 45% report a sleep onset latency >30 minutes. The mean (SD) SHI score was 21.9 (4.5). The 38% had a score >10 for the the FSS, the mean (SD) score was 21.8 (11.73). The prevalence of OSAS was 38%. Out of the drivers analyzed, 35.5% had obesity, 41% were overweight, 25% had glycemia >110 mg/dl and 15% had hypertension. These results show that the prevalence of risk factors for MS and sleep disorders in this study was higher that the observed in other studies for general population. As different health problems were identified, we are working in the design of a systematic education about these disorders as well as health promotion interventions. Keywords: sleep patterns, metabolic syndrome, public transport drivers

160

P038 MORNINGNESS–EVENINGNESS, USE OF STIMULANTS, AND MINOR PSYCHIATRIC DISORDERS AMONG UNDERGRADUATE STUDENTS 1Giovana Dantas, 2Marcia L. M. Schneider, 2Denise C. Vascincelos, 1Rosa Levandovski, 1Wolnei Caumo, 3Karla V Allebrandt, 2Marlene Doring, 1Maria Paz L. Hidalgo. 1Universidade Federal do Rio Grande do Sul/ Hospital de Clínicas de Porto Alegre, Brazil. 2Universidade de Passo Fundo, Brazil. 3Ludwig-‐Maximilians-‐University, Germany. Morningness–eveningness dimension in humans have been indicated to influence social behavior and individual health. The aim of the present study was to investigate the association of the morningness–eveningness dimension with behavioral and health aspects in a sample of undergraduate students. We assessed demographic data; the Pittsburgh Sleep Quality Index was used to evaluate sleep quality; the Morningness/ Eveningness Questionnaire to determine morningness–eveningness, and the Self-‐Reporting Questionnaire to assess minor psychiatric disorders. A total of 372 students (66.7% females), on average 21.6 years old, participated in this study. Among them, 92.2% did not smoke, 58.9% engaged in physical activities, and 19.7% were night-‐shift workers. In regard to morningness–eveningness, 55.9% of the participants were intermediate between evening (39.5%) and morning (4.6%) types. Poor sleep quality (OR=1.89), minor psychiatric disorders (OR=1.92), and tobacco consumption (OR=3.65) predominated among evening types. Evening types were predominantly males (OR=1.72). This study suggests that evening types are more vulnerable to sleep and psychiatric disturbances, and tend to smoke more than morning types Keywords: Chronotype, Morningness/eveningness, Minor psychiatric disorders

P039 CIRCADIAN CLOCK GENE EXPRESSION IN BRAIN REGIONS OF ALZHEIMER'S DISEASE PATIENTS AND CONTROL SUBJECTS Nicolas Cermakian, Elaine Waddington Lamont, Philippe Boudreau, Diane B. Boivin. Douglas Mental Health University Institute, McGill University, Montreal, QC, Canada. Circadian oscillators have been observed throughout the rodent brain. In the human brain, rhythmic expression of clock genes has been reported only in the pineal gland and little is known about their expression in other regions. We sought to determine whether clock gene expression could be detected and varies as a function of time of day in the bed nucleus of the stria terminalis (BNST) and cingulate cortex, areas known to be involved in decision-‐making and motivated behaviors, as well as in the pineal gland, in the brains of Alzheimer's

161

disease (AD) patients and aged controls. Relative expression levels of PERIOD1 (PER1), PERIOD2 (PER2), and BRAIN AND MUSCLE ARNT-‐LIKE PROTEIN-‐1 (BMAL1) were detected by quantitative PCR in all three brain regions. A harmonic regression model revealed significant 24-‐hour rhythms of PER1 in the BNST of AD subjects. A significant rhythm of PER2 was found in the cingulate cortex and BNST of control subjects, and in all three regions of AD patients. In controls, BMAL1 did not show a diurnal rhythm in the cingulate cortex but significantly varied with time-‐of-‐death in the pineal and BNST, and in all three regions for AD patients. Notable differences in the phase of clock gene rhythms and phase relationships between genes and regions were observed in the brains of AD compared to those of controls. These results indicate the presence of multiple circadian oscillators in the human brain, and suggest altered synchronization among these oscillators in the brain of AD patients. Funded by the Canadian Institutes of Health Research Keywords: Human brain, Alzheimer's disease, clock genes

P040 DO NOT MESS WITH YOUR BIOLOGICAL CLOCK: SEIZURE AGGRAVATION AFTER THE PHASE SHIFT 1Magdalena Kinga Smyk, 2 Anton Coenen, 1Marian Henryk Lewandowski, 2Gilles van Luijtelaar. 1Department of Neurophysiology and Chronobiology, Chair of Animal Physiology, Institute of Zoology, Jagiellonian University, Krakow, Poland. 2Donders Centre for Cognition, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, The Netherlands. The light-‐dark cycle is one of the most prominent synchronizer of biological rhythms. Sudden changes in the timing of the light phase is a factor responsible for internal desynchronization between distinct circadian rhythms. It is also thought to cause the jet lag syndrome, fatigue and impaired performance after rapid changes of time zones. Absence epilepsy is characterized by reduced consciousness and generalized, synchronous, bilateral, 3 -‐ 4 Hz (humans) and 7 -‐11 (rats) spike-‐wave discharges (SWD) in EEG. The circadian rhythm of SWD in WAG/Rij rats, a well known, validated animal model of human absence epilepsy is generated endogenously by the circadian timing system. Moreover, under the constant condition of dim light internal desynchronization between the rhythm of SWD and general motor activity occurred (Smyk et al., 2010). The goal of the present study was to investigate the course of the entrainment of the rhythms of SWD and general motor activity to a phase shift of the light-‐dark cycle. Additionally, the number and duration of the SWD were compared pre-‐ and post-‐shift. Chronic EEG and motor activity recordings were made in adult WAG/Rij rats kept in 12:12 light-‐dark cycle. After 4 baseline days, the onset of light was delayed by 8 h and the recordings were made during 10 consecutive days. Preliminary

162

analysis of the results showed an immediate effect of the phase shift on both rhythms. On the first post-‐shift day the acrophase of SWD and motor activity rhythms was advanced of about 7.5 h in comparison to the baseline. The course of the entrainment over the consecutive days was faster for the motor activity and slower for the rhythm of SWD. The phase shift caused a significant increase in the number of SWD starting from the second post-‐shift day, differences in the mean duration were not observed. In conclusion, 8 h phase shift of the light-‐dark cycle alters circadian rhythms of SWD and general motor activity. The timing of the acrophase of both rhythms is immediately shifted in response to the delay of the light phase. However, the time course of the entrainment to the shift is different for the rhythm of SWD and general motor activity, suggesting internal desynchronization between oscillators controlling these rhythms. Disturbances in the circadian physiology caused by the abrupt phase shift increase the number of SWD. These findings suggest that circadian factors play an important role in absence epilepsy. Keywords: absence epilepsy, phase shift, WAG/Rij rats

P041 6-‐SULFATOXIMELATONIN AS A PREDICTOR OF CLINICAL OUTCOME IN DEPRESSIVE PATIENTS TREATED WITH INHIBITORS 1Maria Paz Hidalgo, 2Wolnei Caumo , 1Giovana Dantas, 1Daiane Franco, 1Iraci Trres, Regina P Markus. 1Laboratório de Cronobiología-‐HCPA-‐UFRGS, Brazil. 2Departamento de farmacología UFRGS, Brazil Clinical response to antidepressant is related to increased noradrenergic activity, it is important an exploration of physiological gauge of over-‐all noradrenergic function. Pineal gland production of melatonin is well suited to serve this purpose. The aim was to establish the predictive value of aMT6 urinary concentration as indicative of therapeutic response to noradrenaline uptake inhibition in depressive patients. This study was conducted in Hospital de Clínicas de Porto Alegre in south of Brazil. Twenty-‐two female depressive patients aged between 18 and 60 years old were selected. Depressive symptoms were assessed by Hamilton Depression Scale before, two and eight weeks after treatment. Urine samples were collected one day before and 24h after starting nortriptyline (25 mg, 21h00). The means comparison was made by paired or independent student “t” test. Size effect was calculated to analyze the variation between aMT6s excretion in nighttime before and after nortriptyline. The correlations were analyzed by Spearman’s Rank Coefficient of Correlation. MANOVA and Bonferroni test were used to identify differences between size effects groups at each time point. Statistical significance was set at P = 0.05. aMT6s excretion was higher between midnight and 06h00 a.m. (F= 4.04; P= 0.01). It was observed a positive correlation between aMT6s excretion before and after nortriptyline (r= 0.76; P= 0.0), but not between

163

aMT6 excretion before drug and fractional increase (r= -‐0.33; P= 0.88). There was an interaction between size effect of treatment and depressive symptoms (F= 185.92; P= 0.04). Both groups improved depressive symptoms (F= 4.63; P< 0.01), but a high size effect identified subjects who responded in two weeks of antidepressant, which presented higher level of clinical improve in symptoms throughout the time compared to patients with low size effect. Since melatonin production is directly related to sympathetic outflow, our results reinforce the hypothesis that aMT6s excretion is a predictor of clinical outcome. Keywords: melatonine, depression, pharmacology

P042 CIRCADIAN RHYTHM OF ENERGY EXPENDITURE AND CONSUMPTION OF OXYGEN BY INDIRECT CALORIMETRY IN ENTERAL NUTRITION THERAPY 1,2Marlene Pooch Leuck, 2Rosa Maria Levandovski, 1Claudia Will Machado, 3Maria Paz Loayza Hidalgo. 1Hospital Nossa Senhora da Conceição/RS, Brazil. 2UFRGS/Laboratorio de Cronobiología, Brazil 3UFRGS/Departamento de Psiquiatría/Laboratorio de Cronobiología, Brazil. Introduction: The importance of enteral nutrition has grown in recognition resulting in new methods of administration. That leads to many questions such as: what are the chronobiologic effects of continuous or intermittent nutrition therapy? Objectives: The aim of this study was to evaluate the use of enteral nutrition as a Zeitgeber of biological rhythm. Energy expenditure and oxygen consumption were measured by indirect calorimetry in continuous or intermittent nutrition patterns. Methods: A randomized controlled clinical trial was conducted from December 2009 to November 2010. Thirty four neurological patients received through the same kind of calibrated nasogastric tube the standard protein and energy intakes calculated for each subject. The continuous group (15 patients) received continuous feeding throughout 24 hours; the intermittent group (19 patients) received as follows: 8, 12, 16 and 20 hours during 2 hours infusion. Nutritional support was identical for both groups. The measures were performed during 30 min, with 12 measurements for each patient during 3 days, Measure A: 7:30 am (fasting group intermittent), B: 10:30 C: 14:30; and D: 21:30 hours. Results: The mean age was 69.5±8, 50% were male; BMI 22±3.9kg/m2 (men), 25±5.6 kg/m2 (women). The total energy expenditure showed no significant difference between groups. Oxygen consumption showed a significant difference between continuous and intermittent groups (212±117 ml/min; 257±125 ml/min (p=0.048), respectively.) The variables energy expenditure and oxygen consumption were analyzed by ANOVA-‐one way in different times during the day and some statistically difference was found among all times (A,B,C and D) in both nutritional groups. Comparing energy expenditure and oxygen consumption between the groups (continuous and intermittent) by Mann-‐Whitney , there was a statistically significant difference in time B and C (p=<0.01). In

164

the intermittent group, the average of the test (fasting) was 1798 kcal/24h, in subsequent tests there was a decrease of 1% and 6% on thermogenesis in test B and C, and an increase of 4% in test D. In the continuous group the average of the test during 24 hours was 1532 kcal/24h. We observed a decrease of 11.8% and 13.87% in tests B and C, and 11.4% of increase in test D. Conclusion: We observed in this study a circadian variation of energy expenditure and oxygen consumption in continuous and intermittent method of enteral nutrition, suggesting that only one measurement per day may not necessarily show the patient energy consumption. Moreover, the energy expenditure and oxygen consumption was higher in intermittent groups in all times. Keywords: Circadian Rhythm, Indirect Calorimetry, Enteral Nutrition

P043 PRETERM INFANTS HAVE IMPROVED GROWTH IN LIGHT/DARK CYCLE COMPARED WITH CONTINUOUS BRIGHT LIGHT 1Samuel Vásquez, 2Manuel Ángeles-‐Castellanos, 2Carolina Escobar. 1Hospital General "Benito Juárez" SSA., México, 2Facultad de Medicina, UNAM, México. The neonatal intensive care unit (NICU) environment, including continuous bright light levels, may have negative effects on the growth and development of preterm infants. Continuous bright light has been related to infant stress as manifested in increased activity levels, decreased sleep, and bradycardia. Yet, LD has the potential to promote circadian rhythms with health benefits including hormonal regulation, activity-‐rest cycle, and vital sign regulation. Thus, growth might either be decreased with the stress related to exposure to bright light or increased with the development of circadian patterns and rest-‐activity rhythms when exposed to LD. The purpose of this study was to evaluate the benefits of light/dark cycle (L/D) versus continuous light (CL) on health in preterm infants born at 32 weeks’ gestational age. Study design: Randomized, interventional study comparing infants receiving LD from birth, and infants that receiving continuous light, as is normal in the NICU. Results: Infants receiving LD at birth and 25 weeks’ post-‐conceptional age gained weight faster than infants in constant light, newborns under LD, had a rapid weight gain and improved feed tolerance formula, plus we saw an improvement in oxygen saturation and a reduction of hospital stay of approximately 20 days. Conclusions: These findings suggest that L/D cycle has significant weight gain benefits over of infants, and there are short-‐term advantages of L/D cycle for health in preterm infants. Keywords: desyncrhonization, chronoterapy, human

165

P044 CHRONOBIOLOGIC VISCUM ALBUM STUDY IN BREAST CANCER 1Abuín A. Abuin Landín, 2 Luis Menna Barreto. 1Medical Science University Matanzas “Dr. Juan Guiteras Gener”, Cuba. 2Universidade de Sao Paulo Escola de Artes, Ciencias y Humanidades, Brasil. In Cuba breast cancer incidence rate grows alarmingly. To face this challenge of the public health in Cuba new therapeutic resources they are developed that enlarge the conventional therapy. Inside them they are the coming ones of the treatments optimization with chrononcology, the immunotherapy and the natural medicine. In this context it is very diffused the therapy in the entire world with pharmacological natural phytocomplex with Viscum album, a hemiparasit plant with substances that have been demonstrated goods actions as immunemodulater effects on the linf cells implied in the detection, recognition, attack and clearing of transformed cells, cytotoxic and proapoptotic effect on the tumoral cell that opens a road toward a holistic vision that has in it counts the what , how much and how it is administered, but also to who, when and where. One of the most consistent properties in V. album phytocomplex is the presence of biologically active substances with a vectorial polarity that transcends the pharmacological, morphological and ecological dimensions. This dynamics could be represented at molecular grade and in function of the time, for the presence of three macro-‐molecules groups that act as dynamic attractors and that they explain as much its therapeutic effect as resinchronizator of biological rhythms (chronobiotic effect). To characterize and evaluate the clinical effectiveness and temporary optimization of the Viscum album preparations, we mensure in the present work, with the use individual tempactilumi sensors, capable to register the corporal temperature, the movement (actimetry) and intensity light external exposition that enlarge and supplement the clinical possibilities of mensuration of variable as quotient frequency cardiac/respiratory rate in the exhaustive study of human rhythms and metabonomic profile in patient with breast cancer. Keywords: chronopharmacology, breast cancer, immunomodulation

P045 DISCUSSION ABOUT THE INTERACTION BETWEEN DRUGS AND HUMAN BODY BIOLOGICAL CLOCK Feng Xiu Jie, Zhuang Hong Yan. Beijing An Ding Hospital, Capital Medical University Beijing, China.

166

Through literature retrieval, this paper discusses the interaction between the medicine and human body biological clock. Medicine is the important factor to affect the clockwork. Modern research also found that many medicines in the human body process will show their circadian rhythms change. That is to say, the different time of administration shows different clinical curative effect and different adverse reactions, this relates greatly to human body biological clock. This paper discusses the two aspects respectively, the influence of the medicine on human body biological clock and human body biological clock on medicine. Keywords: medicine, biological clock, biological rhythm

P046 CUSTOM-‐MADE SOFTWARE FOR DIGITAL SIGNAL ANALYSIS ON CHRONOBIOLOGY 1,2Arturo Vega-‐González, 1Raúl Aguilar-‐Roblero. 1Department of Neurosciences, Instituto de Fisiología Celular, UNAM, México DF, 04510. 2Current address Department of Physic Engineering, Universidad de Guanajuato, Leon, Gto. 37150, México. Research in the field of biological rhythms requires precise quantitative and qualitative tools for the signal analysis of time series. The methodology for analysing a biological rhythm has been the subject of much investigation. Nonetheless, many issues still have no definitive answer, for example, in the search for periods and phase markers there are various methods and the researcher must be able to determine those that are the most appropriate and reliable in a particular case. Furthermore, it happens that all those methods are not included in a single software package. The present work is aimed to describe a system for the signal analysis of chronobiological data. This custom-‐made software has some innovations in the analysis and display capabilities The custom-‐made software, named DiSPAC, had several options for analysis and display. it includes the following analysis: Spectral density, periodogram, cosinor, auto-‐correlation and phase plane. The graphics and display features include: graphic estimation of phase and period, rhythm architecture, stability analysis and four types of actogram. DiSPAC was tested with real and artificial data sets to determine the reliability of the algorithms used to calculate the outcome measures reported. This allowed the building of a solid and reliable software application. The DiSPAC software was written in C/C++ for MS-‐Windows operating system. At the highest level of operation the DiSPAC software has a graphical user-‐friendly interface and it was able to provide a range of outcome measures from chronobiological data. Supported by grant CONACyT 49740 and FONCICYT 91984 funded by the European Union and CONACyT.PAPIT IN204811 Keywords: Signal analysis, Software, Algorithms

167

168

P047 THEORETICAL INVESTIGATION FOR THE ORIGIN OF THE ASYMMETRIC RESYNCHRONIZATION AFTER AN ABRUPT SHIFT OF THE LIGHT: DARK CYCLE 1Takeshi Asakawa, 2Satoshi Koinuma, 2Koh-‐hei Masumoto, 2Mamoru Nagano, 2Yasufum Shigeyoshi. 1System Technologies Laboratories, SONY Corporation, Japan. 2Department of Anatomy and Neurobiology, School of Medicine, Kindai University, Japan. We theoretically investigate the origin of the asymmetrical resynchronization process of subregions of the suprachiasmatic nucleus (SCN), which is the center of the circadian rhythm, after an abrupt shift of the light: dark cycle. SCN can be anatomically and functionally regarded as distinct two parts, the ventrolateral (VLSCN) and dorsomedial (DMSCN) regions. The optical information through retina immediately reaches to VLSCN via retinohypothalamic tract, but not in DMSCN, which has been shown by induction of immediate early genes in the VLSCN but not in the DMSCN after a light exposure during the night. With the bias of optical information, desynchrony between VLSCN and DMSCN oscillators appears after an abrupt shift of an environmental steady light:dark cycle (LD cycle), which seemed to generate a jet lag (Nagano et al. 2003). In the experiment, asymmetry appeared in the restoration process. It took about 10 days to restore synchronization after a six hour advance of LD cycle but took only about five days after 10-‐hour delay of the LD cycle. In order to investigate the origin of the asymmetry, we constructed a mathematical model consisting of interacting two limit cycle oscillators (LCO) described as Stuart-‐Landau oscillators. We assumed that one LCO representing VLSCN was promptly entrained to LD cycle and that the other representing DMSCN was entrained by afferent input from VLSCN. In order to realize the immediate entrainment of the VLSCN oscillator after the abrupt shift of LD cycle shown by Nagano et al. (2009), we also supposed that VLSCN basically possesses a property of a damping oscillator. As a result, we reproduced the asymmetry associated with the resynchronization process and found that the appearance of the asymmetry depends on the manner of interaction between two oscillators. Keywords: suprachiasmatic nucleus, mathematical model, jet lag

169

P048 Flying Mice: Internal Forced Desynchronization In A Murine Model Of Chronic Jet-‐Lag 1Leandro Pablo Casiraghi, 2Gisele Akemi Oda, 1Juan José Chiesa, 3Wolfgang Otto Friesen, 1Diego Andrés Golombek. 1Laboratorio de Cronobiología, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Argentina. 1Instituto de Biociências, Departamento de Fisiologia, Universidade de Sao Paulo, Brazil. 3Department of Biology, University of Virginia, USA. We studied locomotor activity rhythms of C57/bl6 mice under a chronic jet-‐lag protocol (6 h phase advances of the light-‐dark schedule (LD) every two days (ChrA)). Periodogram analysis indicated two components of the activity rhythm: a short-‐period component (21.01±0.04h) that followed the LD schedule, and an independent long-‐period component (24.68±0.26h). Onsets of free-‐running rhythms after release in constant darkness (DD) were significantly predicted by both components. Our mathematical model of two coupled circadian oscillators subjected to different jet-‐lag protocols states that the system processes chronic jet-‐lag schedules as new zeitgebers with periods equal to 24 + Step (the ratio of shift size, in h, to the intershift interval, in days; i.e. the Step Components), that induce a higher level of desynchronization as Step Components increase. The model predicted a lesser degree of desynchronization under a 21h period zeitgeber (T21; step=+3/1=+3) than under ChrA (step=+6/2=+3), which was confirmed by experimental data. The model also predicted less desynchronization under phase delaying than under phase advancing protocols. Indeed, most mice subjected to a chronic delay of the LD cycle through 6h shifts every two days (Step= -‐6/2= -‐3) displayed synchronous entrainment, showing an activity rhythm with a period of 26.92±0.11h driven by the predicted 27h zeitgeber. All together, our results indicate that the increase of the step components emulates the effect of a decrease in the amplitude of the arising zeitgeber. In this work we present a new model of circadian forced desynchronization through a chronic jet-‐lag protocol, and a mathematical model that not only explains the behavior found but also provides framework for understanding health issues associated with chronic phase shifting of the circadian system. Supported by ANPCyT, CONICET, UNQ and FAPESP Keywords: shift work, mathematical models, forced desynchronization

170

P049 A SUBTERRANEAN RODENT’S “NATURAL ENTRAINMENT” 1Danilo Eugênio de França Laurindo Flôres, 1Barbara Mizumo Tomotani, 1Patricia Tachinardi, 1Gisele Akemi Oda, 2Verónica Sandra Valentinuzzi. 1Instituto de Biociências, Universidade de São Paulo (IB-‐USP), Brazil. 2Centro Regional de Investigaciones Científicas y Transferencia Tecnológica (CRILAR), La Rioja, Argentina. Subterranean rodents are interesting organisms for the study of “natural entrainment” (Hut et al, 1999) for they spend most of the day inside underground tunnels, where there is little daily variation in environmental variables. Therefore, it has been proposed that entrainment depends mostly on aboveground excursions, e.g., during foraging, when there might be contact with more robust cycles. Our field data on the tuco-‐tuco (Ctenomys cf. knighti) indicates that these animals perceive the aboveground light-‐dark cycle by several random bouts of light-‐exposure during the photophase of the day. Summer observations through 11 consecutive days revealed an average peak of exposure bouts between 9 and 10 a.m. In order to access whether such light-‐dark pattern acts as an entraining agent, we first constructed in laboratory the Phase Response Curve (PRC) for 1h light-‐pulses (1000lux). Its shape is qualitatively similar to other curves reported in the literature. How the tuco-‐tuco’s clock, with its associated PRC, responds to irregular light-‐exposure patterns is being investigated by means of computer simulations. Using the software Circadiandynamix (Friesen & Friesen, 2009), two selected oscillator-‐configurations, A (type-‐1 PRC) and B (type-‐0 PRC), were submitted to potentially entraining cycles, consisting of “light pulses” uniformly distributed throughout a fixed phase range of the day. Several simulations were performed by changing this phase range from 2 to 12 hours. Unexpectedly, oscillator A maintained an apparent stable entrainment even under the most irregular cycle (12-‐hours phase range). Oscillator B, in turn, presented a pattern resembling relative coordination under this same regimen. Future studies will be performed using qui-‐square (instead of uniform) distribution of pulses within the phase range, to simulate more precisely our field data. Simulation results are consistent with a nocturnal animal exposed to light during its rest phase, which seems paradoxical for subterranean nocturnal animals that rest inside dark underground tunnels. However, in spite of laboratory evidence of a nocturnally-‐phased oscillator, the field records described above indicate that tuco-‐tucos do express aboveground activity during the photophase. Funding: CAPES, FAPESP, CNPq, CONICET. Keywords: Light-‐dark cycle, Computer simulations, Phase Response Curve

171

P050 PERIOD-‐2 INTEGRATES BOTH CIRCADIAN AND HOMEOSTATIC ASPECTS OF SLEEP REGULATION Thomas Curie, Valérie Mongrain, Stephane Dorsaz, Yann Emennegger, Paul Franken. Center for Integrative Genomics, Switzerland. In the regulation of sleep two processes have to be considered: a homeostatic process that tracks sleep need and a circadian process which gives time context to most physiological processes and behaviors. Both processes have long been considered to operate independently based on various observations; e.g. rats devoid of circadian rhythms in overt behavior after lesioning the SCN still have an intact homeostatic compensatory response to sleep loss as measured by the increase in EEG delta power (Trachsel et al, 1992). At a molecular level these two processes are less easily distinguishable because, besides their well described role in circadian rhythm generation, we have shown that clock genes also play a role in the homeostatic regulation of sleep. Here we focus on the clock gene Per2, because, on the one hand, its expression is widely used as a circadian state variable while, on the other, its expression is robustly increased with sleep deprivation (SD). Imaging Per2::Luciferase knock-‐in mice we found that also PER2 protein increases within the 6h of a SD (ZT0-‐6) in the brain as well as in liver and kidney. Immunohistofluoresence analysis demonstrated that this increase in PER2 in the brain was most pronounced in the cerebral cortex. We then investigated the interaction between the ongoing diurnal changes in Per2 mRNA levels in the brain and the effects of SD by depriving mice of sleep at 4 times of day for 6h starting at ZT0, -‐6, 12, or -‐18. The SD-‐induced increase in Per2 expression greatly depended on the slope of its change during baseline. To better understand this non-‐linear interaction, we successfully simulated the changes in Per2 expression both in baseline and after each of the 4 SDs, using a driven harmonic oscillator function with the sleep-‐wake distribution and corticosterone as driving forces. The results indicate that Per2 gene expression in the brain is controlled both by circadian and homeostatic factors. This gene is therefore well positioned to reconcile homeostatic needs within the constraints of a circadian physiology. Keywords: Per2 gene, sleep regulation, mathematical modelling

172

P051 NON-‐LINEAR DYNAMICS OF HEART RATE VARIABILITY SHOW SLEEP-‐WAKE HOMEOSTATIC PREDOMINANCE DURING SUSTAINED WAKEFULNESS Antoine U. Viola. Centre for Chronobiology, University of Basel, Switzerland. Circadian rhythmicity in heart rate is clearly established while it remains controversial for indices of the autonomic nervous system (ANS). The objective of the current investigation was to characterize variations on ANS during extended wakefulness. The ECG of 12 young healthy subjects were recorded continuously during 40-‐h constant routine protocol. Spectral analysis of the extracted cardiac signal was performed together with a non linear analysis (symbolic analysis; SA). In SA, RR intervals are concerted into sequence of symbols and subsequent construction of series of patterns with three symbols, out of which four patterns could be identified: 0V (no-‐variation), 1V (one-‐variation), 2LV (two-‐like-‐variations) and 2UV (two-‐unlike-‐variations). The first pattern indicates sympathetic activity, while the last indicates parasympathetic modulation. Heart rate (HR) underwent a clear circadian pattern, with nadir at 03:42±00:53. Similar to HR, the absolute spectral indices show a sinusoidal model rhythm. The total power (an index of global variability) showed circadian rhythmicity with an acrophase at 06:46±00:50, very low-‐frequency had an acrophase at 06:23±00:44, low frequency (LF), had an acrophase at 05:47±01:12 and high frequency (HF) had an acrophase at 06:30±01:51. However, a different sigmoid profile was observed in the normalized data, expressed by LF/(LF+HF) ratio. Before 07:05±01:21h (±20h of sustained wakefulness), LF/(LF+HF) remained stable. Afterwards, this index revealed a significant increase, and remained significantly at a higher level throughout the rest of the protocol, indicating increased sympathetic activity during sustained wakefulness. Symbolic analysis confirmed this finding and showed a clearer sleep loss effect, indicated by a striking increase in 0V pattern (06:24h±00:47h) and decrease in 2UV pattern (06:16h±00:42h), during the same time window. The absence of circadian variation in ANS and the clear circadian modulation in HR suggest an intrinsic cardiac circadian control of the heart. The sigmoid pattern of the ANS most likely reflects the influence of the increase in sleep homeostatic pressure during sustained wakefulness. Keywords: circadian, autonomic nervous system, wakfulness

173

P052 CHANGES OF DISTAL SKIN TEMPERATURE AND SUBJECTIVE SLEEPINESS UNDER MIDDAY BRIGHT LIGHT EXPOSURE Nana N. Takasu, Hisayo Nishida, Akiko Nitta, Kana Nishimura, Tomoko Wakamura. Kyoto University Graduate School of Medicine, Japan.

It has been widely accepted that subjective sleepiness is decreased when we are exposed to bright light and also is increased when distal heat loss via increased skin temperature, which causes core body temperature decline, is enhanced. Several previous studies reported that the daytime core body temperature level was lowered when human subjects were stayed under the bright light compared with dim light condition during the daytime. It is interesting to know whether subjective daytime sleepiness is increased even if human subjects are stayed under the bright light exposures during the daytime, or the relationship between distal heat loss and subjective sleepiness are deviated under the daytime bright light exposure. Therefore, the present study was performed to examine the changes in daytime rectal and skin temperatures as well as subjective daytime sleepiness under the daytime bright light exposure. Twelve young healthy men were participated in both of control dim light (10 lx) and bright light experiments (5000 lx). Rectal and skin temperatures as well as subjective sleepiness were continuously measured under unmasking conditions of constant routine protocol in the course of the experiment. Distal skin temperatures were significantly increased under the bright light exposure. However, rectal temperature did not reach statistical significance even though rectal temperature was kept lower during the bright light exposure than the control dim light condition. On the other hands, subjective sleepiness was decreased under the bright light exposure compared with the control dim light condition. The present study observed that the higher daytime distal skin temperature and lower subjective daytime sleepiness were simultaneously produced when human subjects were exposed to the daytime bright light. Even though heat loss via the distal skin regions has been widely accepted to be the key mechanism for inducing sleepiness, our findings suggest that another key mechanism might be involved with decrement of sleepiness observed under the daytime bright light exposure. Keywords: light, sleepiness, temperature

174

P053 ACTIGRAPHY AND SLEEP LOGS IN NEWBORNS: DIFFERENT RESULTS ACCORDING TO AGE 1Clarissa Bueno, 2Luiz Menna-‐Barreto. 1Departamento de Fisiología e Biofísica/ICB USP, Brasil. 2Escola de Artes, Ciencias e Humanidades/EACH-‐USP, Brasil. Introduction: The study of biological rhythms has been improved with new methods of data recording, such as actigraphy. This new method has been used to study the rhythmic expression in preterm newborns, but comparative studies between data recorded by actigraphy and observed behavior are lacking. In this study we compare the evolution of motor activity rhythm and sleep/wake cycle recorded by actigraphy and sleep logs in fullterm and preterm newborns during their stay in neonatal care units (NCU). Material e methods: Nineteen preterm and seven fullterm babies were followed during their stay in NCU since the first week of life until discharge. These babies had their motor activity recorded by an actigraph attached to the left ankle. Sleep-‐wake, feeding and activity diaries were also filled by the staff. The resulting individual time series were used to build actograms and were divided in 7 days series according to gestational age -‐ GA, which were analyzed with the Lomb Scargle periodogram and with the Cosinor method. Results: We found an ultradian pattern for motor activity for most preterm babies until the 34th week of GA, when a circadian rhythm becomes more evident. Babies who were born with more than 34 weeks of GA and the fullterm ones showed a dominant circadian rhythm since the beginning of data recordings. Otherwise, sleep/wake and feeding behavior recorded by diaries have a dominant 3 hours rhythm for all preterm babies. Sleep/wake behavior and staff/mother procedures observed for fullterm babies exhibit a circadian rhythm. Conclusions: Both actigraphy and sleep logs identified an ultradian pattern in rest/activity and sleep/wake cycle for preterm babies. Actigraphy revealed a more precocious circadian rhythm for this population (present at 34 weeks of GA) than sleep logs. Otherwise, both in sleep and actigraphy idata a circadian rhythm for fullterm babies was identified. Keywords: sleep/wake cycle, human, newborn

175

P054 CHARACTERIZATION OF THE SLEEP-‐WAKE CYCLE IN KINDERGARTEN CHILDREN THAT ATTENDED SCHOOL IN THE MORNING IN NATAL, BRAZIL 1Aline Silva Belísio, 1Fernanda Fernandes Kolodiuk, 1Jane Carla Souza, 1Zoélia Moura Bessa, 1Deyse Silva Bezerra, 1Geilson Lima Araújo, 1Ivanise Sousa Guimarães, 2Fernando Mazzilli Louzada, 1Carolina Macedo Azevedo. 1Universidade Federal do Rio Grande do Norte, Brazil. 2Universidade Federal do Paraná Brazil. Introduction: During the childhood occur biological changes in the sleep/wake cycle. These changes are influenced by social factors such as school starting time, and are compensated by behaviors usually performed at bedtime, called rituals of bedtime. Thus, the purpose of this study was to characterize the sleep-‐wake cycle of children that attended to school on the morning in kindergarten children. Methods: Participated 85 children that attended to school on the morning (40 boys and 45 girls), aged 4-‐6 years. The research was conducted in two stages: 1st -‐ meeting with parents, delivery of the consent forms and characterization of the habits of sleep, with the application of sleep habits questionnaire and economic classification, and 2nd -‐ characterization of patterns of sleep by sleep log by 7 days and observation of behavior in the classroom by 5 days (first observation: 07:35-‐08:20 a.m.; second observation: 09:45-‐10:35 a.m.). There were observed the frequency of yawning, rubbing the eye, stretching and stooping on the desk. The questionnaires were filled by parents. Results: The children performed the bedtime behaviors in different frequencies in the week. Most children performed the co-‐sleeping with their brothers and shared the room with parents less than two times a week. As long as children went to the bathroom and required the presence of parents in the room more than 2 times a week at bedtime (X2, p<0.05). It was observed that over the weekend, the children went to bed and woke up later. Furthermore, they had a tendency (test t, p=0.07) to spend more time in bed compared to week (test t, p<0.05). With regard to the nap, the frequency of children who took a nap and the duration of the nap decreased compared to week (X2, p<0.05). The children showed a higher frequency of yawning, eye rubbing and stretching the 1st observation (Wilcoxon, p<0.05). Moreover, the frequency of these behaviors varied depending on the activity performed on the 1st and 2nd observation, with attention to the activity that the children showed a higher level of sleepiness (Friedman, p<0.05). Conclusions: The kindergarten children showed signs of sleep partial deprivation related to extension and reduction of sleep associated to higher levels of sleepiness during week, probably due the social factors, for example, the school starting time. Acknowledgements: Capes and UFRN. Keywords: Sleep-‐wake cycle, Children, Sleep habits

176

P055 CHARACTERIZATION OF SLEEP-‐WAKE CYCLE, DAYTIME SLEEPINESS AND SLEEP QUALITY OF BRAZILIAN HIGH-‐SCHOOL TEACHERS Carolina Virginia Macédo de Azevedo, Jane Carla Souza, Aline Silva Belãsio, Zoília Camila Moura Bessa, Ivanise Cortez de Sousa Guimarães. Laboratorio de Cronobiologia, Departamento de Fisiologia, Programa de Pós-‐graduação em Psicobiologia, Universidade Federal do Rio Grande do Norte Natal/RN., Brazil. The teacher’s work schedule is differentiated from the majority of workers because they have work inside and outside the classroom. Thus, the excess of extra-‐class tasks can promote partial sleep deprivation which can compromise the health and damage the personal and professional life. Therefore the aim of this study is characterize the sleep-‐wake cycle (SWC), daytime sleepiness and sleep quality of Brazilian high school teachers. The research was conducted in 5 public schools and 5 private schools with 98 high school teachers (public= 50; private=48) of both sexes, aged between 27 and 57 years. Data collection was performed with the use of questionnaires in two stages: 1st -‐ "health and sleep", Epworth Sleepiness Scale (ESS) and the Index of Pittsburg Sleep Quality (IQSP), 2nd -‐ the characterization of patterns of sleep by sleep log for 14 days. The majority of the teachers were married (62%) with children (65%). During the week the teachers showed earlier bedtime and wake-‐up time, and a reduction in time in bed (+ 42 min). In addition, the wake-‐up time on Saturdays was earlier than Sunday, which suggests days of work until Saturday (ANOVA, p <0.05). The irregularity in the SWC was around 57 ±30 minutes for wake up time and 53 ±27 minutes for bedtime. Almost 46% of the teachers showed excessive diurnal sleepiness and 51% poor sleep quality (population mean score of IQSP= 6.4 ±3.0, X2; p > 0.05). Therefore, the teachers SWC is different between week and weekend, accompanied by a reduction in time in bed during the week, suggesting a partial sleep deprivation on working days. Moreover it is important highlight that half of the teachers were diagnosed with excessive daytime sleepiness and poor sleep quality, which can compromise the health and quality of life of these professionals, as well as causing damage in their performance in the school. Keywords: sleep-‐wake cycle, sleep quality, teachers

177

P056 EFFECTS OF 24-‐H SLEEP DEPRIVATION ON ATTENTION Juventino Cortez Diana Juárez, Aida García, Candelaria Ramírez, Pablo Valdez. Universidad Autónoma De Nuevo León, México. Sleep deprivation produces sleepiness and tiredness, as well as impairment in the performance of many activities. These effects on performance could be due to a decrease in attention, which is the capacity to respond efficiently to the environment. This cognitive process has four different components: tonic alertness, phasic alertness, selective attention and sustained attention. The objective of this work was to assess the effects of 24-‐h sleep deprivation on the components of attention. Participants were 7 undergraduate students (3 male, 4 female), mean age=18.12 ±1.24 (16-‐20) years. All participants kept a sleep diary during eleven days. They were registered in the laboratory the day before sleep deprivation at noon (12:00 h); spent the night without sleeping in the laboratory, and were registered the day after sleep deprivation at 06:00 h, 08:00 h, 10:00 h and 12:00. At all these intervals they responded a continuous performance task (CPT), designed to assess the components of attention. Participants slept 5:52 ±1:33 h the night before sleep deprivation. After sleep deprivation there were not significant differences among measures taken from 06:00 h to 12:00 h, so an average of these measures was used for the statistical analysis. Participants showed less efficiency after sleep deprivation in tonic alertness (before=98.78 ±0.94 correct responses, after=85.28 ±8.78 correct responses; T=0, p<0.05), selective attention (before=86.24 ±13.49 correct responses, after=67.59 ±14.43 correct responses; T=0, p<0.05), phasic alertness (before=94.70 ±5.06 correct responses, after=74.11 ±15.71 correct responses; T=0, p<0.05) and sustained attention (before=-‐0.23 ±0.14 linear regression of correct responses, after=-‐0.44 ±0.13 linear regression of correct responses; T=1, p<0.05). There were not differences in the reaction time of any of the components. In conclusion, 24-‐h sleep deprivation impairs all components of attention. These effects could explain the reduction in efficiency of many activities observed in sleep deprived persons. Keywords: sleep deprivation, attention,

P057 EFFECTS OF SLEEP REDUCTION ON THE COMPONENTS OF ATTENTION Diana Juárez, Martha Guerrero, Layla Arroyo, Juventino Cortez, Aida García, Candelaria Ramírez, Pablo Valdez. Universidad Autónoma de Nuevo León, México. Most of the people living in cities reduce their sleep during workdays, compared to weekends. Sleep reduction produces sleepiness, tiredness and a decrease in school and

178

working performance. The decrement in performance could be due to a lower level in a basic cognitive process, such as attention. This cognitive process has four components: tonic alertness, phasic alertness, selective attention and sustained attention. The objective of this study is to determine the effects of sleep reduction on the components of attention. Participants were 32 undergraduate students, (age = 18.03±1.23 years, mean ± standard deviation). They were divided in two groups: an Unrestricted Sleep Group (USG, N=21) and a Restricted Sleep Group (RSG, N=11). The USG slept free and was registered in the laboratory at noon (12:00). The Restricted Sleep Group arrived to the laboratory at 06:00 h and was registered at noon (12:00 h). The night before the laboratory session, the USG slept 10:15±1:15 h, while the RSG slept 5:04±0:19 h. The sleep reduction group showed lower efficiency in tonic alertness (correct responses USG=99.03±0.82%, RSG=96.03±4.08%, U=50, p<.01), and longer reaction times in tonic alertness (USG=366.87±77.43 ms, RSG=431.00±70.16 ms, U=60, p<.05), selective attention (USG=455.16±66.87 ms, RSG=551.26±67.70 ms, U=27, p<0.001) and phasic alertness (USG=374.24±68.95 ms, RSG=453.48±50.08 ms, U=20, p<0.001) compared to the unrestricted sleep group. There were no significant differences in sustained attention. In conclusion, sleep reduction produces a lower level of tonic alertness and an increase in reaction time. These effects could explain the decrease in school and working performance. Keywords: sleep reduction, attention,

P058 EFFECTS OF SLEEP DEPRIVATION ON WORKING MEMORY Jacqueline Del Ángel, Ana Gabriela Iracheta, Diana Juárez, Juventino Cortez, Aida García, Candelaria Ramírez, Pablo Valdez. Universidad Autónoma de Nuevo León, México. Sleep deprivation affects performance of many activities. Working memory is crucial for performance; this cognitive process is the capacity to maintain information for brief periods (seconds). It has two storage components: phonological, essential for verbal information processing, and visuospatial, essential for visual information processing. The objective of this study was to analyze the effects of sleep deprivation on the components of working memory. Participants were 10 undergraduate students, age 17-‐18 years (mean = 17.5 ± 0.53 y). They responded phonological and visuospatial tasks at 20:00 h and 06:00 h, during this period they stayed awake all night in the laboratory. The phonological storage component showed lower percentage of correct responses at 06:00 h (20:00 h = 86.64 ± 1.99 %, 06:00 h = 47.76 ± 9.46 %; T = 0.00, p < 0.01). Also, the visuospatial storage component showed at 06:00 h, lower percentage of correct responses (20:00 h = 83.66 ± 3.03 %, 06:00 h = 69.08 ± 5.43 %, T = 7 p < 0.05) and higher reaction times (20:00 h = 901.88 ± 47.49 ms, 06:00 h = 1042.85 ± 42.55 ms, T = 8 p< 0.05). Sleep deprivation affects the phonological and

179

visuospatial components of working memory, crucial for verbal processing required to reading, and visual information processing required for driving a car or solving arithmetic problems. Keywords: Sleep deprivation, Working memory,

P059 APPLY TCM CHRONOBIOLOGY TO REGULATE SLEEP RHYTHMS Feng Xiu Jie, Zhuang Hong Yan. Beijing An Ding Hospital, Capital Medical University Beijing, China. Poor sleep and sleep rhythm obstacle are prominent clinical manifestations of various degrees of mental disorders. They embody the disorder of biological rhythm, and reflect the corresponding brain spirit and spirit disorders of viscera function. The essence of sleep and sleep rhythm obstacle is the pathological evolution of viscera unionizing and the ups and downs of Yin and Yang. It reflects the flowing, reflowing, speed and cycle of Qi, pathological circumfluence, Zang-‐fu organs and meridians. It forms Viscera of Yin and Yang, unionizing ups and downs that the macro and micro of the cyclical of syncretic and viscera activities, the timeliness of life activities and spatial. Adjusting sleep is the basic method and key measure for psychiatric to aftercare brain. Conforming to and respect of the zang-‐fu organs and physiology characteristics are key point by adjusting the viscera to improve sleep (circadian) for the treatment of various spiritual obstacle. We should emphasize the balance of Yin and Yang, use of Chinese and western medicines and other means flexibly. Keywords: Poor sleep and sleep rhythm ob, the treatment of mental disord, viscera operation

P060 SLEEP HABITS, CHRONOTYPES AND OBESITY/OVERWEIGHT IN MEDICAL STUDENTS AT UNAM. 1Eduardo González, 2Donají Heredia, 2Carolina Escobar. 1Departamento de Salud Pública, Facultad de Medicina UNAM., México. 2Departamento de Anatomía, Facultad de Medicina UNAM., México. Adolescents and young adults have the capacity and the preference of staying up late at night and reducing the sleep hours. Recent studies indicate that short sleep leads to metabolic disturbance and propensity to overweight and obesity. Poor sleep is a common feature among medical students and a high incidence of overweight and obesity is reported among medical professionals. The aim of this study was to examine the chronotypes, sleep

180

habits (quantity and quality) of medical students inscribed in the first two years in the Medical Faculty and to explore the association between obesity/overweight and sleep habits. We used a cross sectional design at the Faculty of Medicine UNAM, Mexico City. We assessed sleep quality with the Pittsburgh Sleep Quality Index (PSQI) and chronotypes with the Horne and Östberg questionnaire. We calculated the Body Mass Index (BMI) by auto-‐report of weight and height. We applied 1006 questionnaires and obtained 966 complete questionnaires for sleep quality, 984 for chronotypes and 986 for height and weight auto-‐report. The median of the subjective quantity of sleep hours during the week days was 5 hour for students in the morning groups and of 6 h for the afternoon schedules. With the PSQI we obtained a proportion of 73.9% bad sleepers. Men had lesser “bad sleepers” than women, 67.6% vs. 76.8% respectively (p<0.05) and the afternoon schedule have lesser bad sleepers than the morning students, 61.3% vs. 80.7% respectively (p<0.05). The more prevalent chronotype was the intermedium(76.8%), then the morning (16.2%) and finally the evening one (7.1%). There was a difference by sex, the proportion of women with early chronotype was 17.9% and that of men was 12.4% (p<0.05). The mean BMI was 23.25 kg/m2(+ 3.33 S.D.) Only 3.8% of the students had a BMI below normal, 71.9% was normal, 20.4% was overweight and 4% indicated obesity. We observed that early schedule students had lesser proportion of overweight/obesity than those of late one19.8% v.s. 28%, (p<0.05). There was an association between chronotypes and obesity/overweight (p = 0.003). Conclusion: medical students have poor sleep quality and it’s poorest in women and early schedule. The chronotype was associated with overweight/obesity. Supported by CONACyT 82462 and PAPIIT IN224911. Keywords: chronotypes, sleep, students

P061 DETECTION OF CHRONO PREMETABOLIC SYNDROME IN DIFERENT MEXICAN POPULATIONS 1Salvador Sánchez-‐de la Peña, 2Franz Halberg, 1Jonathán Levi Rito-‐Medina, 1Irene Mendoza-‐Lujambio, 2Germaine Cornélissen, 1Alfonso López-‐Fiesco. 1Escuela Nacional de Medicina y Homeopatía-‐IPN-‐FICRÓN, México, 2Universidad de Minnesota, EUA. Introduction. Five years ago the cost of health care for expected cases of type 2 diabetes and hypertension in Mexico had reached over $140,000,000. There have been many efforts to reduce both chronic diseases, but still a long way to go. Among attempts to reduce the prevalence of hypertension and diabetes have been developed preventive sequences: pre-‐diabetes and pre-‐hypertension. Since such chronic pathologies conduced to renal and cardiovascular irreversible damages and death. Under a chronomic perspective the study of temporal structure or chronome of blood pressure (BP) or glucose metabolism is possible to detect circadian patterns, variable vascular disorders (VVD) or syndromes (VVS). We had

181

found in normal healthy persons circadian hyper amplitude tension (CHAT) and intolerance to glucose (hyperglycemia). Both factors constituted a pre-‐metabolic syndrome. Now after confirmed in different age and gender groups, it has been defined as chrono-‐pre-‐metabolic syndrome (CPMS), to differentiate from classical health lineal approaches related to the study of the metabolic syndrome itself. Methodology. At the Chronomic Research Center (CRC) of Escuela Nacional de Medicina y Homeopatia-‐IPN was the reception center of data coming from ten different populations (P) s where was mapped the presence of CHAT and CPMS. P1) Guadalajara, JAL; P2) Chetumal, Qroo; P3) Mixcoac; P4) Tacubaya; P5) Ticomán; P6) Tlacojalpan, Ver; P7) Los Cabos, BC; P8) CD. Nezahualcoyotl en DF; P9) San Ignacio en BC y P10) Ciudad Mante Tamps. A total of 450 subjects of both genders gave the consent approval to participate in the present study. In each place special training of BP monitoring was performed with ambulatory automatic instruments (A&D Japan Co.) programmed at 30 and 60 min intervals during day-‐night, respectively during 2 to 7 consecutive days. And was complemented with glucose tolerance test (GTT) test performed with digital glucometer. Circadian rhythm of each subject was detected least-‐squares fit of a 24h cosine. Rhythm detection was considered significant if p<0.05. It was proposed that the presence of CHAT of the mexican double Amplitude was defined at 28 mm of Hg. Results. It was observed circadian rhythm of systolic (S) BP over 70% of studied populations. Incidence of CSPM showed in P1 (5.26%); P2 (7.32%)P3(10.6%); P4(8.82%);P5(19%);P6(22.22%);P7(30.04%);P8(11.76%);P9(12%) y P10(4.17%). Discussion. Detection of CHAT and CPMS seems to be useful chronomic predictive tools that must be wide extended in preventive and general medicine, in such manner that might reduce the progress to irreversible cardio and metabolic syndromes ending in cardio-‐renal vascular damages. Acknowledgment: ICyT-‐DF Grant: PICDS08-‐82. Keywords: chrono-‐premetabolic syndrome, hypertension, diabetes

P062 SLEEP COMPLAINTS AND ITS RELATIONSHIP TO DAYTIME SLEEP AND ACTIGRAPHIC PARAMETERS IN CIRRHOTIC PATIENTS: AN EXPLORATORY STUDY 1Montserrat Concepción Reséndiz Garcìa, 1Aldo Torre, 1Violeta Alejandra Castaño Meneses, 1Andrés Duarte Rojo, 1Maria Victoria Santiago Ayala, 1Demian Gil Aldeco, 2Arturo Vega González, 1Guillermo Garcìa Ramos, 1Matilde Valencia Flores. 1Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubiran, México. 2Instituto de Fisiología Celular, UNAM, México. Several sleep disorders have been reported in cirrhosis patients. There is only one report regarding daytime sleepiness in this type of patients. Actigraphy is a method that utilizes a miniaturized computerized wristwatch-‐like device to monitor and collect data generated by body movements and allows indirect estimation of sleep and wakefulness based on motor

182

activity over extended period of time. The aim of this exploratory study was establish the relationship between sleep complaint, daytime sleep and circadian parameters of sleep-‐wake behaviors. Methods In total sixteen consecutive female outpatients with cirrhosis from the Gastroenterology Clinic at a tertiary Health Institute in Mexico City were included. Patients were excluded if they were under medication, had work conditions or psychiatry problems that could affect sleep-‐wake cycle. The patients completed the following questionnaires: personal and demographic data, sleep habits and sleep disorders questionnaire, Epworth Sleepiness Scale (ESS) and Beck Depression Inventory(BDI). Actigraphs were programmed by zero crossing method and they recorded seven consecutive days for all patients who completed sleep-‐logs simultaneously. Results The mean (SD) age was 52±11 years old and the Body Mass Index was 25.8±7.4 Kg/m2. Self-‐reported sleep time was 6.05 hrs, and 73.3% of the patients reported taking naps. The majority (63.6%) considered that they slept more than a person of the same age. The EES mean score was 10±6 and for BDI was 18±11. All patients reported nocturnal sleep problems, the major complaint was ‘snoring’ (87.5%), following for 73.3% with ‘difficulty for onset sleep’, 43.8% ‘waking up early’ 12.5% ‘having nightmares’. Daytime sleepiness was present in 62.5%. There were significant correlations between ‘difficulty for onset sleep’ and ‘daytime sleepiness’ (rho= 0.53, p=0.04) and ‘nightmares’ (rho=-‐0.66, p=0.008). The circadian parameters of sleep-‐wake behaviors measured by actigraphy were: Total Sleep Time 7.1±2.3 hrs, Sleep Latency 13.2±7.5 (min), Sleep Efficiency 80.5±11.8%, Nocturnal Wake Time 98.2±47.4 (min), Daytime Sleep 35.1±31.6 (min) and Nap-‐Sleep Efficiency 64.4±28.8%. Cosinor analysis showed that the period was 23.9±0.3, mesor 111.8±21.3, acrophase 15.75±1.8 and the amplitude was 79.3±18.9. There was a negative correlation between ‘difficulty for onset sleep’ and amplitude parameter (r=-‐0.53, p=0.04). Conclusion Cirrhotic patients present nocturnal sleep complaints, over 50% complain of daytime sleepiness. Patients underestimate the total sleep time. Objective actigraphic data showed poor sleep efficiency in both, nocturnal and nap sleep. Interestingly, the complaint of ‘difficulty for onset sleep’ negatively correlated with the amplitude of the rhythm, suggesting that the subjective report of sleep difficulty may be related to a circadian alteration of the amplitude of the sleep-‐wake cycle. Keywords: cirrhosis, sleep, actigraphy

P063 DETERIORATION OF SLEEP QUALITY AMONG IN HABITANTS OF CHHATTISGARH Ms. Chaynika Nag, Dr. R. K. Pradhan. Pt Ravishankar Shukla University, Raipur, India. Internal time keeping machinery of living organism is naturally set to do work during daytime and to sleep at night. However the biological clock can be affected by our sleep-‐

183

wake patterns and other factors. Aim of this present study was to evaluate sleep-‐wake pattern, prevalence of sleepiness and sleep deterioration among human populations with different socio-‐economic status and habitat. Prevalence of sleepiness was measured with ESS scale through self reported questionnaire from 2105 subjects of three distinct populations living in urban, rural (electrified villages) and remote area (villages without electricity) of Chhattisgarh, India. Sleep parameters of persons having excessive sleepiness during daytime (EDS subjects) were assessed by Mini-‐Mitter Actiwatch (AW64, Mini Mitter Co. Inc., USA) continuously for four days. Actiware sleep software version 3.0 and excel tool pack was utilized for statistical analysis of data. Result shows that peoples, irrespective of habitat and socioeconomic status experiencing excessive sleepiness during day time spent significantly less time in bed (TIB) and have poor sleep efficiency (SE) with higher level of fragmented sleep (FI) as compared to non-‐EDS peoples (control subjects). Further, comparison of fragmentation index in different population as a measure of sleep deterioration amply suggests that the sleep quality of peoples living in urban area is deteriorated and highly fragmented. Keywords: Actigraphic study, Epworth Sleepiness scale, Excessive Daytime Sleepiness

P064 RELATIONSHIP BETWEEN SOCIAL RHYTHM, SLEEP PHASE AND MINOR PSYCHIATRIC SYMPTOMS IN HEALTHY WORKERS Regina Lopes Schimitt, Rosa Levandovski, Maria Paz Loayza Hidalgo. Laboratório de Cronobiologia-‐HCPA-‐UFRGS, Brazil. Social rhythm is a behavior resulting from the regular exposure to social zeitgebers which are defined as exogenous synchronizers of biological rhythms. The objective of the present research was to determine the relationship among social rhythm, Münich Chronotype Questionnaire (MCTQ) variables and minor psychiatric disorders in healthy workers. The investigation was designed as a cross-‐sectional study involving 143 regular healthy workers from Clinical Hospital. Minor psychiatric disorder was assessed by the validated version of SRQ-‐20, and social rhythm was assessed by the Brazilian version of SRM-‐17 instrument. Each question of MCTQ was analyzed as a independent variable: social jetlag was calculated by the difference in hours of the mid-‐sleep between free and work days. MSF, the mid-‐sleep phase on free days, is the mid point of sleep on free days evaluated by local time, and MSFsc, the mid-‐sleep phase on free days is than corrected for the sleep deficit accumulated during the work-‐week. The assimetric variables were transformed by square root. The correlation was analyzed by Pearson and to control colinearity among variables and potential counfoundigs effects, was used Multivariariate Regression analysis. In univariate analysis, the variables that showed correlation with SRM were age (r=0.34; p=<0.01); Sleep duration in free-‐days (-‐0.25; p<0.01), MSF (r=-‐0.34; p<0.01); MSW (-‐0.16; p=0.05); sleep

184

duration (0.17; p=0.04) MSF-‐scn (-‐0.17; p=0.05); Sun light exposure in free-‐days (0.25;p<0.01); Social jet lag (-‐0.23,p<0.01) SRQ (-‐0.17; p=0.04). In the multiple regression, the variable that were maintained in the model were MSF (β=1.4; p 0.05), Sleep duration in Work days (β=2.1; p=<0.01), PMSW (β=-‐2.1;p=<0.01) and Sun light exposure in free days (β=0.18; p=0.04). The model presented a R2 =43.8%. Moreover, ALI were correlated to schooling (r=0.23;p<0.01); MSF (r=-‐0.17;p=0.05); MSW (r=-‐0.18;p=0.04); Sleep duration (r= 0.2;p=0.02); SRQ-‐20 (r=-‐0.27p=0.01). In multiple regression only SRQ-‐20 (β=-‐0.27; p=0.01) was maintained in the model. The model explained 13% of the variance. Its was no correlation between ALI and SRM. The variables that explain the regularity of the activities were related to the phases and the duration of sleep and Light exposure only in free days. The concept of social rhythm has been used as a possibility to increase quality of life and Heath in particular mood disorder. In this study, in spite the limitations of a cross-‐sectional study, we only observed the relation with minor psychiatric disorder in the univariate analysis. When the confounding and colinearity were control, using multivariate analysis, this relation disappear. SRQ-‐20 was only related with the quantity of activities showed a inverse relationship. Also the regularity of activities was explained by sleep variable and sunlight exposure in 43.8% which was a higher perceptual of variance. Keywords: social rhythm, minor psychiatric disorder, MCTQ

P065 DECREASE OF EVENING PLASMA PROLACTIN IN THE DELAYED SLEEP PHASE SYNDROME. IS A HYPERTONIC DOPAMINERGIC SYSTEM INVOLVED? Bruno Claustrat, Helène Bastuji, Laure Peter-‐derex, Thierry Petitjean, Françoise Borson-‐Chazot, Jocelyne Brun. Hospices Civils de Lyon; Universté Claude Bernard, France. Introduction: The role of dopamine in the regulation of the sleep-‐wake cycle needs clarification. The nigrostriatal pathway participates to the maintenance of behavioral arousal, and to cognitive process and selective attention. Also, the regulation of the prolactin (PRL) secretion is mainly mediated by the tubero-‐infundibular pathway. Some studies, however, have linked PRL regulation to striatal dopaminergic activity. In humans, the study of the 24h plasma PRL profile in the Delayed Sleep Phase Syndrome (DSPS) could give an insight into the involvement of dopamine in both pathophysiology and regulation of the sleep-‐wake cycle. Subjects and Methods: the 24h patterns of plasma melatonin, PRL and cortisol levels, temperature and actimetry were studied in six 14–28 year-‐old male DSPS patients, and seven healthy matched controls, neither morning, nor evening type. In order to detect time and group effects (controls/patients) and interaction, plasma PRL concentrations were submitted to a two-‐way ANOVA for repeated measures, followed by multiple comparisons. Results: as expected, the temperature and melatonin profiles were

185

phase delayed, compared with controls (mean temperature nadir at 06:26 and 04:07 and mean melatonin onset at 00:37 and 21:00 respectively), whereas cortisol profiles did not displayed major delay. Individual PRL profiles revealed very low, indeed even undetectable levels in the evening, and a quick rise with sleep onset. ANOVA performed on data gathered in 3 hour blocks showed a time (p=0.004) and a group effect (p=0.007), without interaction (p=0.128). Also, patients PRL levels were decreased at 20-‐22h (p<0.05), 23-‐1h (p<0.05) and 2-‐4h (p<0.01), compared with controls. Conclusion: plasma PRL profiles observed in the DSPS could reflect an evening dopaminergic hypertony, with consequence the maintenance of late wakefulness. Finally, since an interaction between dopamine and clock genes has been showed, the global hyperdopaminergic climate could contribute to reinforce the clock delay. Keywords: prolactin, DSPS, dopamine

P066 PHARMACOLOGICAL TREATMENT ASSESSMENT WITH MARTAZAPINE ON SLEEP QUALITY OF GERIATRIC PATIENTS WITH MAJOR DEPRESSION: AN ACTIGRAPHY TEST 1Betty Marjorie Rothschild-‐Fuentes, 2Jairo Muñoz-‐Delgado, 3Alejandro Jiménez-‐Genchi, 2José Carlos Sánchez-‐Ferrer, 1Andrés Roche-‐Bergua. 1Psychogeriatrics Services, Hospital Psiquiátrico Fray Bernardino Álvarez, México. 2Chronoecology and Human Ethology Group, Instituto Nacional de Psiquiatría Ramón de la Fuente, México, 3) Sleep clinic, Instituto Nacional de Psiquiatría Ramón de la Fuente, México. INTRODUCTION: Depression in Mexico has an overall prevalence of 9.5% in females and 5% in males older than 60 years. In the elderly, depression can be disguised by somatic symptoms as insomnia, which has a much higher prevalence in depressed people with percentages beyond 60% and about 80% of the depressed individuals undergo some changes in their sleep quality and quantity. Mullaney showed that the data obtained by actimetry had a high accuracy degree as compared with the polysomnography measurements with advantages such as cost, easy management and use outside the laboratory. Some antidepressants with hypnotic effect, such as Mirtazaprine, have been used to improve the depressed patient’s sleep pattern. OBJECTIVE: To assess the changes in sleep quality before and after a pharmacological treatment with Mirtazapine in geriatric patients with major depression. PROCEDURE: It is a prospective study of association. 10 outpatients in Fray Bernardino Alvarez Hospital have been selected, 9 women and 1 man. The patients agreed to participate in the study. Inclusion criteria were the following: minimum age -‐60 years old, literate, with Major Depressive Disorder according to DSM-‐IV-‐TR, and having not received antidepressant treatment in the previous three months. Patients with psychotic symptoms, diagnosis of dementia according to DSM IV-‐TR, or clinical

186

diagnosis of Delirium have been excluded. RESULTS: Results obtained as for the depression degree before and after the treatment (Z=-‐2.80; p=0.005). It was found that daily activity after the treatment displayed significant differences (F(1,18)=7.56;p=0.013). The analysis by age groups also presented differences. Activity in patients aged between 60 and 69 increased. There was no change in patients between 70 and 79 years old and patients aged between 80 and 89 showed an activity decrease (F(2,18)=138.09;p=0.000). The results of the sleep parameters measured with actimetry were individually analyzed. Sleep latency after treatment revealed no significant difference (F(1,18)=0.175;p=0.68). Analysis of real time sleep after treatment displayed differences (F(1,18)=20.88;p=0.000). Likewise, sleep efficiency before and after treatment presented differences (F(1,18)=19.334;p=0.001. Results obtained from the number of awakenings before and after treatment also revealed differences (F(1,18)=10.465;p=0.005). Finally, the sleep fragmentation index after treatment showed a difference of (F(1,18)= 37.76;p=0.000). CONCLUSIONS: Mirtazapine is a useful medication for the elderly people’s depression treatment also associated with improvement in sleep subjective parameters that are correlated with objective measurements by actigraphy. Keywords: actigraphy, sleep parameters, depression in elderly

P067 GLYCEMIC CONTROL AFFECTS SLEEP INITIATION AND SLEEP QUALITY IN T1DM 1Mark Thomas Ugliara Barone, 2D R Franco, 3M K Carra, 3Fabiola Schorr, 3Geraldo Lorenzi, 4Luiz Menna-‐Barreto. 1Instituto de Ciencias Biomedicas da Universidade de São Paulo (ICB-‐USP), Brasil. 2Associação de Diabetes Juvenil (ADJ), Brasil. 3Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo (InCor-‐FMUSP), Brasil. 4Escola de Artes, Ciencias e Humanidades, Universidade de São Paulo (EACH-‐USP), Brasil. Barone, MTU1,2; Franco, DR2; Carra, MK3, , Schorr, F3, Lorenzi, G3. and Menna-‐Barreto, L4. The association of sleep impairment and diabetes has been widely studied in the last years. Some causes and effects have already been identified, e.g. obstructive sleep apnea leading to obesity and inflammation, contributing to the development of type 2 diabetes. Our objective in the present study was identifying possible associations of mean glycemia (MG) and glycemic variability (GV) with sleep patterns. Data was collected from 18 subjects with type 1 diabetes (T1DM), without complications, during 10 consecutive days. The subjects filled out a Sleep Diary and performed 6.41±1.5 (mean ± SD) tests of glycemia per day. The standard deviation of the glycemia is considered here as GV. The correlation between MG and GV was r=0.73 (p<0.001). Sleep latency showed to be positively correlated with GV (r=0.65, p<0.004), and with MG (r=0.60, p=0.013). Splitting the subjects in two groups according to their MG or GV, we observed that the ones with lower MG (below 154mg/dL)

187

or lower GV (SD<73.9mg/dl) presented significantly shorter sleep latency duration (KW-‐H, p=0.024 and p=0.036 respectively). Whereas no significant correlation is observed between sleep quality and MG or GV, considering only the individuals with lower MG or GV, a negative correlation and a tendency appear (r=-‐0.85, p=0.004; r =-‐0.66, p>0.05, respectively). Thus, our data show that subjects with higher MG also present higher GV. Besides that, the time individuals with T1DM take to sleep is correlated with their glycemic control. At the same time, it seems that among the ones with better glycemic control, the lower their MG or GV, the better their sleep quality. Although there is still much to be elucidated about the influence of diabetes on sleep, and vice-‐versa, our study is the first one to show the correlation between T1DM mean glycemia and glycemic variability and sleep latency and quality. Keywords: diabetes, sleep/wake cycle, glycemia

P068 THE ASSOCIATION AMONG SLEEP QUALITY AND MID-‐SLEEP PHASE WITH USE OF ANTIHYPERTENSIVE DRUGS 1Rosa Maria Levandovski, 2Till Roenneberg, 2Karla Viviani Allebrandt, 3Maria Paz Loayza Hidalgo. 1UFRGS, Faculdade de Medicina, PPGCM, Laboratório de Cronobiologia do Hospital de Clínicas de Porto Alegre, Brazil, 2Ludwig-‐Maximilians-‐University of Munich, Department of Medical Psychology, Germany. 3 UFRGS, Faculdade de Medicina, Departamento de Psiquiatria -‐Laboratório de Cronobiologia do Hospital de Clínicas de Porto Alegre, Brazil. Hypertension is one the most important clinical disorders in public health and has become a global epidemic disease. It is also a risk factor for a series of pathological conditions such as cardiovascular diseases, which are responsible for 17 million deaths per year (WHO). Recent studies have indicated that low sleep quality is a risk factor for adult hypertension. Objective: The objective of this study was to investigate the association of sleep quality and mid-‐sleep phase with the use of antihypertensive drugs. Design: Cross-‐sectional study. Participants: A total of 1047 subjects (351 men and 696 women) with an average age of 44 ± 12 years, living in a essentially rural area in south of Brazil (latitude 30). Assessments were performed in a period of one year. The population is a community of German descendants, significantly homogeneous in terms of culture, socio-‐economic level, biological factors, and daily exposure to environmental light. Main outcome measures: Sleep quality was evaluated using the Pittsburgh Scale (PSQI), and mid-‐sleep phase using the Munich Chronotype Questionnaire (MCTQ). An antihypertensive drug was measured by self-‐reported information through the use of standard drugs according to pharmacological ATC codes (adrenergic receptor agonists and antagonists, calcium channel blockers, beta blocking agents and diuretics). Results: In this study, 20.4% (N = 214) of the participants reported use of antihypertensive drugs. Subjects using antihypertensive drugs showed

188

significantly lower mid-‐sleep phase (t test = -‐ 4.82: p< 0.0001), poor sleep quality (Man Whitney test; p< 0.0001), and higher BMI (t test 10.82; p<0.0001), and age (t test 14.69; p<0.0001) than those not using antihypertensive drugs. There were no difference between genders (chi-‐square; p=0.224), smoking status (chi-‐square; p=0.487), and sleep duration on free (t test -‐1.36: p=0.174) and work days (t test 0.35: p=0.582). A multilinear regression model was used to control colinearity and potential confounding factors with self related antihypertensive drugs as a dependent variable. BMI, PSQI and mid-‐sleep phase were independent factors predicting the use of hypertension drugs (r2 = 0.25, F = 57.60, p<0.0001). Conclusion: In this population we found a higher prevalence of the use of antihypertensive drugs, which points to the probability of an over diagnoses of hypertension. In spite of the fact that this was a cross-‐section study, there was a clear association of poor sleep quality, and an advanced mid-‐sleep phase with the use of antihypertensive drugs. This work was supported by PROBRAL/CAPES/Brazil, PNPD/CAPES, FIPE/HCPA, UFRGS (for M.P.H., R.M.L., and G.D.), UNIVATES (undergraduate scholarships), EUCLOCK and DAAD/Germany (KA,TR).

P069 THE IMMOBILITY EPISODES OF TAIEP RATS HAVE ULTRADIAN EXPRESSION 1Ma. del Carmen Cortés, 1Manuel Lara, 1,2José R. Eguíbar. 1Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, México, 2Secretaría General. Benemérita Universidad Autónoma de Puebla, México. Taiep rat is a myelin mutant with a progressive motor syndrome with tremor, ataxia, immobility episodes, epilepsy and paralysis along the first year of life. All the symptoms are due to a hypomyelination followed by demyelination, caused by an abnormal accumulation of microtubules in oligodendrocytes. During immobility episodes (IEs) polysomnographic recordings, the cerebral cortex was desynchronized associated with theta rhythm in the hippocampus suggesting REM sleep-‐like pattern and disorganized sleep-‐wake cycle pattern that supports taiep rats as a model of narcolepsy-‐cataplexy. In this study, we analyze the circadian rhythm of induced and spontaneous IEs in 8-‐month-‐old male taiep rats. The animals were housed under a 12:12 light-‐dark cycle (lights on at 0700 hrs) and free access to rodent pellets and water. The subjects were implanted for EEG and EMG with electrodes and fixed to the cranium with screw sand dental acrylic. EEG/EMG sleep-‐wake patterns were recorded under normal light-‐dark (LD) conditions. The immobility was induced gripping-‐it at the base of the tail. The spontaneous IEs were recorded during the 24-‐h couples to videocamera. Our results showed that IEs start at 6.5 with a peak frequency between 8.5 and 9.5 months. IEs have two peaks, one in the morning (0800–1000 h) and a second peak in the middle of the night (2300–0100 h). However, spontaneous IEs are evenly distributed throughout the circadian period with a mean frequency of 3 IEs every 2 h. In conclusion, the gripping-‐induced IEs showed an ultradian rhythm suggesting that the

189

excitability of the ensemble of neurons that triggers REM sleep and atonia oscillates along the day, but there is not the case with spontaneous IEs in which such oscillation does not occur. This research was supported by Dr. E. Agüera-‐Ibañez Rector-‐BUAP, VIEP-‐BUAP SAL/G/2010, CONACyT 106694 grants to JRE. ML is fellowship from CONACYT No. 229855. Keywords: sleep, narcolepsy, orexins

P070 THE SPIKE-‐WAVE DISCHARGES IN THE MYELIN MUTANT TAIEP RAT HAVE A CIRCADIAN RHYTHM 1Jackeline Corona, 1Ma. del Carmen Cortés, 1,2José R. Eguíbar. 1Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, México, 2 Secretaría General. Benemérita Universidad Autónoma de Puebla, México. The myelin mutant taiep rat show an initial hypomyelination followed by a progressive demyelination. The name is the acronym of the motor syndrome that characterized them: tremor, ataxia, immobility, epilepsy and paralysis. These mutants developed spike-‐wave discharges (SWD) similar to that showed in absence seizures, starting at 3 months of age and progressively increased to 12 months of age. All rats were maintained under standard conditions with 12:12 light: dark cycle (lights on 0700). Rats were implanted using standard conditions for chronic recordings of the EEG and simultaneously video filmed using the Harmonie system (Canada). During 24 h continuous recordings showed that the frequency of absence seizures is stable along the circadian cycle with a mesor of 68 ± 2.2 spike-‐wake discharges per hour. However the majority of them occurred in the awake period, less discharges happen in the slow-‐wave sleep and only few during rapid eye movement (REM) sleep (H= 243.2; p<0.001, followed by Dunn´s test, P<0.05). Importantly, the mean duration of the SWD is higher in the dark phase with 6.0 ± 0.25 s respect to the light phase with just 4.9 ± 0.15 s (ANOVA F(1)=30.1; P<0.001, followed by Holm-‐Sidak test, P<0.05). In conclusion, the hyperexcitability in the thalamo-‐cortical circuit in taiep rats is similar along the circadian cycle, but not its mean duration. The relationship among awakenings with SWD showed that during this phase the excitability of the circuit is higher than during sleep phases. Partially supported by CONACYT No. 106694 and VIEP-‐BUAP No. SAL/G/2011 grants, and by Dr. E. Agüera-‐Ibañez, Rector BUAP. JCC is fellowship of CONACYT No. 348846. Keywords: Absence, Epilepsy, Sleep

190

P071 DEPENDENCE ON TIMING OF 2-‐AG ADMINISTRATION INTO THE LATERAL HYPOTHALAMUS TO MODIFY THE SLEEP WAKING CYCLE OF RATS Mauricio Marcel Pérez-‐Morales, Seraid Caynas, Ilia Alvarado, Mónica Méndez-‐Díaz, Oscar Prospero-‐García. Laboratorio de Canabinoides, Departamento De Fisiología, Facultad De Medicina, UNAM, México. The lateral hypothalamus (LH) has been suggested to induce waking and arousal. Several studies have described the existence of a pair of neuropeptides located in the LH, in intermingled separated populations of neurons: orexins and melanin -‐concentrating hormone (MCH). Initially, both peptides were identified as enhancers of food intake. Later, it was suggested that the neurons that synthesized these peptides influence differentially the regulation of the sleep-‐waking cycle (SWC). In addition, it has been demonstrated that orexin knockout mice exhibit a sleep pattern strikingly similar to the one exhibited by narcoleptic patients, suggesting that this neuronal lineage is somehow necessary to promote and maintain wakefulness (W). By means of immunohistochemistry approaches it has been demonstrated that orexinergic neurons are active during W, and that MCHergic neurons are active during rapid eye movement sleep (REMs), in rats. By means of neuronal unit activity approaches, it has been shown that orexinergic neurons discharge during W, and are silent in both slow wave sleep (SWS) and REMs, in freely moving rats, and in head-‐fixed rats; and that MCHergic neurons discharge in REMs, discharge moderately in SWS, and are silent in W. Additionally, pharmacological studies have demonstrated that orexins increase W and decrease both SWS and REMs; whereas MCH increases both SWS and REMs, thereby reducing W, when administered icv, into rats. On the other hand, several studies support the participation of endocannabinoids (eCBs), the endogenous ligands to cannabinoid receptors (CB1, CB2), in the regulation of sleep. The systemic administration of SR141716A, a CB1 antagonist, increases W and decreases both SWS and REMs, in rats; icv administration of arachidonoyl ethanolamide (ANA) or oleamide (OLE), some of the most studied eCBs, increase REMs, in rats, as well as the intrahippocampal administration of ANA. Complementarily, there is evidence that WIN55 212 2, a synthetic CB1 agonist, depolarizes MCHergic neurons and hiperpolarizes orexinergic neurons in vitro, suggesting that eCBs could interact with MCH and orexins, locally into the LH, and in this way regulate sleep. 2-‐arachidonoylglycerol (2-‐AG) is another one of the most eCBs studied, but it has not been studied so far in relation to SWC. In the present study we explored the effects of the administration of 2-‐AG, a CB1 agonist, directly into the LH, on the SWC of rats: a) at the beginning of the Light Phase (LP) of the cycle, when rats spend more time asleep, and b) at the beginning of the Dark Phase (DP) of the cycle, when rats spend more time awake. The results indicate that 2-‐AG increases REMs when injected into the LH at the beginning of the

191

DP, but not when injected at the beginning of the LP of the SWC, suggesting that the activation of the CB1 into the LH could interact with MCH neurons and orexinergic neurons, in a cycle dependent manner. Also these results support the notion that eCBs are key modulators of the mechanisms involved in REMs. This work was supported by Grant number IN212908 from DGAPA-‐UNAM to OPG.

P072 DIFFERENCES IN SLEEP AND DAILY METABOLIC PROFILES BETWEEN OVERWEIGHED AND NORMAL MICE NEOTOMODON ALSTONI Citlalli Fuentes Granados, Pilar Durán, Manuel Miranda Anaya. Universidad Nacional Autónoma de México, Facultad de Ciencias, México. Obesity is a public health problem of great importance in emerging countries, related to other disorders such as metabolic syndrome (MS), which leads to cardiovascular problems and type II diabetes. The causes of obesity are complex and recent research has found a link between the circadian clock, sleep patterns and energy balance. The volcano mouse (Neotomodon alstoni) is an endemic rodent from Mexican Neovolcanic Transversal belt. This species, in captivity and fed regular laboratory rodents diet, develops obesity in a high percentage of mice, as well as some signs characteristic of MS. Since previous studies on sleep and circadian rhythms in this species, makes it a suitable model where to study the affections of overweight upon circadian physiology. The aim of the present work is to analyze daily profiles of some metabolic-‐related blood parameters (leptin, insulin, triglycerides, corticosterone and glucose), and the main electrocorticographic characteristics of sleep-‐wake cycle, between normal (CTL) and overweighed (OW) volcano mice. In CTL mice, the results of this study showed that there are differences were found between the highest and lowest concentration in the daily profiles, of all the blood parameters (except the leptin). OW mice however did not show daily differences, although the average concentration in almost all the hours tested were higher in OW than in CTL mice (except the corticosterone). The analysis of the sleep-‐wake cycle evaluating the temporal distribution of the vigilance states, indicate a poly-‐phasic architecture. A trend was observed in OW animals to increase the percentage of Slow Wave Sleep and decreasing time Wakefulness compared to CTL. The percentage of Rapid Eye Movements Sleep was similar between groups. The differences observed in this species are consistent with other reports in which leptin deficient or obesity induced rodents, shows alteration in the sleep-‐wake pattern. From this and other related studies, we propose the volcano mice as a good model for studying the daily rhythms and metabolic disorders related to obesity, mainly from the cyclical patterns of metabolites associated to energy balance. Supported by CONACyT 220274 CFG fellowship and PAPIIT IN202808 Keywords: volcano mouse, metabolic daily profiles, sleep-‐wake cycle

192

P073 EFFECT OF TOTAL SLEEP DEPRIVATION ON THE EXPRESSION OF NEUROGLOBIN IN THE ADULT RAT BRAIN Montserrat Alhelí Melgarejo, Eva Acosta Peña, Arturo Venebra Muñoz, Fabio García García. Instituto de Ciencias de La Salud, Universidad Veracruzana.

Several studies have reported that total sleep deprivation (TSD) increases the levels of reactive oxygen species (ROS) in different brain areas. For that reason it has been suggested that sleep plays an antioxidant role in the brain. Neuroglobin (Ngb) is a recently discovered protein in the mammal’s brain, which is involved in oxygen transport and ROS scavenging inside neurons. Interestingly, Ngb is expressed in the peduncule pontine tegmental nucleus (PPT), laterodorsal tegmental (LDTg) nucleus, locus coeruleos (LC) and suprachiasmatic nucleus (Sch). These entire brain regions are involved in the sleep regulation. Therefore, Ngb may be regulating the ROS levels in the brain of sleep deprived animals. The aim of this study was to determine the effect of TSD on Ngb expression in the brain of Wistar rats. For this purpose male adult rats (250-‐300 g body weight) were implanted for standard sleep recording, after 10 days of surgery recovery animals were divided into two experimental groups. Control group [n = 8], in which animals were sleep recorded during 24-‐h without any other manipulation. TSD group [n = 8], TSD was carried out for 24-‐h by gentle handling starting at 09:00 A.M. (lights-‐on). Rats were kept awake by gentle touching in tails or whiskers or by gentle shaking of their cages to prevent falling sleep. Electroencephalographic (EEG) and electromyographic (EMG) activity were recorded during all time of sleep deprivation (SD assuring animals did not show bouts of microsleep). A rotatory shift was scheduled so as to assure any particular researcher would only conduct the SD for 3 h at a time [8 shifts/24h]. SD was done in batches of three rats at a time (randomly chosen from all the TSD group) allowing researchers pay close attention to the animal’s behavior. Animals from both groups were perfused at 09:00 A.M., brains were obtained and processed for Ngb immunohistochemistry. Results showed that the total number of Ngb positive cells was decreased in the TSD group in all brain areas analyzed compared to control group [PPT, TSD group 18.50 ± 3.37 cells, control 35.77 ± 3.91 cells p <0.02; hippocampus, TSD group 4.00 ± 0.70 cells, control 24.60 ± 4.00 cells p <0.001; SCN, TSD group 4.11 ± 0.58 cells, control 18.00 ± 4.206 cells p <0.002]. These results suggest that sleep deprivation reduce Ngb expression in brain areas related with sleep regulation. Therefore, it is plausible that Ngb plays an important role in ROS regulation in cerebral areas with high metabolic demand during sleep. Keywords: Oxidative Stress, Suprachiasmatic, Neuroglobin

193

P074 CEREBRAL ELECTROPHYSIOLOGICAL ACTIVITY INDUCED BY SYSTEMIC INFLAMMATORY RESPONSE SYNDROME (SIRS) 1,2Francisco Nachón García, 3Juan Santiago García, 4Alberto de la Herrán Arita, 3Armando Martínez Chacón, 4René Drucker Colín, 2Fabio García García. 1Programa de Doctorado en Ciencias Biomédicas Universidad Veracruzana, México. 2Instituto de Ciencias de la Salud, Universidad Veracruzana, México. 3Instituto de Investigaciones Biológicas. Universidad Veracruzana, México. 4Departamento de Neurociencias, IFC, UNAM, México. Introduction. Brain levels of pro-‐inflammatory substances such as interleukins (IL) are affected in sleep disorders. It is known that IL’s are recognized by the CNS and induce alterations in neurotransmission. An increased level of IL´s in the brain produces encephalopathy, when that is associated particularly with infections is called Encephalitis Induced by Sepsis (EIS). We believe that changes in sleep patterns associated with inflammatory processes could be the result of the effect of signaling proteins produced during SIRS. Methodology. Twelve male Wistar rats were implanted with 4 electrodes for EEG recording and two for muscle activity recording, and left for 10 days to recover. Then animals received LPS from E. coli at two different doses (i.p. 0.5 and 1 mg / kg), in order to induce SIRS, and were recorded for 24 hrs with a polygraph. Scoring of the recordings was performed at each stage of sleep, and the EEG signal was processed for spectral analysis. Data (means and SD) were compared with those obtained from control animals that received a saline injection. Statistical analysis was performed with a t-‐test. Results. LPS-‐treated animals showed slow cortical activity, but not a physiological condition of slow wave sleep. Spectral analysis of sleep during this phase showed that subjects treated with both doses of LPS significantly reduced the power of delta band, compared to the control group (p <0.001). It was not possible to distinguish a physiological state of REM sleep in animals treated with LPS. In addition, the spectrum of power, during this phase, showed a reduction between 4.5-‐8.5 Hz (theta band) compared to controls (p <0.001). Conclusion. These results suggest that SIRS produced by i.p. injection of LPS induce slowing of cortical activity and abolish the power of slow waves. Keywords: sleep, inflammation, EEG

194

P075 CIRCADIAN CLOCK AND CELL CYCLE COUPLING IS REQUIRED FOR CORRECT WOUND HEALING 1Elzbieta Kowalska, 1Pascal Bruegger, 1Dominik Hoegger, 2Juergen Ripperger, 1Thorsten Buch, 3Anke Mueller, 3Achim Kramer, 2Urs Albrecht, 1Thomas Birchler, 1Claudio Contaldo, 1Steven A. Brown. 1University of Zurich, Switzerland. 2University of Fribourg, Switzerland. 3Charite Universitatsmedezin, Berlin, Germany. Circadian clocks restrict cell proliferation to defined time windows in adult mammals, but both the mechanism and the utility of this process are not well understood. We show here that functional circadian clock/cell cycle coupling is required for correct wound healing. Although we identified the NONO protein previously as a circadian clock component interacting with PER proteins, lack of NONO also eliminated circadian cell cycle gating, as well as increasing cell doubling rate and decreasing cellular senescence in vitro. All of these phenotypes were based on a loss of circadian activation by NONO of the p16-‐Ink4A gene. In vivo, deletion of the NONO gene in mice resulted in defective wound repair in vivo due to overproliferation of dermal fibroblasts without concomitant secretion of collagen. This phenotype is recapitulated in mice lacking core circadian clock components. Therefore, circadian control of cell division may serve to temporally segregate cell proliferation from tissue organization, and be critical to the healing process. Keywords: circadian, cell cycle, healing

P076 ACUTE COCAINE IMPAIRS ADULT CIRCADIAN CLOCK PHASE REGULATION: INVOLVEMENT OF THE PER2 CLOCK GENE 1Adam C. Stowie, 1Allison J. Brager, 2Rebecca A. Prosser, 1Dave J. Glass. 1Kent State University, US. 2University of Tennessee, US. The mechanisms by which cocaine disrupts the adult circadian system are not understood. In these experiments we sought to elucidate these mechanisms by utilizing both wild-‐type (WT) mice and a strain deficient in the circadian clock gene Per2 (Per 2 KO). In WTs, systemic treatment with cocaine (20 mg/kg i.p.) at midday (ZT 6) caused phase-‐advance shifts of 60 minutes, as assessed using an Aschoff type II protocol, involving release to constant darkness. In the Per2 KO mutants, this treatment caused much larger phase-‐advance shifts of 120 minutes (p<0.05 vs. WTs). In a second experiment, similar systemic treatment with cocaine in WTs inhibited the phase-‐delaying effect of a light pulse delivered during the night (ZT 16) by ~60%. In Per2 KOs, cocaine completely blocked this shifting

195

effect of light (p<0.05 vs. WTs). In a third experiment, reverse-‐microdialysis perfusion of the suprachiasmatic nucleus (SCN) with cocaine (50 µM) at ZT 6 induced a 3.5 hour phase-‐advance, vs. no effect in saline infused controls (p<0.01). In complementary experiments, application of cocaine at ZT 6 to the WT SCN in an in vitro slice preparation induced 3.0 hr phase-‐advance shifts that were blocked by application of the 5-‐HT antagonist, metergoline, implicating a cocaine-‐sensitive SCN serotonergic clock-‐resetting mechanism. Cocaine also blocked in vitro glutamate-‐induced (photic) nighttime shifts. These results confirm that the SCN clock is a direct target for photic and non-‐photic actions of cocaine. Collectively, these data point to multiple disruptive effects of cocaine on circadian timing regulation, that are registered directly within the SCN clock, and that are strongly influenced by the mPer2 circadian clock gene. Keywords: SCN, Cocaine, Clock Genes

P077 DAILY TIMING OF CHRONIC INTERMITTENT ALCOHOL EXPOSURE AND WITHDRAWAL DIFFERENTIALLY DISRUPTS CIRCADIAN BODY TEMPERATURE RHYTHMS IN C57BL/6 MICE Susan Amanda Sinning, Michael R. Gorman. University of California San Diego, USA. The acute response to alcohol depends critically on the timing of its administration. Little research, however, has directly examined the withdrawal response as a function of time of day. Additionally, repeated withdrawal from ethanol can lead to potentiated responses via a kindling-‐like mechanism that may have significance for alcohol addiction. If the withdrawal response differs as a function time of day, then control of the circadian cycle across multiple withdrawal periods may mitigate the progression of alcohol dependence. The present work examines how alcohol withdrawal (measured by changes in body temperature [Tb]) differs both as a function of time of day and multiple withdrawal periods. Two groups of C57Bl/6J mice (n =8 per group), implanted with radio-‐telemeters, were entrained to opposite light: dark periods (14:10 LD cycle) so that their active phases were 12 h apart. The animals were simultaneously exposed to three daily cycles of 14 h of EtOH vapor inhalation followed by a 10 h withdrawal period. During this time the two control groups (n = 4 per group), that had been similarly entrained and implanted, were exposed to air vapor and handled in a comparable manner. After the third day of EtOH vapor, the animals were left undisturbed for 11 days to recover. The 14 day protocol of EtOH vapor inhalation and withdrawal was repeated an additional 3 times. The total amount of circadian disruption was calculated by summing the absolute deviation from the baseline body temperature rhythm. In the intoxication phase, animals exposed to alcohol in their active phase showed greater disruptions overall in the Tb rhythm. Initially, during the acute withdrawal phase, Tb was more disrupted in the inactive phase, but animals in their active

196

phase progressively showed greater disruption across rounds compared to the inactive phase. By the fourth round there was a trend for animals in their active phase to be more disrupted in their Tb rhythm than animals in their inactive phase. We conclude that both alcohol exposure and withdrawal differs as a function of time of day, but the disruption during the withdrawal period becomes more pronounced when withdrawal occurs repeatedly during the inactive phase of the circadian cycle. These findings suggest that controlling for the circadian phase may mitigate the progression of symptomatic withdrawal. Keywords: alcohol, circadian, withdrawal

P078 CIRCADIAN GENE MODULATION AND BRAIN SITES OF ACTION IN ACAMPROSATE SUPPRESSION OF ALCOHOL INTAKE AND CRAVING 1Allison Joy Brager, 2Rebecca A Prosser, 1J David Glass. 1Kent State University, USA, 2University of Tennessee, USA. Acamprosate is widely used to reduce ethanol craving and relapse in recovering alcoholics, but its central site(s) of its action are unknown. Here, we mapped brain areas sensitive to acamprosate suppression of ethanol intake and preference using stereotaxically-‐targeted acamprosate micropellets. The experiment was undertaken in PER2-‐mutant mice, which have elevated ethanol intake and craving compared to wild-‐types (WTs) as a partial consequence of their advanced phase-‐angle of entrainment and increased duration of wakefulness. Male mPER2 mutants and WTs under a 12L:12D (LD) photocycle were introduced to free-‐choice 15% ethanol/water for 3 wks. Ethanol was withdrawn and reintroduced 3 weeks later to facilitate relapse. Four days before ethanol reintroduction, mice received bilateral blank (control) or acamprosate-‐containing micropellets releasing 50 ng/day into reward (ventral tegmental [VTA], penduculopontine tegmentum [PPT] and nucleus accumbens [NAc]) and circadian (intergeniculate leaflet [IGL]; suprachiasmatic nucleus [SCN]) areas (n=6, each). The hippocampus was also targeted. Circadian locomotor activity was measured throughout using infrared sensors interfaced with a Clocklab data acquisition system. Baseline levels of ethanol intake and preference were greater in mPER2 mutants vs. WTs (27 g/kg/day vs. 13 g/kg/day and 70% vs. 50%, respectively; both p<0.05). In WTs, acamprosate implants in all areas except the hippocampus suppressed ethanol intake and preference by ~40% over 3-‐4 wk of reintroduction. In mPER2 mutants, acamprosate implants in the VTA, PPT, and SCN suppressed ethanol intake and preference by ~20% over 1-‐2 wk of reintroduction (all p<0.05 vs. blank implants). Overall higher levels of ethanol intake and preference were observed in the mPER2 mutants vs. wild-‐types throughout acamprosate treatment (p<0.05). All mPER2 mutants began their nighttime activity period ~2 hrs before that of wild-‐types (p<0.05), and alpha was extended by a

197

similar duration (p<0.05). The acamprosate implants in the VTA and NAc that inhibited ethanol intake in wild-‐types produced a 300% increase in locomotor activity during the dark-‐phase of the LD cycle, but not in mPer2 mutants (p<0.05). Nighttime activity was increased by 200% in WTs and mPer2 mutants by acamprosate pellets in the SCN (p<0.05). Collectively, these results are evidence that the modulatory actions of acamprosate on ethanol intake, craving and circadian activity are manifest through its actions in multiple drug reward and circadian areas. The less robust suppressive effect of acamprosate on drinking in the PER2 mutants vs. WTs suggests that the mutant’s innate drive to drink could render them less responsive to acamprosate. Individual differences in alcohol craving could thus be a factor determining the varying degree of clinical efficacy of this drug. Acknowledgements: NIAAA grants AA-‐015948 and AA-‐017898 to R.A. Prosser and J.D. Glass Keywords: mPER2, alcohol, mouse

P079 HYPOTHALAMIC CONTROL OF BLOOD PRESSURE: A ROLE FOR THE BIOLOGICAL CLOCK 1Frederik Buijs, 1Mari Carmen Basualdo, 2Carolina Escobar, 1Ruud Buijs. 1Instituto de Investigaciones Biomedicas, 2Dept Anatomia Fac Medicina. The hypothalamus integrates information from the brain and the body. As a result, countless functions are regulated by neuroendocrine and autonomic output in concert with the appropriate behaviour. Within the hypothalamus de suprachiasmatic nucleus (SCN) imposes its rhythm onto the body via three different routes of communication: 1.Via the secretion of hormones; 2. via the parasympathetic and 3.via the sympathetic autonomous nervous system. The SCN uses separate connections via either the sympathetic or via the parasympathetic system not only to prepare the body for the coming change in activity cycle but also to prepare the body and its organs for the hormones that are associated with such change. The hypothesis for our present work is that in view of the role of the SCN in determining the set point of these physiological day-‐night levels, the SCN also needs to be informed about the accurate values of these variables. Apart from light, activity and melatonin little is known about the information that is provided to the SCN in order to execute its functions. Recently we demonstrated that the SCN receives input from the arcuate nucleus as well as from other circumventricular organs. Here we investigated in rats whether the nucleus tractus solitarius (NTS) which receives visceral sensory input may provide information to the SCN. Injections of the tracer CtB into the NTS resulted in the demonstration of projections into the SCN, these projections are especially aimed at the ventral part of the SCN. Next we examined under which conditions these projections might be functional. We have shown that an increase in blood pressure induces an activation of the SCN especially in the part where the input of the NTS enters the nucleus. This observed activation disappears after a unilateral lesion of the NTS. At present we investigate the consequence of lesioning the SCN on blood pressure control with the hypothesis that the

198

SCN will reduce blood pressure via an action on the DMH. The role of the SCN in controlling cardiovascular functions is discussed in relation with the finding that the activity of the SCN is diminished in people who died after a long history of hypertension. Acknowledgments: This study was supported by grants from CONACyT 79797, DGAPA PAPIIT IN215308-‐3 UNAM, Mexico Keywords: Hypertension, NTS, Hypothalamus

P080 EXPERIMENTAL CHRONIC JET LAG PROMOTES GROWTH AND LUNG METASTASIS OF LEWIS LUNG CARCINOMA IN C57BL/6 MICE Mingwei Wu, Jing Zeng, Zhaolei Zeng, Lijian Xian. Cancer Centre, Sun Yat-‐Sen University, China. Circadian rhythm has been linked to cancer genesis and development, but the detailed mechanism by which circadian disruption accelerates tumor growth remains unclear. The purpose of this study was to investigate the effect of circadian disruption on tumor growth and metastasis in male C57BL/6 mice, using an experimental chronic jet lag model to induce circadian disruption by advancing light onset eight hours every two days. Lewis lung carcinoma cells were inoculated into both flanks of the mice following 10 days of exposure to experimental chronic jet lag or control conditions. The effects on tumor growth and lung metastasis were assessed, and the effect on gene expression was detected using a cDNA microarray. Tumors grew faster in the experimental chronic jet lag mice compared to the control mice (P = 0.004). Lung metastases were found in 10 out of 24 mice in the chronic jet lag group, but only 3 out of 24 mice in the LD group (P = 0.023). Microarray data showed that in both liver and tumor circadian disruption altered the expression of genes, including those related to cell cycle, apoptosis, immune response, and metastasis suppressor genes. We conclude that circadian disruption can promote tumor progression and metastasis by affecting the expression of both tumor-‐related genes and metastasis suppressor genes. Keywords: tumor, Jet lag, gene microarray

199

P081 EFFECT OF LIGHT/DARK CYCLE ON SEIZURE SEVERITY AND HIPPOCAMPAL NEURONAL CELL DEATH INDUCED BY STATUS EPILEPTICUS IN THE DEVELOPING RAT Dulce Mariely Álvarez-‐Croda, María Leonor López-‐Meraz. Programa de Neurobiología, Posgrado en Neuroetología, Universidad Veracruzana, México. Status epilepticus (SE) is a neurological condition characterized by sustained or intermittent epileptic seizures during a prolonged period without the full recovery of consciousness. SE is most common in the young children and data from clinical and basic investigation show that SE produces neuronal cell death in the developing hippocampus. On the other hand, there is evidence showing that circadian cycle influences seizures progression in both, humans and experimental models of epilepsy. The goal of this study was to determine the effect of light/dark phases on SE progression and SE-‐induced hippocampal damage. SE was induced in 14 days old rats, either during light or dark phase of the day, by using the lithium-‐pilocarpine model. Rat pups were given 3 mEq/kg lithium chloride i.p. on the day before the induction of SE, which was carried out at P14 by subcutaneous injection of 60 mg/kg pilocarpine hydrochloride. Control animals were given an equal volume of saline subcutaneously. Seizure severity was evaluated and 24h following SE, hippocampal neuronal cell death was assessed by hematoxylin-‐eosin staining. Results showed that incidence, onset and severity of SE was similar if induced during light or dark phase of the day. SE produced neuronal cell death in subiculum-‐CA1 and dentate gyrus fields of hippocampus independently of the cycle phase during which seizures were induced. Results suggest that light/dark cycle modifies neither severity of SE nor SE-‐induced hippocampal damage in the developing rat. Supported by CONACYT (grant 106402 to MLLM and scholarship 249772 to DMAC) and PROMEP (grant PTC-‐474 to MLLM). Keywords: status epilepticus, Hippocampal damage, Light/dark cycle

P082 EFFECTS OF PRENATAL EXPOSURE TO VALPROIC ACID ON ULTRASONIC VOCALIZATIONS OF RAT DURING LACTANCY PERIOD Paul Saft, Jorge Manzo, Consuelo Morgado-‐Valle, Luis Beltrán-‐Parrazal, María Elena Hernández, Brenda Brug, Luis Isauro García, Rebeca Toledo. Programa de Neurobiología. Universidad Veracruzana, México. Recent epidemiological studies reveal that thalidomide (THAL) or valproic acid (VPA) exposure during the first gestational trimester in humans causes higher incidence of autism in the offspring. Morphological abnormalities similar to those found in autism (e.g.

200

cerebellar anomalies, reduced motor neuron numbers) have been reported in animals exposed to these teratogens prenatally, suggesting this manipulation as an experimental model of autism. However, the validity of this model based on anatomical data has yet to be established in behavioral studies. We recorded neonatal rat ultrasonic vocalizations (40 kHz) as a behavioral test to study the teratogenic effect of prenatal exposure to VPA. Vocalizations were recorded every other day during lactancy period (21 days). Recordings were obtained after a brief (5 min) isolation period from the mother. We established the temporal course of the number of vocalizations. We found that rats from all groups (sham, control and VPA treated) show the highest number of vocalizations in day 10 and a pronounce drop followed by extinction of the behavior at day 12. While this pattern of activity is preserved in all groups, we found an increase in the number of vocalizations in VPA treated neonates with respect to sham and control groups. We suggest that, despite the ontogenic changes produced by VPA, the vocalization behavioral pattern remains unaltered. Supported by: PROMEP/103.5/07/2753, (T.R.), Beca-‐CONACyT-‐41704 (S.P.), Proyecto CONACyT-‐106531 (H.M.E.) Keywords: Autism, vocalizations, valproic acid

P083 DIURNALLY OR NOCTURNALLY ELICITED SYSTEMIC INFLAMMATION DOWN REGULATES AANAT GENE EXPRESSION IN THE CHICKEN PINEAL GLAND 1Krystyna Skwarlo-‐Sonta, 1Urszula Kedzierska, 1Aneta Piesiewicz, 1Alicja Olesiejuk, 2Maria Waloch, 1Pawel Marek Majewski. 1Department of Animal Physiology, Faculty of Biology, University of Warsaw, Poland. 2Department of Medical Paraistology, National Institute of Hygiene, Warsaw, Poland. Recently, we have demonstrated in chicken bidirectional relationships between activated immune system and the pineal gland function. Namely, experimental peritonitis developing during darkness inhibited nocturnal elevation in melatonin (MEL) biosynthesis leading to the disappearance of the circadian rhythm of the pineal AANAT activity while MEL treatment blocked the development of peritonitis estimated as an increase in peritoneal leukocyte (PTLs) number. The aim of present work was to compare the influence of the experimental peritonitis evoked at the beginning or towards the end of light phase on the transcription of genes encoding particular enzymes involved in MEL biosynthetic pathway in the chicken pineal gland. Experiments were performed on 16-‐day-‐old male Hi-‐Line chickens reared from the day of hatch under controlled conditions (L:D 12:12). Peritonitis was evoked by ip. injection of thioglycollate solution (TG) 2h after start or 2 h before the end of light phase. TG treated and intact control chickens were sacrificed 4h after TG injection and development of peritonitis was verified by the number of retrieved PTLs. Pineal glands were isolated under dim red light every, immediately frozen and subsequently used for analysis

201

by real-‐time PCR the level of mRNA of genes encoding following enzymes: tryptophan hydroxylase 1, aromatic L-‐amino acid decarboxylase, N-‐acetyltransferase (AANAT) and hydroxyindole-‐O-‐methyl transferase (HIOMT). Additionally, in the pineal glands were measured tryptophan and serotonin content as well as the AANAT protein content and activity and its chaperone 14-‐3-‐3 protein. Moreover, serum MEL concentration was measured in chickens with nocturnally developing peritonitis. The diurnal changes in transcription profile of genes encoding all the enzymes participating in MEL biosynthesis were rhythmic, with acrophases during darkness except that of HIOMT which peaked at light phase. Induction of peritonitis before the beginning of night decreased nocturnal level of MEL in the blood without the effect on the pineal tryptophan and serotonin content. Diminished AANAT mRNA content was observed in TG treated chicken in comparison to control animals regardless of time of peritonitis induction while the expression of the genes encoding other enzymes was not affected. Nocturnal AANAT protein content and enzyme activity were decreased in chickens TG injected 2hs before the end of day without the effect on the level of 14-‐3-‐3 protein. Results obtained clearly indicate that the inflammatory mediators exert the inhibitory effect on the transcriptional level of AANAT gene and this effect seems to be independent of the time of day when inflammation was elicited. Supported by the Polish MSHE grants NN 303 3177 33, NN 303 5034 38, and NN 303 5957 39 Keywords: inflammation, chicken pineal gland, mRNA

P084 A CIRCADIAN CLOCK REGULATES SWIMMING DIRECTION OF ERETMOCHELYS IMBRICATA HATCHLINGS (HAWKSBILL MARINE TURTLE) Kristel Lorena Gopar-‐Canales, Manuel Miranda-‐Anaya. Facultad de Ciencias, UNAM., México.

Navigation in migrating animals may depend on several mechanisms. A particular one consists on a time-‐compensated sun compass for orientation displacement. The location of the sun relative to the earth’s horizon can provide significant directional information so an animal is able to compensate changes in the sun’s position throughout the day. This phenomenon has been studied particularly in migrating flying organisms such as birds and insects; however there are many marine organisms that migrate over the seasons, particularly marine turtles. It is also known that magnetic orientation plays an important role in swimming direction by hatchlings; yet studies on the participation of a circadian clock in marine sea turtles are scarce. At the present study we aim to test whether a circadian clock is involved in the navigational direction of “hawksbill” hatchlings turtles Eretmochelys imbricata. We used two main groups of hatchlings from the same nest. The nest was collected by federal permission at Playa Sisal, Yucatan. After hatching, turtles were kept in controlled conditions of water quality, food and photoperiod 12:12 at the facilities

202

of Multidisciplinary Research Unit (UMDI) of Science Faculty, UNAM. One group (control) was kept during four days in a LD schedule (06:00-‐18:00 photo phase, n=50) and a second group was kept in a delayed LD photoperiod (12:00-‐24:00 photo phase, n= 30). Direction was individually tested in an observation arena consisting on a circular container of marine water (2m diameter) in two shifts of the natural day, morning (07:00-‐11:00 h) and evening (at 16:00-‐19:00 h). From center of the container, a guide line held a turtle at the surface water level, in such way that turtle could swim without reaching the edge. After 10 minutes swimming, six photographs were taken from above every 20 seconds, then at the end of the evening shift each tested animal was released at the seashore. All results were interpreted in printings and position was calculated in grades. Swimming direction was then tested by means of circular statistics (Rayleigh and Marty Watson test). An external group of 7 turtles were set in infrared light crossings for locomotor activity in separate aquaria during 4 days and exposed to natural indirect LD regime. During observation at field, animals from each group were tested with or without a magnet in their plastron, in order to test swimming direction in magnetically disoriented hatchlings at both phases of the natural photoperiod. Results show differences (p<0.05) among hatchlings from control group. A significant swimming position oriented to NNE was observed in hatchlings exposed to a 4 day delayed photoperiod and exhibited an advance near 60° average towards NNW. No clear differences were observed whether using or not a magnet. Locomotor activity monitored by infrared light beams indicates a crepuscular activity when in natural photoperiod and also an anticipate activity to sunrise and sunset. CONACyT fellowship for KGC Keywords: Sun compass, hatchlings, entrainment

P085 SIXTH ABDOMINAL GANGLION MODULATES THE CIRCADIAN RHYTHMS OF LOCOMOTOR ACTIVITY AND ABDOMINAL POSTURE IN CRAYFISH Leonardo Rodríguez Sosa, Gabina Calderon Rosete, Departamento de Fisiología, Facultad de Medicina, UNAM, México, D. F Our previous observations on the electrical activity of the caudal photoreceptor (CPR), in the sixth abdominal ganglion (6th AG), in crayfish have been driven in the context of a distributed circadian-‐pacemaker system. We think that CPR also modulates the motor behavior in crayfish. The objective of this study is to analyze the effects of injury in the 6th AG on two functions: 1) spontaneous locomotor activity (SLA), and 2) abdominal postures (AP), both exhibited by crayfish in an aquarium. Experiments were made at regulated conditions in adult intermolt Procambarus clarkii crayfish with random gender. We formed three groups: 1) control, 2) injured by sectioning connective between fifth and 6th AG, and 3) injured by ablation of the 6th AG. Initially, all the crayfish were collocated in LD condition (12:12) by two weeks. Then, both experimental groups were taken back to LD condition by

203

one or more weeks. Crayfish were then collocated for observation in LL conditions. Data were analyzed along 24-‐h cycle. The main results are the following. 1) For the SLA, the tree groups displayed circadian rhythmicity. Both experimental groups have shown changes of phase, with respect to control group. 2) AP were observed and classified into four categorical abdominal positions. We noticed circadian rhythmicity in the AP behavior in all groups. Both experimental groups showed changes on three of the four abdominal postures, out of phase compared with control group. In some cases ultradian rhythms were observed for both, SLA and AP. Taken together, these data suggest that the 6th AG modulates the locomotion activity and abdominal posture in crayfish, probably through the CPR or some possible neuromodulators. Supported by: Facultad de Medicina, UNAM to LRS. The authors express their sincere appreciation to Mr. Victor Anaya for his assistance in surgery experiments. Keywords: caudal photoreceptor, Procambarus clarkii, ultradian rhythm

P086 SCN DRIVEN CHANGES IN THE ACTIVATION OF ARCUATE NUCLEUS ΑMSHNEURONS 1Mara Alaide Guzmán Ruiz, 1Guadalupe Acosta Galván, 1Daniela Herrera-‐Moro Chao, 1Ma. Carmen Basualdo Sigales, 2Carolina Escobar Briones, 1Ruud Buijs. 1Instituto de Investigaciones Biomédicas, UNAM, México. 2Facultad de Medicina, UNAM, México. The Arcuate Nucleus (ARC) is known to respond to metabolic cues as Glucose, Free Fatty Acids, Leptin, Ghrelin and Insulin, these signals are known to inform neurons within the ARC about metabolic state of the organism. Previous studies, have demonstrated anatomical connections between the ARC and the Suprachiasmatic Nucleus (SCN), suggesting an involvement of the SCN in ARC sensorial function. Therefore, the aim of the present study was to investigate the influence of the SCN on ARC activity, especially with respect to the anorexigenic neurons α-‐Melanocortin Stimulating Hormone (α-‐MSH). This neuronal population is known to be activated after food intake. In order to determine whether the SCN may influence ARCs neuronal activity, c-‐Fos IR was determined at six time points along the LD cycle on two groups, fed Ad libitum and 48h fasted animals. The results show that α-‐MSH neurons from the ARC present a peak in their activity at the end of the dark phase; this activation persists despite fasting. To determine the involvement of the SCN on ARC neuronal activity, bilateral SCN lesion were made and animals sacrificed at the acrofase α-‐MSH neuronal activity. An almost complete loss of the activation within the ARC was observed. To determine how the SCN is driving the activation of α-‐MSH neurons, SCN unilateral lesions were executed and the neuronal activation at the lesioned and non-‐lesioned side of the ARC were quantified. The loss of α-‐MSH neuronal activity at the lesioned site demonstrates that the SCN activates α-‐MSH neurons via neuronal connections,

204

suggesting that temporal information is transmitted to the ARC may affect its function. Acknowledgement: This study was supported by DGAPA project IN215308 and Conacyt 79797 Keywords: Metabolism, Arcuate, Suprachiasmatic Nucleus

P088 HOW CORTICO – BASAL GANGLIA – THALAMOCORTICAL NETWORK CAN INFLUENCE RATE OF "INTRINSIC CLOCK” Izabella G. Silkis. Institute of Higher Nervous Activity & Neurophysiology, Russian Academy of Sciences, Moscow, Russian Federation, Russia. A hypothetical mechanism is suggested for participation of cortico – basal ganglia – thalamocortical network in perceiving subsecond time intervals and influencing a rate of "intrinsic clock”. We postulated that parameters "when" specifying time of stimuli arriving and parameters “what” determining physical properties of stimuli are perceived simultaneously by the same CNS structures. As a result, associations “what – when” are coded in neocortical areas in which properties “what” are processed. Rate of "intrinsic clock” depends on the length of one cycle of repeated excitation of a cortical area. This excitation is provided by a neuronal loop that includes subthalamic, pedunculopontine and thalamic nuclei, and neocortex. Interval between perceived stimuli is proportional to the number and duration of cycles of cortical excitation. We assume that the time of repeated neocortical excitation must depend on dopamine-‐modulated functioning of the cortico – basal ganglia – thalamocortical loop. According to our model, this loop promotes disinhibition of thalamic cells and subsequent contrasted amplification of firing of cortical neurons activated by a stimulus. Time counts could be coded by a number of neuronal discharges and accumulated in the neocortex. Time counting starts involuntary by a stimulus, or voluntary due to activation of the prefrontal cortex. Proposed mechanism must be similar for stimuli of different modalities owing to resemblance of functioning of topically organised cortico – basal ganglia – thalamocortical loops. It follows from our model that the more concentration of dopamine in the striatum, the stronger thalamic disinhibition and higher clock rate, so real time interval is estimated more precisely and perceived as longer. In opposite, the less concentration of dopamine in the striatum, the low clock rate, and real interval is perceived as more short. These consequences of proposed model are in accordance with known role of dopamine in time estimation. Based on our experimental data we assume that the time length of repeated cortical excitation is approximately 20-‐25 ms. This estimation is the result of our observation of bimodal or trimodal distribution of latencies of responses of individual neurons of visual cortical area V2 to a light flash. Interval between first and second peaks was in the range of 20-‐25 ms. Analogous peaks were found for auditory or somatosensory stimuli. These peaks cannot be

205

explained by propagation of signals through the magnocellular and parvocellular subdivisions of the lateral geniculate body since difference in latencies for these pathways is about 7-‐10 ms. Peaks cannot be explained by polymodal cortico-‐cortical or thalamo-‐cortical interactions because in such case distribution of latencies must be flat. Our model and data allow understand why it is difficult to differentiate intervals that do not exceed 20-‐30 ms. This work is supported by Russian Foundation of Basic Research, grant No 11-‐04-‐00252 Keywords: intrinsic clock, basal ganglia, dopamine

P089 THE PARTICIPATION OF THE VAGUS NERVE IN THE REGULATION OF OVARIAN FUNCTIONS DEPENDS ON DAY THE ESTROUS CYCLE AND TIME OF THE DAY Pamela María Everardo, María Guadalupe Gúzman, Carlos Abraham García, Angélica Flores, María Esther Cruz, Roberto Domínguez. Universidad Nacional Autónoma de México.

The vagus nerve participates in the regulation on ovulation and hormone release by the ovaries. Also, it is one of the pathways communicating the ovaries and the CNS. Recent studies have shown that the regulation of ovarian function by ovarian innervation varies during the estrous cycle and daylight hours. To analyze whether the Vagus Nerve involvement in the regulation of spontaneous ovulation depends on the day of the estrous cycle and the time at which the study is performed, at 07:00, 13:00 or 19:00 hours of the day of diestrus-‐2 or proestrus, rats were submitted to ventral laparotomy (LP) or section of the Left (Nv-‐L), Right (Nv-‐R) or both (Nv-‐B) vagus nerves. The animals were sacrificed on the day of expected estrus after surgery. In rats treated at 07:00 on diestrus-‐2, the section of the the Nv-‐L resulted in a lower ovulation rate (3/7 vs. (LP) 7/7, p <0.05) and number of ova shed (2.3±1.1 vs. 9.1±0.6: p <0.01). The Nv-‐R section resulted in a higher number of ova shed by both ovaries (18.2±0.5 vs.14.0±0.8, p <0.05). Nv-‐B resulted in a lower number of ova shed by both ovaries (5.4±0.5 vs. 14.0±0.8, p <0.01). The unilateral or bilateral section of the vagus nerve performed at 13:00 h did not modify the ovulation rate. The number of ova shed by rats with Nv-‐B was higher than in LP rats (16.0±0.8 vs. 12.8±1.0, p <0.05). Rats submitted to surgery at 19:00 h did not modify the ovulation rate, nor the number of ova shed. In rats treated at proestrus, unilateral or bilateral section of the vagus nerve did not modify the ovulation rate nor the number of ova shed. Present results suggest that the participation (inhibitory and stimulatory) of the vagus nerve ovarian innervation on spontaneous ovulation,is asymmetric and depends on the day of the estrous cycle and the time of the day. Keywords: Ovulation, Vagus Nerve, Estrous Cycle

206

P090 THE ROLE OF KISS1 NEURONS IN CIRCADIAN-‐TIMED LUTEINIZING HORMONE SURGES IN SHORT DAY FEMALE HAMSTERS 1Azim R Khan, 2Eric C Ku, 1Michael R Gorman, 2Alexander S Kauffman. 1Department of Psychology, Center for Chronobiology, University of California-‐San Diego, USA. 2Department of Reproductive Medicine, Center for Chronobiology, University of California-‐San Diego, USA. Female Syrian hamsters become reproductively acyclic and anovulatory during short day (SD) photoperiods (i.e. winter months). Despite this phenomenon, SD female hamsters still demonstrate a daily circadian rise in luteinizing hormone (LH) in the late afternoon. This LH secretion profile of short-‐day females resembles the circadian-‐timed LH surge signal that normally initiates ovulation in hamsters housed in long days (LD; i.e. summer months). In LD hamsters, mice, and rats, circadian LH surges (indicative of GnRH surges) are thought to be governed by upstream neural circuits, which include Kiss1 neurons in the anteroventral periventricular nucleus (AVPV). Indeed, Kiss1 neurons in the AVPV of LD females exhibit circadian changes in gene expression and neuronal activation which are in synchrony with the LH surge. Kiss1 neurons are also found in the arcuate nucleus (ARC), but these neurons are not implicated in the LH surge of LD rodents. The LH surges in LD hamsters also require estrogen, as ovariectomized females demonstrate severely blunted or absent circadian patterns in LH secretion and AVPV Kiss1 expression. However, circadian rises in LH are nonetheless observed in ovariectomized SD hamsters, suggesting the SD LH “surge” is both circadian-‐timed and sex steroid-‐independent. To investigate the potential role of Kiss1 neurons in the occurrence of daily LH surges in SD hamsters, the levels of Kiss1 expression in the AVPV and ARC were determined in ovariectomized female Syrian hamsters at four different circadian times (before and during the predicted LH surge). Surprisingly, Kiss1 levels in the AVPV and ARC did not change throughout the day, despite the presence of a significant late afternoon LH surge in these SD females. Further studies will determine whether Kiss1 neurons are selectively activated in the late afternoon or whether the LH surge occurs without detectable changes in the AVPV or ARC Kiss1 systems. This work supported by NIH grant R00 HD 0156157 and NSF grant IOS-‐1025893. Keywords: kisspeptin, seasonal reproduction, luteinizing hormone surge

207

P091 MODULATION OF THE CIRCADIAN RHYTHM OF BODY TEMPERATURE BY INTENSE ACTIVITY IN A SUBTERRANEAN RODENT (CTENOMYS CF. KNIGHTI) 1Patricia Tachinardi, 1Barbara Mizumo Tomotani, 1Danilo Eugénio de França Laurindo Flôres 1José Eduardo Pereira Wilken Bicudo, 2Verónica Sandra Valentinuzzi, 1Gisele Akemi Oda. 1Instituto de Biociéncias, Universidade de Sao Paulo (IB-‐USP), Brazil, 2Centro Regional de Investigaciones Científicas y Transferencia Tecnológica, La Rioja (CRILAR), Argentina. Most mammals show a daily body temperature (Tb) rhythm which is endogenous and entrained by light/dark (LD) cycles. Usually, the highest temperatures occur during the same phase of motor activity, which often may mislead to the conclusion that the Tb rhythm is a mere consequence of the activity/rest rhythm. Nevertheless, several studies have verified that both rhythms persist independently of each other. Intense activity may, however, modulate some parameters of the Tb rhythm. Quantifying this modulation helps dissecting the exogenous and endogenous components of the Tb rhythm when the animal is under different experimental conditions or in its natural habitat. We investigated this issue in a subterranean rodent, the tuco-‐tuco Ctenomys cf. knighti, found in northwest Argentina. First we studied their Tb under a LD cycle and verified a daily Tb rhythm with the highest temperatures occurring during the dark, synchronously to running-‐wheel activity. Furthermore, Tb rhythm persisted under constant darkness (DD), an evidence of its endogenous nature. Interestingly, we observed a much smaller variability in rhythmic patterns (period, phase and amplitude) compared to other subterranean rodents. In order to study the exogenous components of the Tb rhythm due to intense activity, its parameters in the presence and absence of a running-‐wheel were compared. To continuously measure temperature and gross motor activity, telemetric transmitters were implanted intraperitoneally. Three tuco-‐tucos were housed individually in cages equipped with running-‐wheels and exposed to the following lighting conditions: 1) DD (44 days); 2) LD (12:12, 14 days); 3) reestablishment of DD (16 days). The wheels were then removed and the exposure to the lighting conditions repeated. After running-‐wheel removal, mean gross motor activity diminished 54-‐62% in two animals. Free-‐running period (ranging from 23.6 to 24.4 hours) and phase relationship with activity rhythm did not change significantly. The amplitude, in turn, decreased about 0.5oC in 2 animals. These results will be important for further analysis of experiments using running-‐wheels as well as studies of Tb rhythmicity of tuco-‐tucos in their natural habitat, where they show frequent bouts of intense activity while digging. Funding: CNPq, FAPESP, CAPES and CONICET. Keywords: tuco-‐tuco, body temperature, running-‐wheel

208

P092 SOCIAL INTERACTION ALTERS CIRCADIAN RHYTHM MANIFESTATION IN RATS SUBMITTED TO CONSTANT LIGHT Antoni Diez-‐Noguera, Lucia Castejon, Trinitat Cambras. Dept. Physiology. Fac. Pharmacy, Univ. Barcelona, Spain, Spain. In some species, social cues have been found to have a role as synchronizers of the circadian behavior, although in other species the effect of these signals are controversial. Here, the influence of social interaction on rat circadian behavior was investigated, in two experiments. In the first one, we addressed the question of whether cohabitation would produce a delay in the appearance of arrhythmicity under constant light conditions. To this end, the circadian rhythms of male and female rat body temperature were studied for 10 days under light-‐dark conditions, followed by 33 days under constant bright light. Half of the animals were maintained in individual cages, whilst the others were maintained in larger cages in groups of three rats of the same sex. Results showed that individual circadian rhythms under 24 hour light-‐dark (LD) cycles were more stable and with higher amplitude in grouped than in isolated animals, and higher in males than females. In the second experiment, the motor activity and temperature rhythm of 10 pairs of rats were studied. Each pair or rats were formed by one rat with clear circadian behavior and another that was clearly arrhythmic. Each pair was housed in the same cage for 20 days and afterwards they were again isolated for 20 more days. During this time, body temperature was recorded for each animal to test the effect of the cohabitation in their rhythm. Results showed that 6 out of 10 arrhythmic rats recovered a circadian rhythmicity after the exposure to cohabitation. These experiments suggest that living in the same house increases the internal coherence of circadian rhythms, and suggests that social interaction may be an influencing agent contributing to the organization of animal behavior over the day. Keywords: social-‐interaction, arrhythmicity, cohabitation

P093 COMPARATIVE ANALYSIS OF CIRCADIAN RHYTHMS OF THE MOSQUITOES AEDES AEGYPTI AND CULEX QUINQUEFASCIATUS UNDER TEMPERATURE CYCLES Gustavo Bueno Rivas, Carla Gentile, Alexandre Afranio Peixoto. Laboratório de Biologia Molecular, Instituto Oswaldo Cruz FIOCRUZ, Rio de Janeiro, Brazil. Many species of mosquitoes are vectors of important diseases. Their activity and blood feeding rhythms, which are important for pathogen transmission, are under circadian control. However, relatively little is known about the molecular genetics of the endogenous clock controlling these rhythms. We are studying the locomotor activity rhythms and

209

circadian expression of the main clock genes in Aedes aegypti, a diurnal vector of Dengue and Yellow fever and Culex quinquefasciatus, a nocturnal vector of Filariasis and West-‐Nile fever. Our previous work revealed conserved circadian expression patterns between the two species in most genes, except cryptochrome2, under light-‐dark and constant darkness conditions and we are currently examining the effects of temperature cycles. We observed that both species are entrained by temperature cycles in constant light and showed differences in their activity behavior that resemble their patterns in light-‐dark conditions. Ae. aegypti is more restricted to the termophase while Cx. quinquefasciatus has its activity more restricted to the cryophase. At the molecular level, we observed mRNA cycling in temperature cycles, albeit in lower amplitude than in light-‐dark cycles, and some species-‐specific differences in their clock gene expression patterns. Our results suggest that both species use temperature, as well as light, to concentrate their activity in a specific phase of the day. Keywords: circadian clock, mosquitoes, temperature cycles

P094 RAT LIVER GABAERGIC SYSTEM CHARACTERIZACTION DURING FOOD ENTRAINABLE OSCILLATOR EXPRESSION Olivia Vázquez Martínez, Mónica Villalobos Leal, Isabel Mendez, Diego Hernández Saavedra, Mauricio Díaz Muñoz. Instituto de Neurobiología, UNAM, México. It is a few know about the action of GABA outside of the nervous system, firstly in the liver with circadian rhythms implications. This organ has all the GABAergic elements: the molecule, the sintetics and catabolic enzymes, transports, as well the hiperpolarizant action of GABA in hepatic slides and cells. In a previous microarrays experiment, we found many changes in the hepatic transcriptional level of classical neurotransmitors systems, like GABA, glutamate and serotonine, follow an feeding restriction protocol which promote the expression of a biological clock sincronizaced by the food access (FEO). In this proyect we propose that the food restriction will provoke changes in the expression and activity in GABA receptors, as well in the catabolics and sintetics enzyme of GABAergic system in the liver. The general goal is characterized if there are changes in the GABA A (subunit a5) and GABA B (subunit 2) by Western bolt and immunohistochemestry experiments; we want to cuantified the succinate semialdehide deshydrogenase activity, GABA transaminase (catabolic) and glutamate descarboxilase (anabolic) In our protocol we use Wistar male rats in a food restricted schedule by tree weeks in which we present the food from 12:00 to 14:00. The controls are rats with: 1) Ad libitum food, 2) Fasting rats of 24 and 48 h, and 3) Re-‐feeding rats with a 24 and 48 h of fasting but two hours of food before de sacrifies. Keywords: GABA, system, liver

210

P095 BEHAVIORAL AND TEMPERATURE CHANGES THAT PRECEDE FOOD ENTRAINMENT Dulce María Palomares-‐Vázquez, Estefa Espitia, Rodrigo Ivan Osnaya, Carolina Escobar. UNAM, México. Introduction Feeding schedules entrain behavior and physiological circadian rhythms changing their oscillations and organizing their peaks of maximum expression around feeding hours. Food entrainment is achieved when food is restricted to a few hours and is given in a regular and predictable way. Food anticipatory activity (FAA) develops after 4-‐5 days and is strongly expressed after 3 weeks in such protocol. In peripheral clock this process also seems to develop after several cycles although in our group we have reported that with unpredictable feeding schedules modifications in behavior and metabolism can be observed after 24 hours of the last meal. The process and mechanisms underlying the process to develop FAA may lead to uncover pacemakers underlying food entrainment. Purpose: The aim of this study is to characterize the process in the circadian pattern of behavior and temperature in response to the first and second events of food access in a paradigm of food entrainment. Method: Adult male Wistar rats weighing 250-‐300 g at the beginning of the experiment were maintained in a 12:12h LD cycle, controlled temperature and free access to food and water. General activity was monitored with movement sensors under each cage, and temperature loggers (i-‐buttons) were inserted under the abdominal wall of the rats. Rats were divided in three groups; 1. Ad libitum (AL) control group had free access to food and water, 2. Restricted feeding group for one event (RF-‐1p) was fasted on day 6 and 7 and on day 8 the food was supplied for 2h from 12:00 hrs. to 14:00 hrs-‐. 3. Restricted feeding for 2 events (RF-‐2p) was fasted on day 6 and 7 on day 8 and 9 food was delivered for 2h. For both RF groups, the feeding events were followed by 2 days of total food deprivation to evaluate behavior and temperature food-‐entrainment. Results: The following day after the food event the RF-‐1p group showed behavioral activation and an increase of temperature at the same phase as food was given. The RF-‐2p group showed behavioral activation that lasted for two days after the pulses, they also showed a change in temperature corresponding to the phase of the feeding event. On the first day of food deprivation a low amplitude FAA was observed. Conclusion: Data here provided confirm that development of FAA is a gradual process and that it starts developing as early as day 2 of RF. Supported by CONACyT 82462 and PAPIIT IN-‐224911. Keywords: food anticipatory activity, circadian rhythms, food entrainment

211

P096 EXPECTATION FOR PALATABLE FOOD. A PROCESS THAT IS GENERATED GRADUALLY IN THE BRAIN Aurea Susana Blancas Velázquez, Katia Rodríguez Gonzales, Carolina Escobar Briones. UNAM, México. Introduction: Sweet and fatty food is strongly motivational for humans and rats. It has been observed that the animal becomes expectant to the palatable food very quickly (in a matter of days). When the stimulus is withdrawn, the behavioral and neuronal changes persist. Purpose: The aim of this study is to analyze how does the brain develop the activation of different nucleus such as the Nucleus Accumbens (NAcc), Prefrontal Cortex (PFCx) and Suprachiasmatic Nucleus (SCN) in rats that ingest 5 gr. of chocolate and those who are expectant for it. Methods: Male Wistar rats weighing 250-‐300gr were divided in two groups, those who were sacrificed after ingestion of chocolate and those who were sacrificed when they were expecting the palatable food without eating it. Sacrifice days were on days: 1, 2, 3, 5 and 8. Rats were perfused with 4% paraformaldehyde, brains were removed, posfixed and cryoprotected in 30% sacarose. Brains were cut with a cryostat and the slices were marked for Fos immunohistochemistry. Cells were counted for positive Fos immunohistochemistry. Results: The group who was sacrificed after ingestion of chocolate shows more Fos immunoreactive cells than controls, indicating activation of NAcc and PFCx. Within this group there’s not a difference in the activation among days, showing a reactive activation to palatable stimulus. The “expectant” group shows progressive activation that increases depending on the number of days of previous ingestion of chocolate. NAcc and PFCx show a progressive activation in the first days and in day 3 the activation is alike to that of rats that ate chocolate. SCN does not show any changes during ingestion and neither during expectation. Conclusions: The acquisition of a perseverant behavior where the animal looks for the pleasurable stimulus develops gradually and it involves both NAcc and PFC. Keywords: palatable meal, anticipation, motivation

P097 REGULATION OF HEPATIC MITOCHONDRIAL BETA-‐OXIDATION DURING THE EXPRESSION OF THE FOOD ENTRAINED OSCILLATOR Julieta Rivera, Mauricio Díaz. Instituto de Neurobiología, UNAM, México Restricted feeding schedules (RFS) promote numerous metabolic, physiological, and chronobiological adaptations to optimize the handling of nutrients. This condition activates

212

responses in hypothalamic and midbrain areas, as well as in peripheral organs involved in nutrient and energy metabolism that form part of a circadian clock known as the food entrained oscillator (FEO). RFS is associated with marked behavioral arousal previous to food access known as food anticipatory activity (FAA), and an extreme hyperphagia after the 2-‐h mealtime. Food restriction induces adaptations in the metabolic handling of energetic substrates by the liver and the adipose tissue during the FAA. For example, lipolytic release of free fatty acids (FFA) and production of ketone bodies are largely increased, whereas in the liver, the levels of triacylglycerols are reduced, but glycogen is only partially decreased. Fatty acids act as endogenous ligands of peroxisome proliferators-‐activate receptors (PPARalpha, beta, and gamma), which are transcriptional factors that influence peroxisomal and mitochondrial activities. PGC1-‐alpha coactivates PPARalpha starting the transcription of genes that encode for enzymes participating in the mitochondrial beta-‐oxidation of fatty acid, such as carnitine palmitoyltransferase 1 alpha(CPT-‐1 alpha This enzyme controls the transfer of long-‐chain fatty acid-‐CoA molecules from the cytosol into the mitochondrial matrix. The aim of this project was to determine metabolic parameters associated to beta-‐oxidation of fatty acids in liver mitochondria in rats entrained by circadian food availability. Control group: animals fed ad-‐libitum. Experimental group with restricted food access (from 12:00 to 14:00 h) during 3 weeks. At the end of the restricted food protocol, different subgroups of animals were sacrificed at 3 h intervals, starting at 08:00 h, to complete a 24 h cycle. The results showed by ad-‐libitum group: PPAR alpha and PPAR gamma showed a robust diurnal rhythmicity with a peak at 17:00 and 23:00 h respectively. PPAR beta displayed a nocturnal valley at the middle of the light period (17:00 h). FR group: PPAR alpha and PPAR gamma showed similar rhythmicity with a peak at 11:00 h (during FAA) and a valley at 23:00 and 02:00 h respectively. PPAR beta also exhibited a valley, but during the dark period (05:00 h). Perspectives: Explore: 1) complete mitochondrial beta-‐oxidation, 2) the expression of PGC-‐1 alpha and CPT-‐1 alpha and 3) activity of CPT-‐1 alpha. Keywords: Restricted feeding schedule, lipid metabolism, beta-‐oxidation

P098 STUDY OF THE ZONAL DISTRIBUTION OF LIVER PER1 PROTEIN DURING THE EXPRESSION OF THE FEEDING ENTRAINABLE OSCILLATOR Dalia Luz De Ita, Mauricio Diaz. Instituto de Neurobiología, UNAM, México. Biological clocks provide an adaptive advantage by ensuring that internal processes of the organism, both physiological and biochemical, coupled with the behavior are optimally adapted to the local envioronment. In mammals, the main biological clock is located in the hypothalamic suprachiasmatic nucleus (SCN) and es synchronized by day-‐night alternation.

213

The SCN controls the circadian activity, maintaining a time order across the interface with peripheral oscillators. However, when experimental animals are subjected to restrict feeding schedules (2-‐3 h per day) an emergent clock known as the Feeding Entrainable Oscillator (FEO) takes command of the circadian activity. The oscillations of both systems are controlled by "clock genes", among which is the gen per. Recently, it has become clear the intimate relationship between the circadian molecular mechanism and metabolic processes at key points such as energy charge, redox state, the calcium dynamics and nuclear receptors, among other. A central organ in the supply of energy to the body is the liver, functioning in the specialization of many metabolic functions according to the arrangement and anatomical characteristics of the cell populations (periportal and pericentral hepatocytes)of the liver acinus. In this work we elucidated the zonal distribution of the liver protein PER1 by letting the FEO to set. This was done by inmunohistochemistry and fluorescence microscopy. In conclusion, this study revealed that the distribution of PER1 in the periportal and pericentral hepatocytes varies according to metabolic and nutritional status of the organism. Keywords: liver, PER1, FEO

P099 SUB CELLULAR LOCALIZATION AND RELEASE OF THE MITOCHONDRIAL ENZYME ORNITHINE TRANSCARBAMYLASE, IN RAT LIVER DURING THE EXPRESSION OF THE FOOD ENTRAINED OSCILLATOR Braulio Ayala-‐García, Mauricio Díaz-‐Muñoz. Instituto de Neurobiología, UNAM, México. It is well known that the master clock in mammals is the suprachiasmatic nucleus in the hypothalamus which is entrained by the light/dark cycle. Nevertheless, it has been proposed the existence of another oscillator that is elicited when animals are subjected to restricted feeding schedules. This biological clock is known as the food entrained oscillator (FEO). It is believed that complex and dynamic interactions among peripheral oscillators are crucial for its emergence. However, information about the mechanisms of these interactions is still missing in order to achieve a more complete understanding of the FEO. Recently, it has been proposed that the enzyme Ornithine Transcarbamylase (OTC) can act as a signaling molecule during the proliferative phase of liver regeneration. Exploring this novel statement, we have hypothesized that this enzyme could have the same roll during the expression of the FEO, and hence, the aim of this study was to analyze the effects of the FEO on the subcellular localization and the release of this enzyme out of the liver. The qualitative presence and the activity were measured by means of western blot and a colorimetric method, respectively. Samples of serum, total liver homogenate, isolated mitochondrion and cytosol were collected along 24 h (in 3 h intervals) from rats under

214

restricted feeding schedule (from 12:00 to 14:00 h daily), from a control group fed ad-‐libitum, and from a fasting refeeding groups (feeding condition control). Liver fractions were used to detect OTC by immunofluoresence. The results suggest that the FEO has a clear effect in the localization and rhytmicity in the presence and activity of the OTC, and that there is a distinct pattern of the activity and presence within the different cellular compartments. It was also observed that FEO expression caused striking difference in comparison to control groups. Altogether, our results have led us to think that, during the FEO; this enzyme could have a novel function besides its classic catalytic role in urea cycle. Keywords: Food Entrained Oscillator, Ornithine Transcarbamylase, Liver

P100 DIFFERENTIAL INVOLVEMENT OF OREXIN CELLS IN FOOD ENTRAINMENT 1Angeles Jiménez, 2Mario Caba, 3Carolina Escobar. 1Centro de Investigación en Reproducción Animal; Cinvestav-‐Uat. Tlaxcala. Tlax 90000, México. 2Lab. Biología Reproducción Instituto de Investigaciones Biológicas, Universidad Veracruzana, Xalapa, Veracruz, México. 3Facultad de Medicina, Universidad Nacional Autónoma de México, México DF 04360, México. Rats under a restricted feeding schedule develop food anticipatory activity (FAA) 2 – 3 h prior food access, characterized by increased arousal, foraging and exploratory behavior. This FAA disappears when rodents are allowed ad libitum food access and reappears for several cycles when animals are fasted. Previously we reported in rats under a restricted feeding schedule (RFS) an increase in the expression of c-‐Fos protein in the dorsomedial (DMH), in the perifornical area (PeF) and in the lateral hypothalamic area (LH) anticipating and reacting to food intake. Considering that these hypothalamic structures contain abundant orexin (Orx) producing neurons and promote arousal, reward and metabolic balance, we explored the participation of the orexinergic system in rats under RFS and analized the expression of c-‐Fos in Orx cells by double label immunocytochemistry during and after food access, as well as in rats exhibiting persistent activation in fasting after RFS. RFS induced a daily rhythm of activity on Orx cells with highest levels at the time of FAA in the DMH, LH and PeF. In previously food-‐entrained rats, after 48 h of fasting the activation of Orx cells persisted in the PeF and LH with a similar temporal pattern as observed during RFS, however the DMH showed increased activation during fasting in all time points. We conclude that Orx cells in PeF and LH are related to a self sustained oscillator involved in FAA, whereas the DMH is related to the arousal mechanisms during FAA possibly associated with the metabolic condition resulting from the feeding-‐fasting alternation. Keywords: Hypocretins, Food-‐Entrainment, Hypothalamus

215

P101 NURSING TIME SYNCHRONIZES THE EXPRESSION OF PER1 PROTEIN IN THE PREOPTIC AREA AND BED NUCLEUS OF THE STRIA TERMINALIS OF THE FEMALE RABBIT 1José Enrique Meza, 1Rossana Zepeda, 2Mercedes Acosta, 3Stefan Walisewski, 1Mario Caba, 1Centro de Investigaciones Biomédicas, Universidad Veracruzana Xalapa, Ver., México. 2Facultad de Medicina, Universidad Veracruzana, Xalapa, Ver., México. 3Instituto de Medicina Forense, Universidad Veracruzana, Boca del Río Ver., México. The rabbit doe exhibits an unusual pattern of maternal behavior; in the pospartum its maternal care is restricted to a single 3-‐5 minute period to feed their litter once a day. Several studies suggests that neurons from the medial preoptic area (MPOA) and the bed nucleus of the stria terminalis (BST) form a common functional system important for maternal behavior, also these structures are sites where estradiol and lactogenic hormones act to facilitate the onset of this behavior. In the present study we explored by immunohistochemistry the expression of PERIOD1 (PER1) protein product of clock gene Period1 in MPOA and BST, considering their role in the regulation of maternal behavior in the rabbit and other mammals. The aim was to identify the effect of suckling stimulus on the pattern of per1 expression during circadian nursing of rabbit females. Method -‐ New Zealand white female rabbits were housed under controlled light cycle (12:12 h light/dark; light on at 07:00 = zeitgeber time ZT0). On the day of delivery the access to nest was closed and nursing was scheduled at ZT03 (AR-‐ZT03 group) and ZT19 (AR-‐ZT19 group). Besides, nonpregnant, nonlactating adult females were used as controls. After 7 days of lactation females were euthanized and perfused with saline solution and paraformaldehyde every four hours to complete a 24-‐hour cycle. We analyzed labeling of PER1 in brain structures by free floating immunohistochemistry. Preliminary results showed a peak of PER1 in lactating subjects according to their scheduled nursing, to prove whether this peak persists, does were deprived for one or two lactating periods and sacrificed at the time of expected maximal expression. Results-‐ Lactating females had a clear circadian rhythm of PER1 in the MPOA and BST, which peaks at 8h after nursing in both groups. On the contrary, control group had abundant PER1 without a circadian pattern in both brain structures. When the subjects were deprived of nursing the peak in MPOA there was a decrease in PER1 which was not significant in comparison to nursing group in both schedules. On the contrary, in BST the decrease of PER1 was significantly different from nursing subjects only in AR ZT19 group. According with our results probably the suckling stimuli during nursing entrains PER1 pattern in brain areas involved in maternal behavior and could be related to the circadian expression of this behavior in the rabbit. Keywords: Maternal Behavior, Clock gene, Circadian Rhythm

216

P102 FUNCTIONAL ADAPTATION OF THE GHRELIN-‐GROWTH HORMONE-‐IGF-‐1 AXIS DURING THE EXPRESSION OF THE FOOD ENTRAINED OSCILLATOR Elvira Arellanes-‐Licea, Martha Carranza, Maricela Luna, Mauricio Díaz-‐Muñoz. Instituto de Neurobiología, UNAM, México. Circadian food restriction in mammals promotes the expression of the food entrained oscillator (FEO). It is not currently know its anatomic substrate, but it is proposed that it involves the interaction of brain structures and peripheral organs such as the liver. The alternation among fasting (22 h) and feeding (2 h) could be implicated in the synchronization in the FEO, being part in the physiology of this clock endocrine cues whose secretion is regulated by feeding and the nutritional status. The activity of the hormonal axis ghrelin, growth hormone (GH) and the insulin-‐like growth factor-‐1 (IGF-‐1) could play a role in the FEO because ghrelin secreted during fasting promotes GH secretion, and in turn this last one promotes IGF-‐1 synthesis. We propose that in the FEO, hepatic biochemical adaptations before and after feeding, as well as the hyperphagia and posprandial stomach distention, will lead to changes in the secretion and the metabolic function of this axis. In a circadian protocol, comparing rats fed Ad libitum (AL) and rats with restricted feeding schedule (RFS 2 h from 12:00 to 14:00 h) we determined serum levels of total and active ghrelin, GH and IGF-‐1; hypophysis morphometry and GH content in addition to immunodetection of GH in adenohypophysis. We found that the secretion pattern of total ghrelin in the RFS group showed changes in its rhythmicity, as well as an increase in the 24-‐h average level. In contrast a decrease of circulating GH in the RFS group occurs, except immediately in the posprandial time point (14:00 h). Hyphophyseal weigth was lower during RFS, but surprisingly it had a higher GH signal per cell than the AL group. Finally, IGF-‐1 production showed changes in its rhythmicity and a decrease in the total levels during FEO expression. The changes in the temporal pattern of secretion in the components of this axis could lead to a differential signalling in its target tissues and be part of the physiological and metabolic rheostasis observed during FEO manifestation. Keywords: FEO, Hormones, Hypophysis

217

P103 SCHEDULED FEEDING DOES NOT REENTRAIN THE ESTROUS CYCLE IN FEMALE WISTAR RATS MAINTAINED IN CONSTANT LIGHT Donají Heredia García, Mariana Pliego Caballero, Isabel García-‐Peláez, Carolina Escobar Briones. UNAM, México. Introduction: A large number of women are exposed to night-‐work that alters circadian rhythms, disturbs menstruation, pregnancy and leads to early menopause and polycystic ovary (PCO) syndrome, characterized by menstrual irregularity, hirsutism, anovulation, and infertility. The rat estrous cycle is tightly regulated by the circadian system, under constant bright light (LL), rodents become arrhythmic and the estrous cycle stops, follicles develop to the preovulatory stage and secret estrogen, the ovary contains no corpora lutea nor ova. Constant secretion of estrogen results in continuous vaginal cornification, high uterine weight, and continuous sexual receptivity, constituting a state of persistent estrus (PE) that leads to PCO. Restricted feeding schedules (RFS) are strong entraining stimuli for locomotor activity and peripheral oscillators in intact and SCN lesioned animals. Thus, it is possible that RFS can be used to reestablish circadian rhythms, resynchronize the estrous cycle and prevent and/or treat PCO. Methods: 12 female Wistar rats (weighing 150-‐250g) divided in 2 groups. Group A: n=4, experiment length 12 weeks and group B: n=8, experiment length 15 weeks. All rats were housed in individual acrilic transparent cages placed over movement sensors, under controlled LD cycle (lights on at 07:00–19:00 h), temperature between 20-‐22°C, water and food ad libitum. During the experimental phase rats were exposed to LL, and restricted food access. The estrous cycle was determined with daily vaginal smears in baseline and in each experimental condition for 3 weeks. Group A. The effects of 6 weeks in LL and the recovery in a 12-‐h LD cycle were tested. Group B. After 8 weeks in LL, rats were exposed to a 12 h cycle of scheduled food access (SF) for 4 weeks (food from 20:00-‐8:00), vaginal smears were taken during the last 10 days. Results: In an LD cycle all rats were cycling (4-‐5 days cycle). Actograms and activity analysis indicated a synchronized rhythm with a period of 24 h. LL induced loss of rhythmicity in general activity, confirmed by no-‐peak on the periodogram and an equal activity percentage during subjective day and night, vaginal cytology indicated loss of rhythmicity and PE. In group A, when LD was reestablished, estrous cycle reappeared after 4 days in 50% of the rats while 50% remained in diestrus. In group B, general activity entrained slowly to SF, after 4 weeks exhibited a significant period of 24 h with a low amplitude, and rats remained in PE. Conclusion: The estrous cycle depends of the light-‐dark alternation. In LL when circadian rhythms are altered, persistent estrus appears. SF for 12-‐h synchronizes circadian rhythms of behavior, but not the estrous cycle. Supported by CONACyT 82462. Keywords: reproduction, food entraiment, desynchrony

218

P104 DAYTIME RESTRICTED FOOD SCHEDULE CHANGED THE HEPATIC ZONATION OF THE PHOSPHOENOLPYRUVATE CARBOXYKINASE 1 Moisés Pérez-‐Mendoza, Mauricio Díaz-‐Muñoz. Instituto de Neurobiología, UNAM, México. When the glycemia decreases, like in fasting or between meals, circulating glucose recovers by two principal processes: glucogenolysis and gluconeogenesis. If the fasting period is too long, the organism obtains energy from acetyl-‐CoA produced by the degradation of the free fatty acids (FFA). Animals in a restricted food schedule (RFS) expressing the food entrained oscillator (FEO) showed a temporal profile of 24 h characterized by hypoglycemia and increased phosphoenolpiruvate carboxykinase (PEPCK) activity with a period close to 12 h. It has been reported that in RFS before food access high levels of circulating FFA and ketone bodies, increased ATP and an oxidized redox state in the liver, partial reduction of hepatic glycogen and a high level of glucose. It has been suggested that the liver metabolism adopt a rheostatic adaptation during the RFS. As a consequence, our hypothesis is that RFS promotes an upregulated response of hepatic gluconeogenesis (even as a higher glycemia, or an increased glycogen production in the periportal hepatocytes). Hence, the aim of this project was to explore the process responsible of maintaining the glycemia and the regulation of the hepatic gluconeogenesis during the expression of FEO. We analyzed the gluconeogenic response and the presence and distribution of key enzymes of the gluconeogenesis such as PEPCK in the liver by alanina tolerance test and immunohistochemical localization (in liver slides). We found significant differences in the circulating glucose formed from intraperitoneally injected alanine. RFS rats showed a similar response in comparison to the animals fed AL, whereas the fasting group increased the level of glucose 20 min postinjection. By other way, the hepatic zonation changed in the animals under RFS, increased the presence of PEPCK in the periportal hepatocyte versus pericentral hepatocite before food access. These data suggest that PEPCK could be active synthesizing glycogen.in this type of hepatocytes. Therefore our results suggest that gluconeogenesis is upregulated in the RFS condition than in AL. Hence, the metabolic changes detected in the liver also strengthen the notion that RFS promotes a rheostatic adjustment in liver physiology during FEO expression. We are thankful to the CONACYT for financial support (project U-‐49047), PAPIIT (201209) and also to the CONCYTEQ. The technical assistance of Dra.Olivia Vázquez–Martínez is gratefully recognized. Keywords: PEPCK, gluconeogenesis, glycogen

219

P105 GLUCOCORTICOID RECEPTOR SIGNALING IS MODIFIED BY RESTRICTED FEEDING Alma Dalia Luna, Mauricio Díaz. Instituto de Neurobiología, Campus UNAM-‐Juriquilla, México. Under protocol of restricted food, the circadian rhythmicity is controlled not by the suprachiasmatic nucleus but by a different biological clock known as the Food Entrained Oscillator (FEO). There is a behavioral and physiological arousal previous to food access that characterized the FEO. During this stage a rise in plasma corticosterone is detected. This hormone is very important in the synchronization of the timing system and is one of the principal factors that modulate glucose (gluconeogenesis), protein (ureogenesis) and lipid metabolism. It also plays a significant role as anti-‐ inflammatory agent. These actions are mediated by intracellular glucocorticoid receptor (GR). The aim of our project is to characterize the corticosterone signaling in the liver during the expression of the FEO. We have studied the presence and cellular distribution of GR as well as urea formation and the activity of several enzymes of urea cycle. The results obtained by Western immunoblot showed that restricted feeding led to a significant increase in cytosolic and a decrease in nuclear GR levels compared to ad libitum animals. Carbamoyl-‐phosphate synthase I (CPS1) is the rate-‐ determining enzyme in urea cycle, this enzyme and Ornithine transcarbamylase (OTC) are located within the mitochondrial matrix. Urea levels and both CPS1, OTC activities were determined by enzymatic method. There were no significant changes in average values of urea cycle parameters during the FEO expression. However, important modifications were observed in the rhythmic patterns of urea and CPS1 and OTC activities. Preliminary conclusions suggest that FEO induces profound changes in liver corticosterone signaling that influence the rhythmicity of the urea cycle. Keywords: glucocorticoid receptor, urea cycle, anticipatory activity

P106 INDUCTION OF FOS AND PER1 PROTEINS IN THE MAIN OLFACTORY BULB OF RABBIT PUPS IN RELATION TO SCHEDULED NURSING 1Nahum Nolasco, 2Enrique Meza, 2Mario Caba. 1Doctorado en Ciencias Biomédicas, México. 2Centro de Investigaciones Biomédicas, México In nature and under laboratory conditions, rabbit pups suck milk once a day with circadian periodicity. Previously we reported that between postnatal days 3-‐4 rabbit pups develop food anticipatory activity (FAA) as indicated by a significant increase in locomotor behavior 3-‐4 h before mother arrival. Besides that several metabolic, hormonal and neural parameters shifts in parallel to the timing of nursing. Olfactory cues play a decisive role in

220

the search and suckling of milk as anosmic pups are unable to suck milk and die of starvation. In the present contribution we explored the role of olfactory cues on entrainment of FAA in rabbit pups. To this aim we determined the expression of FOS and PER1 proteins, the product of the Per1 clock gene by immunohistochemistry in the main olfactory bulb (MOB). Pups were nursed either at 10:00 or 02:00 h and were sacrificed at postnatal day seven either just before their scheduled time of nursing or at 1.5, 4, 8, 12, 16 or 20 h after nursing. All pups were entrained to the nursing schedule as evidenced by the expression of FAA. FOS and PER1 were observed at all times in the bulb however we determined a peak associated to their nursing schedule. There was a sharp increase of FOS 1.5 h after suckling of milk, significantly different than remaining time points. In contrast there was a significant increase of PER1 8 h after suckling of milk. In both cases, peak induction of proteins shift in parallel to nursing schedule. These patterns were observed in the periglomerular, mitral and granular cell layers of the MOB. We also observed FOS and PER1 in the accessory olfactory bulb but not a clear pattern was determined. In the suprachiasmatic nucleus both FOS and PER1 have a rhythm which did not change in relation to the nursing schedule. We conclude that the induction of FOS and PER1 proteins may play a key role in entraining of FAA of rabbit pups. This work was supported by CONACYT (Ref. 215326 to N. N.) Keywords: circadian rhythms, clock genes, olfactory cues

P107 REFEEDING AFTER FASTING ELICITS INSULIN-‐DEPENDENT REGULATION OF PER2 AND REV-‐ERBA WITH SHIFTS IN LIVER CLOCK Yu Tahara, Makiko Otsuka, Yuta Fuse, Akiko Hirao, Shigenobu Shibata. Department of Physiology & Pharmacology, School of Advanced Science and Engineering, Waseda University, Japan. In nocturnal animals, the daytime restricted feeding paradigm induces a peripheral circadian clock entrainment to participate the feeding time and also to make a preparation of food metabolism by an induction of clock-‐controlled gene expression. Recently, it is suggested that the food entrainable peripheral oscillator (FEPO) was there in the peripheral tissues, because this food entrainment of the liver could be observed within 2-‐3 days before appearance of food anticipation (FAA) which is induced by food entrainable oscillator (FEO) also in the brain (Hirao et al., 2009). However, the entrainment mechanism of this FEPO is not fully understood. In the present study, we focused on the first input of liver clock gene expression rhythm in a fasting-‐refeeding condition. To elucidate this, we searched the circadian clock gene(s) which show both phase advance and acute change of gene expression during the early term of the daytime refeeding schedule in mice, and the genes showed both changes are thought to be a key factor of liver clock phase-‐change. The up-‐

221

regulation of Per2 gene and down-‐regulation of Rev-‐erba gene were observed 2 hr after refeeding at ZT6, and these expression rhythms after refeeding were significantly phase-‐advanced. Additionally, the up-‐regulation of Per2 mRNA and down-‐regulation of Rev-‐erb mRNA were induced within 2 h by insulin injection in intact mice. These expression changes by food intake were not revealed in streptozotocin-‐treated insulin-‐deficient mice, but insulin injection was able to recover the impairment of Per2 and Rev-‐erb gene expression. Therefore, we suggest that a feeding induced insulin secretion is important for the early signal to the food entrainment of FEPO. Furthermore, 2 days insulin injection to intact mice or streptozotocin-‐treated mice could phase advanced of the liver Per2 expression rhythm in vivo, and insulin itself induced the phase-‐shift of Per2::luc mouse embryonic fibroblasts (MEFs) with up-‐regulated Per2 expression and down-‐regulation of Rev-‐erb in vitro. The data strongly suggests that insulin directly affects the peripheral clock, and MEFs may be a possible material for the peripheral food entrainment study. So we are now investigating the additional nutrient signal effects, such as amino acid or vitamin or and so on, for the insulin inducible phase shift in vitro. Keywords: liver clock, insulin, restricted feeding

P108 RABBIT NURSING SHOWS CIRCADIAN PERIODICITY AND IS MODULATED BY SUCKLING STIMULATION CHARACTERISTICS 1Gabriela González-‐Mariscal, 2Ana Celia Lemus, 3Raúl Aguilar-‐Roblero. 1CINVESTAV-‐UAT, México. 2UAT, México. 3Instituto Fisiología Celular, Universidad Nacional Autónoma de México, México.

Nursing rabbits kept under a 14:10 (L:D) photoperiod were video-‐monitored continuously across lactation days 1-‐15. The time and duration of nursing were determined in mothers provided with 1, 2, 4, or 6-‐8 pups inside the nest box (Exp 1). A Rayleigh analysis revealed that mothers given 6-‐8 pups nursed them once/day and provided a vector with a phase angle=58°, rho=0.78 and p<0.001. Nursing bouts lasted ca. 3 min, which is the norm for rabbits. Mothers given 1, 2, or 4 pups showed multiple entrances into the nest box throughout the day (range=2-‐13) and stayed inside it for 0.85-‐61 min. These findings suggest that a threshold of suckling stimulation is necessary to maintain the circadian periodicity of nursing and a normal duration of the suckling bout. To investigate if lactation per se is necessary for this process we used the model of anosmic virgins (Exp 2), as such rabbits show “pseudo-‐nursing” behavior towards foster pups. Providing litters of 4 young (that were exchanged daily to be nursed by their own mothers) provoked in anosmic virgins an irregular, unpredictable number of entrances into the nest box/day and the adoption of a nursing posture over the pups that lasted longer than a normal suckling episode. In Exp 3 (in progress) we are exploring the effect of providing larger litters to anosmic virgins. The

222

results from such experiment will reveal if the differences between anosmic virgins and nursing mothers persist under equal amounts of suckling stimulation and will, consequently, indicate the relevance of lactation per se for the circadian display of rabbit maternal behavior. Keywords: nursing, rabbit, circadian

P109 THE SUPRACHIASMATIC NUCLEUS IS NECESSARY FOR CHOCOLATE ENTRAINMENT Katia Rodríguez González, Aurea Susana Blancas, Manuel Ángeles Castellanos, Carolina Escobar. UNAM, México. Rats exposed to daily access to 5 grams of chocolate develop brief and precise anticipatory activity and in D/D conditions. Daily scheduled chocolate access entrains locomotor activity and Per1 in the SCN (Mendoza et al, 2005). It is suggested that the SCN may be interacting with limbic areas and might be necessary for motivational cues entrainment. In order to determine how the SCN may contribute to chocolate entrainment, rats bearing a complete bilateral lesion of the SCN were evaluated on daily rhythms of general activity and core temperature under a protocol of daily chocolate access. Neuronal activation during the interval of chocolate anticipation was also evaluated. Rats, between 200 to 250 g, were housed in individual cages placed on plates with movement sensors in soundproof lockers with controlled lighting conditions (L/D 12:12). After 2 weeks of acclimation to the monitoring system rats were randomly assigned to an intact SCN control group (CTRL), or to an SCN lesion group (SCNX). After surgical procedures rats were allowed to recover for at least a month before starting the study. General activity was evaluated to confirm loss of rhythmicity. Rats that exhibited a daily rhythm were assigned to a partial lesion group (PLX). CTRL and SCNX (n=4) rats underwent surgery in order to place intra-‐abdominal temperature sensors, (iButton Sensor-‐Temperature Logger; Maxim Integrated Products, Dallas, Semiconductor, USA). In order to determine neuronal activation during anticipation to, CTRL and SCNX rats (n=4) were anesthetized and perfused at the time of chocolate delivery (ZT6). During baseline the control group showed robust 24 h rhythms in locomotion activity and core temperature. Rats in the SCNX group showed complete loss of rhythmicity in both variables and at the end of the experiment the lesion was verified with Nissl staining and c-‐fos inmunohistoquemistry. The PLX group showed a similar temporal pattern of general activity as the control group. In the CTRL group chocolate entrainment produced anticipatory activity in general locomotion, his temporal pattern persisted for 3 days after interrupting the chocolate delivery protocol. Interestingly core temperature did not exhibit a chocolate entrained pattern. The SCNX group maintained its arrhythmic pattern and did not develop anticipation to chocolate as observed with general activity and core

223

temperature, while the PLX group developed a similar response as the CTRL. . Neuronal activation in the Accumbens, was low in SCNX as compared to CTRL and PLX rats. Present data indicate that the SCN is relevant for chocolate entrainment and possibly modulates temporal responses to motivational zeitgebers. Supported by PAPIIT IN 203 907 and CONACYT 82462 Keywords: palatable meal, SCN lesion, motivational zeitgeber

P110 DAILY RHYTHM OF BLOOD GLUCOSE INCREASE BY HIGH OR LOW DIGESTIBLE STARCH INTAKE IN NORMAL MICE AND MICE WITH RESTRICTED FEEDING PARADIGM Akiko Hirao, Misa Itokawa, Hiroki Nagahama, teiji Outsu, Takuma Imanishi, Ayako Shinozaki, Yu Tahara, Shigenobu Shibata. Waseda University School of Advanced Science and Engineering Physiolosy and Pharmacology, Japan. Blood glucose level is influenced by two main factors such as digestive-‐absorption rate and insulin secretion. Basal secretion of insulin may be controlled by circadian rhythm, and also phased secretion of insulin is controlled by feeding-‐induced increase of blood glucose level. On the other hand, digestive systems such as digestion of food and absorption of nutrients are also controlled by circadian rhythm. Daily restricted feeding (RF) entrains the circadian rhythm of mouse clock gene expression in the central nervous system excluding the suprachiasmatic nucleus (SCN), as well as in the peripheral tissues such as the liver, lungs, and heart. Therefore, digestive systems are controlled by light-‐entrainable SCN circadian oscillator (LEO), and also by food-‐entrainable circadian oscillator (FEO). In our previous paper we demonstrated that combination food of carbon hydrate plus casein was good entrainable nutrients, and that high digestible starch has stronger entrainable signal than slow one (Hirao et al., 2009). The purpose of present study is to elucidate whether LEO and/or FEO control blood glucose and insulin increase induced by high or low digestible starch injection. After 24 hrs starvation, AIN-‐93M(0.1g/ml, 1ml per mouse) (substitution of corn starch by beta corn starch or beta potato) was administered orally to ICR male mouse at ZT6 or ZT18, and then blood sample was collected 0, 15, 30, 60, 90, and 120 min after administration. The contents of glucose and insulin were measured. Similar experiment was applied to mouse with restricted feeding during ZT0-‐ZT4 or ZT12-‐16. Increase of blood glucose was stronger in corn starch-‐treated mouse than in potato-‐treated mouse suggesting that digestion/absorption process of starch is important for increase of blood glucose after food injection. In comparison of food injection with ZT6 or ZT18, increase of blood glucose is stronger at ZT6 than at ZT18 in both corn starch-‐treated and potato-‐treated mice, suggesting that insulin secretion response is faster and stronger at ZT18 than at ZT6. Increase of blood glucose by corn starch or potato at ZT0 for ZT12-‐ZT16 RF mice was similar

224

to that at ZT12 for ZT0-‐ZT4 RF mice, suggesting that circadian rhythm of food intake-‐induced glucose increase is under control of FEO rather than LEO. Further experiments using STZ-‐treated insulin deficit mice and butyl-‐scopolamine-‐treated digestion deficit mice are planned to demonstrate the mechanism of circadian change of high or low digestible starch-‐induced glucose increase under the control of FEO. Keywords: Blood glucose level, FEO, restricted feeding

P111 DO NEWBORN RABBITS BEARING SUPRACHIASMATIC NUCLEI LESIONS ANTICIPATE TO NURSING? 1Oscar Hernández-‐Campos, 2Rodrigo Montúfar-‐Chaveznava, 1Ivette Caldelas. 1Depto. Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, 2División de Tecnologías Urbanas, Instituto de Ciencia y Tecnología del Distrito Federal, México. In mammals the circadian pacemaker is located in the suprachiasmatic nuclei (SCN) of the hypothalamus; single neurons of this nucleus are able to generate and maintain self-‐sustained circadian oscillations and regulate several physiological outputs. It’s well known that SCN lesions abolish the rhythmic expression at behavioural and physiological level, but does not affect the emergence of food anticipatory activity in adult rodents maintained under schedules of food access. However, there is a lack of evidence about the importance of this hypothalamic area in the anticipatory phenomena during early stages of development. An animal model unusually suited for the study of biological rhythms during early mammalian development is provided by the European Rabbit (Oryctolagus cuniculus), this is due to the rabbit’s unusually limited pattern of maternal care; during the first weeks of life the newborns are maintained in a burrow, under complete darkness and largely isolated from potentially entraining environmental signals other than the vital, once-‐daily nursing visit of their mother and they anticipate and prepare themselves for it. For the first time, we determine the effect of SCN lesions on the anticipatory phenomena in newborn rabbits. For this reason newborn rabbits were separated from their mothers at birth and maintained in L:L, from the postnatal day 1 (P1) to P19. Rabbits were randomly assigned to one of three conditions: intact, sham or SCN lesion groups. In P4 the electrolytic lesions were performed estereotaxically and also were implanted telemetry sensors for the continuous recording of locomotor activity and core body temperature. At the end of the experiment the rabbits were deeply anesthetized and perfused for the histological verification of the SCN lesions. The locomotor activity and temperature time series were analyzed by Fourier transform. The newborn rabbits of the three groups under study exhibited diurnal patterns in locomotor activity and core body temperature; in all the cases the anticipatory component was evident, even if the rabbits were isolated from the mother

225

during 48 hours. The present data indicate that other circadian oscillators than the SCN are possibly involved in the generation of the anticipatory component. Supported by ICYTDF/261/2009, PAPIIT IN219510-‐3 and CONACyT 131314. Keywords: SCN lesion, food anticipatory activity, circadian oscillator

P112 DOES THE OLFACTORY BULB OF NEWBORN RABBITS EXHIBIT TIME DEPENDENT RESPONSES TO THE MATERNAL PHEROMONE 2MB-‐2? 1Lucero Anabel Trejo-‐Muñoz, 2Rodrigo Montúfar-‐Chaveznava, 1Ivette Caldelas. 1Depto. Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México. 2División de Tecnologías Urbanas, Instituto de Ciencia y Tecnología del Distrito Federal, México. An animal model unusually suited for the study of biological rhythms during early mammalian development is provided by the European Rabbit (Oryctolagus cuniculus); this is due to the rabbit’s remarkably characteristic maternal care pattern. During pre-‐visual stages the newborn rabbits are usually maintained in a dark nursery burrow and the lactating female only visits their young for a few minutes once every 24 hr. Under such conditions, pheromonal cues such as the 2-‐metyl-‐2but-‐enal (2MB-‐2), play an important role for the location of nipples allowing efficient suckle. In addition, recent studies of our group, demonstrate that it is possible to synchronize the locomotor activity and core body temperature rhythms by daily exposition to the maternal pheromone 2MB-‐2. Furthermore, previous studies of our group demonstrate that the clock genes Per1, Bmal1 and Cry1 exhibit diurnal oscillations in the main olfactory bulb (OB) of the newborn rabbits, indicating that the molecular clockwork develops earlier in the OB than in the SCN. In order to determine whether the olfactory bulb of newborn rabbits exhibit phase responses to the maternal pheromone 2MB-‐2, newborn (n=48) rabbits were kept in constant light and fed by a lactating female every 24-‐h, from the postnatal day 1 to 6 (P1-‐ P6). On P7 rabbits were isolated from maternal cues and exposed to one of three odor stimuli -‐ 2MB-‐2 pheromone, Ethyl isobutyrate or water pulse-‐ for 5 minutes, at different times (ZT00, 06, 12 and 24). Pups were killed 1h after pulse exposure, the brain was removed and the tissue was prepared for the c-‐Fos detection by immunohistochemistry in the olfactory bulb and in SCN. Keywords: Olfactory Bulb, Phase Response Curve,

226

P113 PHOSPHORYLATION-‐DEPENDENT DEGRADATION OF CRY2 REGULATES CLOCK GENE EXPRESSION AND CIRCADIAN PERIOD Arisa Hirano, Nobuhiro Kurabayashi, Yoshitaka Fukada. Department of Biophysics and Biochemistry, Graduate school of Science, The University of Tokyo, Japan. Cryptochrome proteins are critical players for molecular oscillations in the circadian clocks of central and peripheral tissues in mammals. CRY2 is phosphorylated at Ser557 located in the unique C-‐teminal region in the mouse SCN and liver, in both of which Ser557-‐phosphorylation form accumulated in parallel with CRY2 protein (Kurabayashi et al., Chronobiol. Int., 2006; Harada et al., JBC, 2005). The priming phosphorylation of mCRY2 at Ser557 by DYRK1A allows subsequent phosphorylation at Ser553 by GSK3ƒÀ, resulting in proteasomal degradation of CRY2 (Kurabayashi et al., MCB, 2010). To reveal the physiological importance of Ser557 phosphorylation in vivo, we generated mutant mice expressing S557A-‐CRY2 protein. First, we analyzed CRY2 protein level because the mutation site is important for CRY2 degradation. As expected, CRY2 protein level was increased in mutant mouse liver lysate. PER2 protein, a partner of CRY2, was also elevated in mutant mice. It is known that CRY2 inhibits ubiquitination of PER2 in cultured cells (Yagita et al., 2002) and therefore, it is suggested that S557A mutation of CRY2 affected the stability and expression level of PER2. Then, we analyzed the effect of the S557A mutation on expression rhythms of the clock genes in the mouse liver and fibroblast cells. We found that the S557A mutation of CRY2 decreased mRNA expression levels of Cry2, Per1, Dec1 and Dbp. These data indicate that accumulation of CRY2 protein by the mutation caused downregulation of E-‐box-‐regulated gene expressions. Interestingly, the wheel-‐running activity analysis revealed that mutant mice showed significantly longer free-‐running period compared to that of the wild-‐type. Our results demonstrate that Ser557 phosphorylation of CRY2 regulates clock gene expressions and circadian period in vivo. Keywords: cryptochrome, phosphorylation, mammal

227

P114 DETERMINATION OF INTRON MOVEMENT IN PER GENE 1Gabina Calderón Rosete, 2Francisco Martínez Pérez, 3 Juan A. González Barrios, 4 Luis Kameyama, 2Jorge L. Fuentes Lorenzo, 1Leonardo Rodríguez Sosa 1Departamento de Fisiología, Facultad de Medicina UNAM., México. 2Laboratorio de Microbiología y Mutagénesis Ambiental, Escuela de Biología, Universidad Industrial de Santander, Colombia. 3Laboratorio de Medicina Genómica, Hospital Regional 1ero de Octubre, ISSSTE, México. 4Departamento de Genética y Biología Molecular, CINVESTAV, México. The first described clock gene, per (period), was encountered in Drosophila melanogaster and is compose by 9 exons and 8 introns. In mammalian, Per proteins may be transcribed by 3 genes. The difference in the number of introns, the position and the phase among the members from a gene family has been used to propose the origin of intron. An early model for this origin suggests that the introns were present in the ancestral genome and they were eliminated in the prokaryotic cells while in eukaryotic they continue. There is another model which claims that the presence of the introns in eukaryotic cells is recently, because the introns were absent in prokaryotic cells. To per gene, the intron analysis among invertebrates and vertebrate was determined between D. melanogaster and Homo sapiens suggesting that the origin in vertebrate corresponds to the early model. However, the putative intron relation among invertebrates and vertebrates to explain the gene evolution with other invertebrate no has been established yet. In this work, the intron analysis from arthropods and vertebrates were revised to determine the putative intron evolution. The results showed that the intron position and phase suggest that new introns were obtained in vertebrate, probably were eliminated from the ancestral gene. Some common introns had phase 2 or 3, suggesting the presence of alternative splicing in this regions in arthropods. In addition, we investigated the possible exon skipping in per mRNA RT/PCR from the crayfish Cherax quadricarinatus with cDNA RACE library from abdominal ganglia obtained of animals adapted to cycles light:dark (12:12 h) shows 3 amplicons. Our experimental results in crayfish C. quadricarinatus are analyzed and discussed in relation to the hypothesis that some introns are ancient in the per gene while others were included in the genetic evolution to participate in alternative splicing. Supported by: Facultad de Medicina, UNAM to LRS. ISSSTE 507.2009 and CONACYT 126646 to JAGB Keywords: Crayfish, Cherxax quadricarinatus, clock genes

228

P115 EXPLORATION OF THE HUMAN MOLECULAR CLOCK IN FIELD STUDIES Marta Novakova, Martin Sladek, Alena Sumova. Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

Functionality of the human circadian system has been mostly assessed by determination of daily melatonin (or its metabolite) levels in bodily fluids like blood, saliva or urine. The melatonin secretion from the pineal gland is controlled by the circadian clock located in the suprachiasmatic nuclei of the hypothalamus and may thus serve as a reliable hand showing the time of the internal clock. Melatonin levels are also sensitive to light at subjective night and, therefore, exposure to environmental light may affect endogenous melatonin levels especially in field studies when circadian system is explored under real-‐life conditions. Therefore, introduction of an additional marker of human circadian system for its usage in field studies is advisable. Recently, a new non-‐invasive method for determination of clock gene expression in buccal mucosa samples collected from subjects under constant posture protocol has been introduced (Cajochen et al., 2006, Eur. J. Neurosci., 23:1082¨C1086). The aim of this study was to adapt this method for its applicability in studies of circadian system under real life conditions. To achieve this goal, we have compared daily profiles of melatonin in saliva and those of expression of clock genes in samples of buccal scrubs obtained from healthy children and adult volunteers. 9 males and 13 females, aged 3 ¨C 56 years participated in the study. The subjects were asked to keep their habitual sleep-‐wake schedule for 1 week before the study and not to use bright light on the day of sampling. Adult subjects were also chronotyped using adapted Munich chronotype questionnaire. On the day of the study, the subjects provided samples of saliva and buccal mucosa in their home every 4 h starting from 11:00 till 11:00 on the next day. The melatonin levels were determined using direct double-‐antibody radioimmunoassay (B¨_hlmann Laboratories, Allschwil, Switzerland). The Per1, Per2 and Rev-‐erb_Á expression profiles were determined by RT-‐PCR. The results demonstrated high correlation between the phases of the melatonin and clock gene expression profiles in individual subjects. Therefore, we have proven a suitability of the method for determination of basic characteristics of the circadian system, namely of its phase, in individual subjects in studies under real-‐life conditions. Sponsored by the Internal Grant Agency of the Ministry of Health of the Czech Republic, grant no. NT11474-‐4/2010, the Grant Agency of the Charles University in Prague, no. 22810, the Grant Agency of the Czech Republic, no. 309/08/H079 Keywords: human, melatonin, clock gene

229

P116 DAILY FLUCTUATIONS IN THE BIOSYNTHESIS OF PHOSPHATIDYLCHOLINE AND ACTIVITY OF ITS KEY ENZYME CTP: PHOSPHOCHOLINE CYTIDYLYLTRANFERASE IN SYNCHRONIZED CULTURES OF FIBROBLASTS Victoria América Acosta Rodríguez, Sebastián Márquez, Mario Eduardo Guido. CIQUIBIC-‐Department of biological chemistry, faculty of chemical science, National University of Cordoba-‐CONICET, Argentina. An open question in biology is how the lipid components of the biological membranes are synthesized and assembled to maintain membrane homeostasis and the diverse cellular processes. Circadian clocks regulate diverse biochemical functions, whereas cell cultures make up a model of peripheral oscillators to investigate metabolic oscillations. We previously reported that the phospholipid biosynthesis oscillates daily in synchronized fibroblast cultures; however, the way phosphatidylcholine (PC) synthesis is daily regulated in cell cultures, is unknown. PC is mainly synthesized through the Kennedy’s pathway with CTP: phosphocholine cytidylyltranferase (CCT) as key enzyme. In mammals, there are two genes encoding for CCT: Pcyt1a that encodes the CCTa protein from alternative transcripts termed CCTa1 and CCTa2 and Pcyt1b gene that encodes the CCTb2 and CCTb3 proteins from differentially alternative spliced mRNA CCTb2 and CCTb3. Here, we investigated possible temporal changes in PC biosynthesis and CCT activity and expression in quiescent NIH 3T3 fibroblasts after a 2 h-‐horse serum shock synchronization. We found that the PC labeling with [3H]glycerol or [32P]phosphate in the cultures exhibits a daily variation with levels peaking at 4-‐6 h after serum stimulation and decreasing by 29-‐32 h. Significant daily variations were also seen in CCT activity with higher levels at 6 and 35 h during the first and 2nd cycles respectively. Moreover, CCT expression across time for the most abundant isoforms, CCTa1 and CCTb2, at mRNA and protein levels displayed complex pattern of expression. Both CCTa1 transcript and protein peaked at 3 h post stimulation, while CCTb2 mRNA was higher at 9-‐12 h. We also detected increased levels of CCTb isoforms during 9-‐18 h using an antibody that recognized both CCTb2 and CCTb3 proteins. Results demonstrated that synchronized fibroblasts exhibit temporal variations in PC biosynthesis which could be due, at least in part, to changes in CCT activity and/or to a concerted and differential expression of different CCT isoforms. Supported by: ANPCyT-‐FONCyT (PICT 04 967 – PICT 06 898), CONICET, SeCyT-‐UNC and MinCyT-‐Cba. Keywords: phospholipid, peripheral clocks, NIH 3T3

230

P117 OPSIN EXPRESSION AND LIGHT RESPONSES IN RGC-‐5 CELLS Paula Sofía Nieto, Diego Javier Valdez, Victoria América Acosta-‐Rodríguez, Mario Eduardo Guido. CIQUIBIC-‐Department of biological chemistry-‐faculty of chemical sciences, National University of Córdoba-‐ CONICET, 5000 Córdoba, Argentina, Argentina. The vertebrate retina contains at least three classes of photoreceptor cells, cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-‐visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this work we investigated the potential capability of the rat RGC line (RGC-‐5) as intrinsic photosensitive cells by assessing the presence of visual and non-‐visual opsins as well as the responses to brief light pulses on the expression of the immediate-‐early gene protein c-‐Fos, and intracellular Ca2+ mobilization. We observed that cultured RGC-‐5 cells express opsin mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin but not melanopsin (Opn4) or rhodopsin. Furthermore, the expression of the OPN5 in RGC-‐5 cells was confirmed by inmunocitochemistry. In addition, we report for the first time the localization of this opsin within the mammalian (rat) retina: OPN5 is expressed in some neurons of the inner nuclear layer and in the ganglion cell layer. Strikingly, functional assays in RGC-‐5 cells showed significant changes in intracellular Ca2+ levels in subsets of RGC-‐5 cells after bright white light pulses of 30 to 60 sec of duration while cells exhibit the induction of the c-‐Fos protein by a 30 min-‐light pulse. In summary, our results indicate that RGC-‐5 cells express diverse putative functional photopigments and display the intrinsic photic induction of c-‐Fos protein and changes in intracellular Ca2+ mobilization. Supported by: ANPCyT-‐FONCyT, (PICT 04 967/PICT 06 898), SeCyT-‐UNC and MinCyT-‐Cba. keywords: opn5, rgcs, rgc-‐5

P118 CIRCADIAN REGULATION OF CYTOPLASMIC MRNA-‐GRANULES. Juan Ignacio Lescano, Mario Eduardo Guido, Eduardo Garbarino Pico. CIQUIBIC-‐Dpto. de Química Biológica, fac. Ciencias Químicas, Universidad Nacional de Córdoba., Argentina. Gene expression is modulated by clocks, 5-‐20% of the mRNAs expressed in a given tissue show daily oscillations. Presumably, this is largely generated by changes in the transcription of those genes; however, the relative importance of mRNA post-‐transcriptional processing has not been established. Stress granules (SG) and P-‐bodies are cytoplasmic subdomains

231

involved in the regulation of translation, mRNA decay and storage. They are formed by RNA and a number of factors involved in mRNA cytoplasmic processing. Considering that some of the SG and P-‐body components are expressed under circadian basis, we have analyzed whether these foci show rhythmic changes. NIH3T3 cells were synchronized by serum shock and fixed at different times. SG and P-‐bodies were detected by ICC. P-‐body per cell (number) showed fluctuations peaking 28 h after synchronization, and reaching highest areas 14 h after the shock. We applied arsenite (oxidative stress) for inducing SG. The number of SG/cell did not change through time; however the area and perimeter of them showed daily variations with peak 14 h after serum shock. Further studies will be carried out with other markers to confirm these results; however they suggest that these foci, or a subpopulation of them, are regulated by a circadian clock. Supported by: IBRO, CONICET, FONCyT, SECyT-‐UNC y Ministerio de Ciencia y Tecnología de Córdoba. Keywords: Post-‐translational regulation, Processing bodies, Stress granules

P119 JNK PHOSPHORYLATES BMAL1-‐CLOCK COMPLEX AND CONTROLS OSCILLATION SPEED AND PHOTIC REGULATION OF THE CIRCADIAN CLOCK 1Yoshitaka Fukada, 1Hikari Yoshitane, 2Shin-‐ya Nishide, 3Hiroto Nakajima, 2Daisuke Ono, 1Hiroshi Kiyota, 1Kiyomichi Imamura, 3Naoya Shinozaki, 3Hirokazu Matsuki, 3Naoya Wada, 4Hirofumi Doi, 2Toshiyuki Hamada, 2Ken-‐ichi Honma, 2Sato Honma. 1Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Japan. 2Department of Physiology, Hokkaido University Graduate School of Medicine, Japan. 3R&D Division, Daiichi Sankyo Co., Ltd., Japan. 4Celestar Lexico-‐Sciences, Inc., Japan. In mammalian clockwork, BMAL1-‐CLOCK complex plays a central role for E-‐box-‐dependent circadian transcription, and the complex is subject to several types of posttranslational modifications for normal oscillation of the molecular clock. We previously found that circadian phosphorylation of the complex negatively regulates E-‐box-‐mediated transcription (Yoshitane et al., 2009). Molecularly, BMAL1 phosphorylation is regulated by CKI (Eide et al., 2002), CKII (Tamaru et al., 2009) and GSK3 (Sahar et al., 2010), while our previous study demonstrated in vitro phosphorylation of chicken BMAL1 by ERK2 at Ser527, Thr534 and Ser599 (Sanada et al., 2002). On the other hand, treatment of NIH3T3 cells with a specific inhibitor of ERK activation caused no significant inhibitory effect on the up-‐shift of intrinsic BMAL1 protein band. Here we found that JNK highly responsive to external stimuli confers dynamic regulation on the clock by phosphorylating BMAL1-‐CLOCK complex. Alanine mutations at the three phosphorylation sites largely reduced CLOCK-‐dependent up-‐shift of BMAL1 protein band in NIH3T3 cells. Inhibition of JNK activity attenuated phosphorylation rhythms of BMAL1 protein and lengthened circadian periodicity in cultured cells. On the

232

other hand, acute activation of JNK by hyperosmotic stimuli caused type 0-‐resetting of the cellular rhythm, and activated JNK phosphorylated BMAL1 and CLOCK proteins. Jnk mutant mice exhibited behavioral rhythms of prolonged circadian period in DD with compromised phase shifts in response to light pulse given at subjective night. Intriguingly the locomotor rhythm in LL became insensitive to changes in the light intensity, while WT mice obeyed Aschoff´s rule that empirically indicates light intensity-‐dependent lengthening of the free-‐running period in nocturnal species. Collectively, JNK plays an active role in dynamic regulation of the circadian clock by responding to external signals. Keywords: phosphorylation, JNK, BMAL1

P120 PHOTOTRANSDUCTION IN ZEM-‐2S EMBRYONIC CELLS OF ZEBRAFISH DANIO RERIO Bruno Ribeiro Ramos, Leonardo Ribeiro Lima, Maria Nathália Magalhães Moraes, Maristela Oliveira Poletini, Ana Maria Lauro Castrucci. Universidade de São Paulo, Brasil. Melanopsin, a photopigment found in the retina of all classes of vertebrates, has been related to photoentrainment of circadian rhythms. Until recently, it was believed that the melanopsin found in the various classes of vertebrates was orthologue to that originally found in Xenopus. However, comparisons between the deduced amino acid sequences of the opsins led to a study that showed that non-‐mammalian vertebrates have two melanopsin genes, Opn4m (mamalian-‐like) and Opn4x (Xenopus-‐like), whereas in mammals only Opn4m remains. The melanopsins, despite being found in vertebrates, present greater homology with invertebrate opsins (rhabdomeric photoreceptors) than with vertebrate opsins (ciliary photoreceptors). A study conducted in our laboratory with photosensitive Xenopus melanophores showed that light-‐activated melanopsin triggers melanosome dispersion through phospholipase C (PLC) pathway, corroborating the literature data for rhabdomeric photoreceptors. Like Xenopus melanophores, ZEM-‐2S embryonic cell line of the zebrafish Danio rerio is also an ideal model for phototransduction studies. We have previously shown that this cell line is responsive to light stimuli, displaying a differential proliferation under different lengths of light period and exhibiting rhythmic Clock, Per1 and Cry1b gene expression. In addition to this fact, the co-‐expression of Opn4x and Opn4m genes makes this cell line particularly interesting in the investigation of the melanopsin function as a mediator of photoentrainment. Preliminary data obtained with the stimulation of ZEM-‐2S cells by blue light (450-‐475nm), lasting 1 and 10 minutes, indicate that the clock genes Cry1b and Per1 respond to light, with the latter gene exhibiting a more robust response. To confirm these results, new experiments with PLC pathway inhibitors are presently being conducted. Not less important in this work is the determination of the light signaling initiated by the photoactivation of melanopsin: so far, our data indicate an

233

intracellular increase in NO production as well as cAMP decrease. Experiments for determination of cGMP level and PKC activity are under way. Work partially supported by FAPESP, CNPq and CAPES. Keywords: Clock genes, Danio rerio, Phototransduction

P121 EFFECTS OF LIGHT ON PER1 AND PER2 TRANSCRIPTION: MODULATION AND SYNCHRONIZATION IN Xenopus laevis MELANOPHORES Maria Nathália Carvalho Moraes, Bruno Ribeiro Ramos, Maristela Oliveira Poletini, Leonardo Ribeiro Lima, Ana Maria Lauro Castrucci. Universidade de São Paulo, Brasil. Introduction: Many biological rhythms are clearly associated with the environmental light-‐dark cycles, which are of great importance for the species that have some type of photopigment. Xenopus laevis melanophores are photosensitive, and respond to light with dispersion of melanin granules, due to the presence of two melanopsins, OPN4X and OPN4M. In mammals, melanopsin is essential for setting the biological clock to light-‐dark cycles. Previous data from our laboratory demonstrated that the two melanopsins show temporal variation when kept in light-‐dark cycle, and that a medium change associated with this condition is able to synchronize their expression. In X. leavis melanophores, melanosome dispersion in response to light is maximal at 470nm. Based on this evidence, and in previously published data showing that a light pulse promotes increased expression of Per, and that Per can be synchronized by light-‐dark cycle in other models such as Danio rerio,, we investigated the effect of light on the transcription of Per1 and Per2 in X. laevis melanophores. Methods and results: Cultured melanophores of X. laevis were seeded in medium supplemented with 2% fetal bovine serum and 10-‐8M retinaldehyde. Two different protocols were used: (1) Cells were kept in 12 hours of light and 12 hours of darkness (12L:12D) or constant dark for 5 days. Total RNA extraction was performed along 24 hours every four hours starting at ZT0 of the fifth day. The samples were submitted to RT-‐PCR followed by quantitative PCR for mRNA quantification of Per1 and Per2. The data were compared by one-‐way ANOVA, followed by Tukey test (significance at P <0.05). Under this condition we observed a temporal variation, indicating a rhythmic pattern of transcription of these genes, thus demonstrating that mRNA transcripts of Per1 and Per2 are affected by light-‐dark cycle. (2) The cells were kept in darkness for 3 days and were stimulated with blue light for 1 and 10 minutes in the third day. Total RNA extraction was performed 30, 60, 120, 360 and 720 minutes after stimulation. The samples were processed and the data were analyzed as described above. In constant darkness, Per1 and Per2 were responsive to the blue light pulse, demonstrating that this modulation is due to the activation of melanopsin(s) by light. Conclusion: The present data show evidence that X. laevis

234

melanophores behave as peripheral clock, which responds directly to light through the activation of melanopsin(s) leading to changes of the transcription pattern of Per genes. Support: FAPESP (06/03381-‐1 and 09/15912-‐0) and CNPq (473658/2008-‐9) grants (Brazil). MNCMM is a fellow of FAPESP. Keywords: clock genes, melanopsins, light-‐dark cycle

P122 CRUSTACEAN HYPERGLYCEMIC HORMONE mRNA OSCILATION IN THE CRAYFISH PROCAMBARUS CLARKII EYESTALK Janikua Nelson-‐Mora, Julio Prieto-‐Sagredo, María Luisa Fanjul-‐Moles. L. Neurofisiología Comparada, F. Ciencias, Universidad Nacional Autónoma de México, México. The crayfish´s eyestalk X organ-‐Sinus gland system (XO-‐SG) is part of this animal’s neuroendocrine system. It synthetizes and secretes peptidic hormones important for the metabolic regulation. One of them is the crustacean hyperglycemic hormone (CHH), involved in the glycemic regulation and controlled by the circadian timekeeping system. Previous works using Immunochemical and hemolytic essays had demonstrated the CHH’s secretion in the P. clarkii´s in the XO-‐GS complex and the retina, both organs proposed as circadian pacemakers of P. clarkii, however up to now daily and circadian variation of the synthesis of CHH either in eyestalk or retina have not been proven. The objective of this work was to find out whether in the eyestalk a CHH’s synthesis daily rhythm exists and whether CHH mRNA is synthesized in the retina of crayfish. To characterize RNA in both organs, total RNA was extracted from the eyestalk at different times of day: 0300, 0700, 1100, 1300, 1500, 1900 and 2300 hours, and at 0700 h for the retina. Tripure (Roche) reactive was used following manufacturer’s instructions. Oligonucleotides based in the mRNA CHH sequence (GeneBank accession no. AB027291) were designed for the reverse transcriptase-‐PCR reactions (RT-‐PCR). For internal control, B-‐actin oligonucleotides were used. All the amplicons obtained were purified and sequenced. Once the amplicons were identified as being part of the B-‐actin and CHH mRNAs, Semi-‐quantitative RT-‐PCR reactions were carried out with the total RNA extracted from the eyestalk at different hours. Temporal variation was analyzed for the mRNA expression by band quantification in a Gel Logic 200 system (Kodak) and the Molecular Imaging software (Kodak). The results show eyestalk CHH mRNA daily variations with a peak at 0700 h and the lowest value at 1500 h. For the retina it was possible to identify chh mRNA. This work was supported by PAPIIT IN 218811. Keywords: mRNA, crustacean hyperglycemic, eyestalk

235

P123 GENOMIC DISSECTION OF TASK-‐RELATED PLASTICITY IN CIRCADIAN RHYTHMS IN HONEY BEES 1Guy Bloch, 2Sandra L. Rodríguez-‐Zas, 3Bruce R. Southey, 1Mira Cohen, 4Gene E. Robinson. 1The Hebrew University of Jerusalem, Department of Ecology, Evolution, and Behavior, Israel.2University of Illinois, Institute for Genomic Biology, USA. 3University of Illinois, Department of Animal Sciences, USA. 4University of Illinois, Department of Entomology, USA. Honey bee workers care for ("nurse") the brood around the clock without circadian rhythmicity when they are young, but then they forage outside with strong circadian rhythms and a consolidated nightly rest. This chronobiological plasticity is associated with variation in the expression of the canonical “clock genes” that regulate the circadian clock: nurse bees show no brain rhythm of expression, while foragers do. We performed a genome-‐wide survey to determine general patterns of brain gene expression in nurses and foragers sampled around the clock. We found 160 and 541 transcripts that exhibited significant sinusoidal oscillation in nurses and foragers, respectively, with peaks of expression distributed throughout the day in both task groups. Transcripts of genes involved in circadian rhythms and vision oscillated in foragers but not in nurses. The oscillating transcripts also were enriched for genes involved in “development” and “response to stimuli” (foragers), “muscle contraction” and “microfilament motor gene expression” (nurses), and “generation of precursor metabolites” and “energy” (both). This study identified new putative clock-‐controlled genes and suggests that some brain functions show circadian rhythmicity even in nurse bees that are active around the clock. Keywords: plasticity, social behavior, microarray

P124 MEASURE OF THE mRNA OF RYANODINE 2 RECEPTOR AND GENES PER1 AND PER2 IN UNIQUE CELL OF THE SUPRAQUIASMATIC NUCLEUS AT TWO DIFFERENT HOURS (ZT12 AND ZT23) José Luis Chávez Juárez, Daniel Quinto Muñoz, Raúl Aguilar Roblero. Departamento de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México. The supraquiasmatic nucleus (SCN) is an important component of the mammalian circadian system, SCN receives information from different brain regions including retinohypothalamic tract (RHT). Entrainment of rhythmicity to the light-‐dark cycle depends on the RHT input to

236

SCN and the mechanisms involved are well documented, the glutamate is released to initiate 2nd messenger cascades that lead to molecular regulation of clock genes. Nevertheless there is little information about the output of the circadian clock; there is evidence that calcium binding proteins and intracellular calcium are involved in these relevant aspects of SCN. In a previous work we have shown that Ca mobilization through the intracellular calcium channel activated by ryanodine receptor (RyR) and is involved in output pathway between the molecular circadian clock and the electrical output of the SCN. In the present work we set out to demonstrate the levels of RyR type 2 mRNA at different hours and compare it with the mRNA expression of per1 and per 2 in cells that express VIP. Male Wistar rats were exposed to light:dark (12:12 ) conditions and were anaesthetized at Zetigeber (ZT) times 03-‐05 or ZT 18-‐23 , the brain was quickly removed and placed in ice-‐cold extraction solution (oxygenated solution with 95%O2 and 5% CO2 gas mixture). To prevent phase shifts induced by lights the brain slices used during subjective night were prepared between ZT 10-‐ZT 11. Once the neurons were visualized, they were picked up with a boro silicate pipette and transferred to an ependorf tube (0, 2 mililiter), the end of the pipette was broken and quickly freeze at -‐20 °C. cDNA was synthesized according to the kit provider (InVitrogen RT). Real time PCR reaction was made following PCR program on ABI Prism SDS 7000 with SYBR Green PCR master mix (InVitroGen), we measured the levels of the 4 genes in 5 cells in duplicate, we found differences: in vip cells all mRNA`s were higher during the day (zt06) than during the night (zt23) as following: Ryanodine 12 %, per1 40% and per2 94%. Although yet preliminary, this data is consistent with previous reports indicated circadian modulation of RyR2 in SCN neurons Aknowledgements: This work is supported by project No. 91984 of FONCICYT funded by the European Union and CONACyT. PAPIt IN204811. Biblioteca and Unidad de Computo Instituto de Fisiologia Celular UNAM Keywords: Supraquiasmatic Nucleus, Ryanodine receptors, mRNA

P125 DIFFERENTIAL DEVELOPMENT OF THE DIURNAL CLOCK GENE EXPRESSION IN THE OLFACTORY BULB AND SUPRACHIASMATIC NUCLEUS OF EUROPEAN RABBIT 1Oscar Hernández Campos, 2Rodrigo Montúfar Chaveznava, 1Ivette Caldelas. 1Depto. Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México. 2División de Tecnologías Urbanas, Instituto de Ciencia y Tecnología del Distrito Federal., México. In altricial mammals, during pre-‐visual stages of development the olfactory system plays an important role. In the case of the European rabbit (Oryctolagus cuniculus), the newborn were usually maintained in a dark nursery burrow and the lactating female only visit their young for a few minutes once approximately every 24 hr; under this conditions the newborn rabbits depend entirely on pheromonal cues on the mother’s ventrum to locate

237

nipples and suckle efficiently. In addition, the daily exposition to maternal olfactory cues to newborn rabbits, such as the maternal pheromone 2MB-‐2 is possible to synchronize the locomotor activity and core body temperature rhythms. Despite the undoubted importance of the suprachiasmatic nucleus (SCN) as a circadian pacemaker, there is considerable evidence of other circadian oscillators in the mammalian olfactory bulb (OB) with properties similar to and largely independent of the SCN. In order to understand the development of the rabbit’s circadian system, we characterized the 24-‐h pattern of expression of the clock genes in the OB and SCN of pre-‐visual week-‐old rabbits and compare these with the pattern of expression of visual juvenile rabbits several weeks after weaning. In order to characterize the diurnal pattern of clock gene expression in the OB and SCN of pre-‐visual rabbits, 43 newborn were used. The rabbits were maintained in constant darkness and fed by a lactating female every 24-‐h, from the postnatal day 1 to 7 (P1-‐ P7). After nursing on P7, pups were killed in the dark at 3 hours intervals, in order to obtain eight groups (n=6 pups/group) distributed evenly across the 24 hours cycle. The visual rabbits (n=46) were weaned at P25 and maintained in an L:D cycle, at the P45 the juvenile rabbits were sacrificed at the same time points across the cycle. The expression of the clock genes Per1, Cry1 and Bmal1 were determined using in situ hybridization, using oligodeoxynucleotides labelled with S35, in sagital sections containing the OB and coronal sections containing the SCN. The autoradiography films were digitalized and quantified the relative optical density of at least three sections of each subject; the data were analyzed by COSINOR and ANOVA. We report for the first time: 1) that Per1, Cry1 and Bmal1 are expressed in the OB and in the SCN of the newborn and juvenile rabbits, 2) the expression in the OB takes place mainly in the mitral cell layer, and 3) the diurnal pattern of clock genes expression develops earlier in the OB, than in the SCN of newborn rabbits. Our findings suggest that the OB is a potential element of the newborn pre-‐visual circadian system, since the molecular machinery develops earlier than in the SCN. Supported by ICYTDF/261/2009, PAPIIT IN219510-‐3 and CONACyT 131314. Keywords: Olfactory bulb, Development, Rabbits

P126 PARTICIPATION OF INTRACELLULAR CALCIUM-‐RELEASE CHANNELS AND CALCIUM PUMPS IN THE CIRCADIAN RHYTHMICITY OF THE SUPRACHIASMATIC NUCLEUS 1Adrian Baez-‐Ruiz, 2Raul Aguilar-‐Roblero, 3Gabriella S. Lundkvist, 1Mauricio Diaz-‐Muñoz. 1Instituto de Neurobióloga, UNAM-‐Juriquilla, México, 2Instituto de Fisiólogia Celular, UNAM, México, 3Karolinska Institutet, Sweden. The role played by intracellular calcium in the physiology of the principal mammalian pacemaker, the suprachiasmatic nucleus (SCN), is not completely understood. The aim of this project is to study the participation of intracellular calcium-‐handling proteins such as

238

the Ryanodine Receptor (RyR), Inositol-‐trisphosphate Receptor (IP3R) and calcium-‐dependent ATPase pump (SERCA) in the circadian rhythmicity of SCN. Results from Dr. Gabriella Lundkvist‘s laboratory using unilateral SCN cultures from transgenic mice carrying luciferase as reporter gene for PERIOD2, showed a severe damping (period and phase were undetectable after this treatment) after treatment with the SERCA inhibitor cyclopiazonic acid (CPA: 10, 20, 50 and 100 ìM during 3 days). Short term CPA treatment (50 ìM for 2 h) showed a phase delay of ~2 h on PER2, without change in the period. This means that SERCA inhibition by CPA is washable but with an apparent PER2 phase shift. SCN explants were treated for 2 h with 80 ìM (inhibitory) and 100 nM (activatory) of ryanodine at 2 different times: peak and trough of PER2 (early morning and early evening, respectively). No effect on phase and period was detected in these experiments. These results suggest: 1) SERCA seems to participate in the molecular clock machinery; 2) RyR is not affecting PER2 rhythmicity. Further experiments using IP3R inhibitors (i.e. 2-‐APB or xestospongin) are necessary in order to characterize the possible participation of this intracellular calcium release channel in the circadian rhythms of PER2 in SCN cultures. PAPIT IN204811 Keywords: pacemaker, calcium, SERCA

P127 HISTOCHEMICAL CHARACTERIZATION OF ENDOPLASMIC RETICULUM PROTEINS THAT MOVILIZE INTRACELLULAR CALCIUM IN THE SCN Clara Mercado López, Mauricio Díaz Muñoz. Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Campus UNAM-‐Juriquilla, Querétaro, México. Anatomical studies have established that the SCN is a heterogeneous structure and have made an important distinction between the dorsomedial (DM) and ventrolateral (VL) part of this pacemaker on the basis of neurotransmitter content. Afferent and efferent pathways tend to connect with either the dorsal or ventral region: in rat SCN the ventral region enriched with vasoactive intestinal peptide (VIP), whereas the dorsal region contains mainly arginine vasopressin (AVP). A variety of proteins regulating intracellular calcium homeostasis in SCN have been identified. We reported that ryanodine receptor (RyR) is a fundamental output of SCN for expression of behavioral circadian rhythms. However, a detailed study of regional distribution and temporality of proteins that regulate the dynamics of intracellular calcium within the SCN has not been performed. In the present study we use markers immunofluorescent for: 1. Characterize the regional localization of RyR, IP3R and calcium ATPase. 2. Characterize the temporality localization of RyR, IP3R and calcium ATPase. 3. Differentially located neuropeptides within the VL and DM SCN in combination within the RyR, IP3R and calcium ATPase. Slices collected in mid-‐day (ZT 6-‐8) show that RyR, IP3R and calcium ATPase-‐immunoreactivity was found throughout the

239

rostro-‐ caudal extent of the rat SCN. The majority of IP3R expressing neurons are located in part medial of the SCN. RyR and calcium ATPase-‐expressing neurons are distributed homogenous in the SCN and not co-‐located with the IP3R. Regional localization of these proteins in mid-‐night (ZT17-‐19) remains to be determined. This result could contribute to a better understanding of the role of proteins that regulate the dynamics of intracellular calcium within the SCN. We thank Ing. Elsa Nydia Hernández Ríos for skillful technical assistence in Confocal Microscopy and Dra. Olivia Vázquez Martínez for skillful technical assistence in laboratory. This work was supported by the posdoctoral grants from the DGAPA/UNAM. PAPIT IN204811 Keywords: suprachiasmatic nucleus, intracellular calcium, proteins

P128 THE EFFECT OF GATING RYANODINE RECEPTORS ON THE INTRACELULAR CALCIUM CONCENTRATION IN SCN NEURONS IN MICE 1Daniel Quinto-‐Muñoz, 2Stephan Michel, 1Raúl Aguilar-‐Roblero. 1División de Neurociencias Cognitivas, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México. 2Department of Molecular Cell Biology, Laboratory for Neurophysiology, Leiden University Medical Center, Nederlands. The Suprachiasmatic Nucleus (SCN) is responsible for circadian rhythms of physiological and behavioral processes in mammals. The intracellular calcium concentracion ([Ca2+]i) in this neurons shows a circadian oscillation, which is taught to be regulated by the ryanodine receptors (RyRs) widely expressed in neurons in the SCN. In a previous study we found that the spontaneous firing rate (SFR) of these cells increases when the RyRs are activated and decreases when they are blocked pharmacologically. In this study we used calcium imaging with Fura-‐2 AM to see the effect of gating the RyRs on the [Ca2+]i in SCN slices from mice. Recordings were conducted during day and during night. We activated the RyRs applying 100nM of Ryanodine and blocked them with 10µM of Dantrolene. We hypothesized that activating or blocking the RyRs will either increase or decrease the [Ca2+]i, respectively. In the experiments with ryanodine we found that the basal [Ca2+]i was higher in the day than in the night (90.96nM, SEM=6.57; and 68.95nM, SEM=2.41, respectively; t=3.55, P<0.001), as previously reported. We also found that not all cells responded equally to the drugs. When we applied ryanodine in the day (n=98), we found that by activating the RyRs, the [Ca2+]i increases in 42.86% of the cells (t=-‐4.83, P<0.001), it decreases in 23.47% (t=6.2, P<0.001) and in 33.67% it did not change (t=1.77, P=0.087). Moreover when we applied ryanodine in the night (n=240), the [Ca2+]i increased in 52.92% of the neurons (t=-‐11.50, P<0.001), decreased in 12.5% (t=7.52, P<0.001), and in 34.58% it did not change (t=-‐1.46, P=0.148). The experiments with dantrolene were all conducted in the day (n=337) and in these, the [Ca2+]i decreased in 54.01% of the cells (t=12.79, P<0.001), while in 25.52% it

240

increased (t=-‐8.65, P<0.001) and it did not change in 20.47% of the cells (t=0.036, P=0.97). In the day experiments with ryanodine, the mean basal [Ca2+]i was very similar among all cells. Nevertheless in night experiments with ryanodine, in those cells in which the [Ca2+]i increased, the mean basal calcium concentration was 57.31Nm (SEM=2.46) and it went to 91.68 (SEM=3.89)after the drug; and in the cells in which the [Ca2+]i decreased, the mean basal concentration was 97.99nM (SEM=11.61) and it went to 68.37nM (SEM=9.74)after the ryanodine. Similarly, in the experiments with dantrolene, those cells in which the [Ca2+]i decreased had a mean basal concentration of 124.94nM (SEM=7.97) and it went to 33.84nM (SEM=3.26), while those in which the [Ca2+]i increased had a mean basal concentration of 52.99nM (SEM=.17) and it went to 110.08nM (SEM=7.31). In conclusion, the RyRs can directly affect the [Ca2+]i in SCN neurons an thus probably regulate the membrane excitability throughout the day, for example, modifying the SFR as we described before. Aknowledgements: This work is supported by project No. 91984 of FONCICYT funded by the European Union and CONACyT. PAPIT IN204811 Keywords: Suprachiasmatic nucleus, Calcium imaging, Ryanodine receptors

P129 MECHANISMS OF RESPIRATORY RHYTHM GENERATION IN VITRO: EFFECT OF INCREASING CA2+ BUFFERING CAPABILITY IN PREBÖTZINGER COMPLEX INSPIRATORY NEURONS Consuelo Morgado-‐Valle, Jorge Manzo, Luis Isauro García, Luis Beltrán-‐Parrazal. Programa de Neurobiología, Universidad Veracruzana, México. The preBötzinger Complex (preBötC) is a neuronal network postulated to be the kernel of respiratory rhythm generation (Feldman and Del Negro, 2006). A brainstem transverse slice preparation that contains the preBötC is used to study respiratory rhythm generation in vitro (Smith et al., Science 1991). The rhythmic command from preBötC inspiratory neurons is transmitted to motoneurons in the hypoglossal nucleus; thus a respiratory-‐like motor output can be recorded from the hypoglossal nerve. The mechanism for rhythm generation is not well understood in either network or single-‐neuron level. In active preBötC inspiratory neurons, intracellular Ca2+ concentration and Ca2+ buffering are intrinsic properties relevant for respiratory rhythm generation (Pace et al, 2007; Morgado-‐Valle et al 2008). To investigate how these properties affect single neuron rhythmic activity we examined the effects of modifying the intracellular Ca2+ buffering capability. We current clamped single preBötC inspiratory neurons using various concentrations of EGTA, BAPTA or CaCl2 in the patch-‐clamp intracellular solution. We quantified the short term effects of altering buffering by analyzing and comparing inspiratory drive, number and shape of action potentials and membrane potential immediately after establishing whole-‐cell recording and 15 min later. We found significant changes in membrane potential trajectories and action potential shape in neurons current-‐clamped with intracellular solutions containing 5 mM BAPTA and 30 mM

241

EGTA. Action potentials in solution containing 11 mM EGTA were indistinguishable from those with 1.1 mM EGTA. These results suggest that changes in intracellular Ca2+ buffering modify membrane properties relevant for both inspiratory drive potentials and action potentials. Supported by UC Mexus-‐CONACyT CN-‐09-‐331 grant (CM-‐V); PROMEP PTC-‐487 (CM-‐V); PTC-‐419 (LB-‐P) Keywords: respiratory rhythm, preBötzinger Complex, intracellular calcium

P130 MODULATION OF APOPTOTIC ACTIVITY IN LIVER CELLS BY THE FOOD ENTRAINABLE OSCILLATOR Christian Molina Aguilar, Olivia Vázquez Martínez and Mauricio Díaz Muñoz. Instituto de Neurobiología, UNAM Campus Juriquilla, Querétaro, México. As a consequence of the Earth’s rotation about its axis approximately every 24 h, most organisms on this planet are subjected to predictable fluctuations of light and temperature. A diverse range of species, from cyanobacteria to humans, evolved endogenous biological clocks that allow the anticipation of these daily variations. This information is transmitted to the suprachiasmatic nuclei (SCN) of the hypothalamus, considered the master clock of the organism. Under normal conditions, endogenous oscillations are synchronized to the environment, and it is generally thought that biological clocks provide an adaptive advantage by ensuring that an organism’s internal biochemical and physiological processes. Cyclic access to food, may also act as an entraining factor, by means of a different circadian oscillator, known as the Food Entrained Oscillator (FEO). The FEO can be expressed even when the SCN is destroyed. When food is restricted to 2 h daily for 2-‐3 weeks, the organism is synchronized to the feeding schedule. This condition increases locomotive activity during the previous hours of mealtime. This characteristic behavior is known as Food Anticipatory Activity (FAA).The nature and location of the FEO are still unknown. Our hypothesis is that the FEO is a distributed system constituted by 1) central nervous structures such as hypothalamic nuclei with the capacity to act in response to signals derived from the nutritional state and energetic metabolism and 2) peripheral organs such as the liver, pancreas, or adipose tissue, in which synthesis, oxidation, and coordinated handling of energy metabolites such as carbohydrates, lipids, and amino acids take place. The time-‐keeping mechanism of the FEO would emerge based in the feedback loops between these two systems and conduced by both neuronal and humoral signals.The liver is one of the most important components in the physiology of FEO, because of it´s role playeds in the processing of nutrients. It has a great capacity to regenerate even losing 2 / 3 of its mass. To prevent tissue mass increases in an uncontrolled manner (hyperplasia), also shows a cell disappearance, usually by apoptosis. Establishing a balance between mitotic-‐apoptotic activity, which is under circadian regulation and is susceptible to modulation by nutrient availability.

242

In this work, we studied apoptotic changes produced in the liver due to food restricted schedule. Experimental groups: 1) food ad libitum 2) food restricted (22 h of fasting and 2 h of food access for 3 weeks). Liver samples every 3 h (to cover the entire day). Methodology: 1) Measurement of Fas-‐L, (extrinsic pathway). 2) Cytochrome c released into cytosol (intrinsic pathway). 3) Measuring the activity and presence of caspase-‐3 which is the effector enzyme for both paths and mesuringe the percent of fragmented DNA, as a marker for apoptosis. The apoptosis dynamic change whit the restriction. The release of cytochrome c is higher in rats with food restriction than rats ad libitum. The activity of caspase 3 is higher when food is restricted than the ad libitum, but the presence is lower than the ad libitum. Our results suggest that food restriction increases the levels of apoptosis in the liver of rats. Key words: Apoptosis, Caspase and liver.

243

244

Indexes

245

246

AUTHOR INDEXA Acosta Mercedes P101 Acosta Peña Eva P73 Acosta Rodríguez Victoria S10.3, P116, P117 Acosta-‐Galván Guadalupe S17.1, P86 Agostino Patricia P23 Aguilar-‐Roblero Raúl P24, P46, P108, P124, P126,

P128 Albrecht Urs S14.4, P75 Alcaraz Víctor P19 Aldeanueva Aguirre José L. P13 Allebrandt Karla S14.1, P34, P38, P68 Allen Charles S7.2 Allison Kelly P35 Almeida Rosas Georgina P24 Alvarado Ilia P71 Alvarez-‐Croda Dulce P81 Álvarez-‐Ude de la Torre Juan P13 Amaya Jorge S12.3 Amir Shimon S6.3, S13.3 An Sungwon S1.5 Angeles-‐Castellanos Manuel S17.1, P109, S12.3, P43 Araujo Nogueira Tatiane S10.2 Archer Simon S21.1 Arellanes-‐Licea Elvira P102 Arranz José Luis P13 Arroyo Layla P57 Asakawa Takeshi P47, P87, S1.4 Aton Sara S1.5 Ayala-‐García Braulio P99 Azevedo Carolina P55, P54

B Bachmann Valérie S21.3 Báez-‐Ruiz Adrián P126 Bardasano Rubio José Luis P13 Barone Mark P67 Bartness Timothy S11.2 Bastuji Helène P65 Basualdo Sigales Carmen S12.4, S15.5, S17.1, P79, P86 Beale Andrew S10.1 Beaver Paura S3.1 Bedos Marie S11.4 Belforte Nicolás P12 Belle Mino S7.3 Beltrán-‐Parrazal Luis P82, P129 Berger Wolfgang S21.3 Bertolucci Cristiano S14.2 Bicudo José P91 Birchler Thomas P75 Blancas Velazquez Aurea P96, P109 Bloch Guy S3.3, P123 Boivin Diane P39 Bolborea Matei S11.1 Borrani Jorge P33

Borson-‐Chazot françoise P65 Boudreau Philippe P39 Bourgin Patrice S21.4 Brager Allison S5.4, P76, P78 Brenda Brug P82 Brown Steven P30, P75 Bruegger Pascal P75 Brugger Peter S21.3 Brun Jocelyne P65 Buch Thorsten P75 Bueno Clarissa P53 Bueno-‐Rivas Gustavo P93 Buijs Frederik P79 Buijs Ruud S12.3, S12.4, S6.5, S17.1, P79,

P86 Bunnell Bruce S9.3 Buonfiglio Daniella S10.2 Bussi Ivana P23

C Caba Mario S8.4, P100, P101, P106 Cajochen Christian S21.1, P30 Caldelas Ivette S2.2, P111, P112, P125 Calderón Rosete Gabina P85, P114 Calmo Wolnei P35 Cambras Trinitat P92 Canales-‐Espinoza Domingo P21 Carmona-‐Alcocer Vania P. P3 Carra M P67 Carranza Martha P102 Casiraghi Leandro P48 Castaño Meneses Violeta P62 Castañon-‐Cervantes Oscar S15.2, P6 Castejon Lucia P92 Castro Afeche Solange S10.2 Castrucci Ana Maria S10.4, P120, P121 Caumo Wolnei P38, P41 Caynas Seraid P71 Cermakian Nicolas S15.4, P39 Chan Sofina S2.1 Chavez-‐Juarez Jose Luis P124 Chellappa Sarah S21.1 Chiesa Juan P48 Cipolla-‐Neto José S10.2 Claustrat Bruno S6.4, P65 Coenen Anton P40 Cohen Mira P123 Colwell Christopher S7.1 Contaldo Claudio P75 Contin Maria S10.3 Cornélissen Germaine P61 Corona Jackeline P70 Cortes ma. Del carmen P20, P69, P70 Cortez Juventino P33, P56, P57, P58 Costa Rodolfo S14.2 Cruz María P89 Curie Thomas S21.2, P50

247

D Dailey Megan S17.4 Damulewicz Milena S18.4 Dantas Giovana P34, P38, P41 Dardente Hugues S15.4 Davidson Alec S15.2, P6 Davis Fred S8.3 De Ita Dalia P98 de la Herrán Arita Alberto P74 de la Iglesia Horacio S3.2, S12.2 de Lima Leonardo S10.4 de Zavala Nuria P12 Del Angel Jacqueline P58 Delgadillo José S11.4 Delgado Herrera Maribel P24 Diaz Nicolas S10.3 Díaz-‐Muñoz Mauricio P94, P97, P98, P99, P102,

P104, P105, P126, P127, P130 Diez-‐Noguera Antoni S20.3, P92 Dijk Derk-‐Jan S21.1 do Nascimento Micaela P23 Doi Hirofumi P119 Doi Masao S6.1 Domínguez Belisario S8.4 Domínguez Roberto P89 Doring Marlene P38 Dorsaz Stephane P50 Drucker-‐Colín René P74 Duarte Gerardo S11.4 Duarte Rojo Andrés P62 Duhart José M; S15.3 Duran Pilar P7, P72 Durán Lizarraga Ma. Elena P37

E Eban-‐Rothschild Ada S3.3 Eckel-‐Mahan Kristin S1.2 Eckert Anne P30 Eguibar Jose P20, P69, P70 Ehlen Chris S15.2 Eichele Gregor S6.2 El-‐Hennamy Rehab S8.1 Elliott Jeffrey P4, P8 Emennegger Yann P50 Escamilla-‐Chimal Elsa P9 Escobar Carolina S12.3, S12.4, S15.5, S17.1,

P43, P60, P79, P86, P95, P96, P100, P103, P109

Espitia Estefa P95 Evans Jennifer P6 Everardo Pamela P89

F Fanjul-‐Moles María P9, P122 Farias Alam Marilene P34 Fernandes-‐Kolodiuk Fernanda P54

Fernandez Diego P12 Fernández Ilda S11.4 Fitz-‐Rodríguez Gonzalo S11.4 Fliers Eric S9.4 Flores Anahí P29 Flores Angélica P89 Flôres Danilo P2, P49, P91 Flores José S11.4 Floyd Z. S9.3 Foley Duncan S1.3 Foley Nicholas S1.3 Foppen Ewout S9.4 Fortier Erin S15.4 Franco Daiane P41, P67 Franken Paul S21.2, P50 Freeman Jr. G. Mark S1.5 Friesen Wolfgang P48 Fu Yue P27, P28 Fuentes-‐Granados Citlalli P72 Fuentes-‐Lorenzo Jorge L. P114 Fukada Yoshitaka P113, P119 Furukawa Kei-‐ichi S1.4, P87 Fuse Yuta P107

G Galeana Garcia Alejandra P24 Gamble Karen S15.2 Gao Tianyan S1.2 Garbarino-‐Pico Eduardo S10.3, P118 García Aída P25, P26, P33, P56, P57, P58 García Carlos P89 García García Fabio P73, P74 García Luis Isauro P82 García Minerva P29 García-‐Peláez Isabel P103 García-‐Tecpan Ana P9 Garcìa Ramos Guillermo P62 Garren Madeleine S19.2 Gaspar do Amaral Fernanda S10.2 Gentile Carla P93 Giebultowicz Jadwiga S3.1 Gil Aldeco Demian P62 Gimble Jeffrey S9.3 Glass J. David S5.4, P76, P78 Golombek Diego S14.3, S15.3, P12, P23, P48 González Eduardo P60 González Barrios Juan A. P114 González-‐Mariscal Gabriela P108 Gopar-‐Canales Kristel P84 Gorman Michael P1, P4, P8, P77, P90 Gorska-‐Andrzejak Jolanta S18.4 Goya María S14.3 Granada Adrian S20.4 Granados-‐Fuentes Daniel S13.1 Guerrero Martha P57 Guerrero Natali S15.5 Guibal Christophe S10.1 Guido Mario S10.3, P116, P117, P118 Guilding Clare S13.4

248

Guinn Jessie S5.4 Gúzman María P89 Guzmán Ruiz Mara P86

H Hagenauer Megan P10 Halberg Franz P61 Hamada Toshiyuki P119 Harb Ana P35 Hardin Paul S4.1 Hardy Marie-‐Pierre S15.4 Harrison Elizabeth P1 Helfrich-‐Forster Charlotte S14.2 Heredia García Donají P60, P103 Hernández-‐Campos Oscar P111, P125 Hernández Horacio S11.4 Hernández María Elena P82 Hernández Saavedra Diego P94 Herrera-‐Moro Chao Daniela P86 Herzel Hanspeter S20.4 Herzog Erik S1.5, S13.1, S18.2 Hidalgo Maria Paz P34, P35, P36, P38, P41, P42,

P64, P68 Hirano Arisa P113 Hirao Akiko P107, P110 Hoegger Dominik P75 Holguin Victoria P19 Honma Ken-‐ichi S1.1, S16.2, P119 Honma Sato S1.1, S16.2, P119 Hood Suzanne S13.3 Houl Jerry S4.1 Hsu Cynthia S2.1 Hughes Alun S13.4 Hut Roelof A. S

I Imamura Kiyomichi P119 Imanishi Takuma P110 Indic Premananda S5.1, S16.5 Iracheta Ana P58 Irwin Robert S7.2 Itokawa Misa P110 Izakovic Jan P30

J Jackson F. Rob S18.3 Jhamandas Jack S17.1 Jie Feng P45, P59 Jimènez Angeles P100 Jiménez-‐Genchi Alejandro P66 Jorge Manzo P82, P129 Josef Gal-‐Ben S13.1 Juárez Claudia S8.4 Juarez Diana P33, P56, P57, P58 Juarez-‐Tapia Cinthia P7

K Kalsbeek Andries S9.4 Kameyama Luis P114 Kankariya Shrutika P16, P17 Kauffman Alexander P90 Kedzierska Urszula P14, P83 Keller Sarmiento María P12 Kent Brianne S2.1 Khan Azim P90 Kiessling Silke S6.2 Kiyota Hiroshi P119 Klosen Paul S11.1 Koinuma Satosh S1.4, P47, P87 Kowalska Elzbieta P75 Kramer Achim S14.4, P75 Krishnan Natraj S3.1 Krock Rebecca S1.5 Kronfeld-‐Schor Noga S3.4 Ku Eric P90 Kurabayashi Nobuhiro P113 Kyriacou Charalambos S14.2

L La Spada Francesco S21.2 Labrecque Nathalie S15.4 Lamont Elaine P39 Landín Abuín P44 Landolt Hans-‐Peter S21.3 Lanzani María P12 Lara Manuel P69 Laran-‐Chich Marie-‐Pierre S11.1 Lee How-‐Jing S4.4 Lee Theresa P10 Leise Tanya S20.2, P6 Leitner Claudia S11.2 Lemus Ana P108 Leone María S15.3 Leonardi Emanuela S14.2 LeSauter Joseph S1.3 Lescano Juan P118 Levandovski Rosa P34, P35, P38, P42, P64, P68 Lewandowski Marian P40 LI Garcia P129 Lima Leonardo P120, P121 Lima Araújo Geilson P54 Lopes Schimitt Regina P64 López-‐Fiesco Alfonso P61 López-‐Meraz María P81 Lorenzi Geraldo P67 Luby Mathew S2.1 Luna Alma P105 Luna Maricela P102 Lundkvist Gabriella P126

M Machado Claudia P42

249

Madrid Juan Antonio P10 Majewski Pawel P14, P83 Mansuy Isabelle S14.4 Marchant Elliott S2.1 Markus Regina P41 Marpegan Luciano S18.2 Marquez Sebastian P116 Martau Betina P36 Martínez Benito P25 Martínez Chacón Armando P74 Martínez Pérez Francisco P114 Mason Moyra S14.2 Masubuchi Satoru S1.2 Masumoto Koh-‐hei S1.4, P47, P87 Mateju Kristyna S8.1 Matsuki Hirokazu P119 Mazzilli Louzada Fernando P54 Mazzotta Gabriela M. S14.2 Meier Fides P30 Melgarejo Montserrat P73 Melo Angel S8.4 Menaker Michael S2.3 Mendez Isabel P94 Mendez-‐Diaz Monica P71 Mendoza Jorge S5.2, S13.2 Mendoza-‐Lujambio Irene P61 Menegazzi Pamela S14.2 Menna-‐Barreto Luis P31, P44, P53, P67 Mercado López Clara P127 Merrow Martha S14.1, 19.3 Meza Enrique S8.4, P106 Meza José P101 Michel Stephan P128 Migliori María S14.3 Miguel Mario P31 Miranda-‐Anaya Manuel P7, P3, P72, P84 Mistlberger Ralph S2.1 Molina Aguilar Christian P130 Mongrain Valérie S21.2, P50 Montúfar-‐Chaveznava R. P111, P112, P125 Moraes Maria Nathália S10.4, P120, P121 Moran Timothy S17.4 Morgado Elvira S8.4 Morgado-‐Valle Consuelo P82, P129 Morriggi Ermanno P30 Moura Bessa Zoélia P54, P55 Mueller Anke P75 Muñoz-‐Delgado Jairo P19, P21, P66

N Nachón García Francisco P74 Nag Chaynika P63 Nagahama Hiroki P110 Nagano Mamoru P47, P87 Nagano Mamoru S1.4 Nakagoshi Hideki S4.3 Nakajima Hiroto P119 Nakamura Wataru S16.3 Nelson-‐Mora Janikua P122

Newton Alexandra S1.2 Ng Fanny S18.3 Nieto Paula S10.3, P117 Nishida Hisayo P52 Nishide Shin-‐ya P119 Nishimura Kana P52 Nitta Akiko P52 Nolasco Nahum P106 Novakova Marta S8.1 P115

O O Neill Audrey S1.2 Oda Gisele P2, P48, P49, P91 Ofir Levy S3.4 Okamura Hitoshi S6.1 Olesiejuk Alicja P83 Oliveira Ceres P35 Ono Daisuke S1.1, P119 Ortiz Xóchitl P29 Osnaya Rodrigo P95 Oster Henrik S6.2 Otalora Beatriz P10 Otsuka Makiko P107 Outsu teiji P110

P Pagani Lucia P30 Page Terry S19.2 Paladino Natalia S15.3 Paliza José P2 Palomares-‐Vazquez Dulce P95 Palomino Garibay M. Angel P37 Pande Babita P22 Parfyonov Maksim S2.1 Parganiha Arti P16, P17, P22, P32 Pati Atanu P16, P17, P22, P32 Patra Pradeep P22 Patton Danica S2.1 Paul Ketema S15.2 Paul Matthew S5.1, S16.5 Paul Saft P82 Pedrazzoli Mario P11 Peixoto Alexandre P93 Peliciari-‐Garcia Rodrigo S10.2 Peres Rafael S10.2 Perez-‐Mendoza Moises P104 Perez-‐Morales Mauricio P71 Perry Gavin S13.1 Peter-‐Derex Laure P65 Petitjean Thierry P65 Pévet Paul S15, S11.1, P15 Pezuk Pinar S2.3 Piesiewicz Aneta P14, P83 Piggins Hugh S7.3, S13.4 Plano Santiago P12 Pliego Caballero Mariana P103 Poletini Maristela S10.4, P120, P121

250

Polidarova Lenka S8.1 Ponce Juan Manuel P37 Pooch-‐Leuck Marlene P42 Pradhan R.K. P63 Prendergast Brian S15.1 Prieto-‐Sagredo Julio P122 Prospero-‐Garcia Oscar P71 Prosser Rebecca P76, P78 Ptitsyn Andrey S9.3 Pyza Elzbieta S18.4

Q Quinto Muñoz Daniel P124, P128

R Ramírez Candelaria P25, P26, P29, P33, P56, P57,

P58 Ramos Bruno S10.4, P120, P121 Raposo Sánchez Miguel P13 Rasri Kamontip S11.1 Rebeca Toledo P82 Reddy Akhilesh S9.2 Refinetti Roberto S5.3 Reséndiz Garcìa Montserrat P62 Reynoso Victor S10.1 Rieger Dirk S14.2 Ripperger Juergen P75 Rito-‐Medina Jonathán P61 Rivera Julieta P97 Robinson Gene P123 Roche-‐Bergua Andrés P66 Rodriguez Gonzales Katia P96, P109 Rodríguez-‐Sosa Leonardo P85, P114 Rodriguez-‐Zas Sandra P123 Roenneberg Till S14.1, P68 Rol Maria Angeles P10 Romanowski Andrés S14.3 Romer Konstanze P30 Rooney Julie S15.4 Rosenstein Ruth P12 Rossi Alessandro S14.2 Rothschild-‐Fuentes Betty P66 Ruiz Contreras Alejandra P24

S Saderi Nadia S12.4 Salgado-‐Delgado Roberto S12.3, S12.4, S15.5 Salido Ezequiel P12 Sánchez-‐de la Peña Salvador P61 Sánchez-‐Ferrer José Carlos P21, P66 Santiago Ayala Maria P62 Santiago Garcia Juan P74 Santillán-‐Doherty Ana P21 Sassone-‐Corsi Paolo S1.2 Scheer Frank S9.1 S12.1

Schmidt Maren P5 Schmitt Karen P30 Schmutz Isabelle S14.4 Schneider Marcia P38 Schorr Fabiola P67 Schöttner Konrad P5, P15, P18 Schwartz William S5.1, S16.5 Segall Lauren S6.3 Sellix Michael S2.3 Seron-‐Ferre Maria S8.2 Shibata Shigenobu S2.4, S17.2, P107, P110 Shigeyoshi Yasufumi S1.4, P47, P87 Shinozaki Ayako P110 Shinozaki Naoya P119 Silkis Izabella P88 Silva Belasío Aline P55 Silva Bezerra Deyse P54 Silva-‐Belísio Aline P54 Silver Rae S1.3 Simon Tatiana S18.2 Simonetta Sergio S14.3 Simonneaux Valérie S11.1, P15 Singh Arati P17, P16 Sinning Susan P77 Skwarlo-‐Sonta Krystyna P14, P83 Sladek Martin S8.1, P115 Smyk Magdalena P40 Song C. S11.2 Sousa Guimarães Ivanise P54, P55 Southey Bruce P123 Souza Jane Carla P54, P55 Souza Rosana P34 Steele Andrew S2.1 Steinlechner Stephan S11.1, P15 Stewart Jane S13.3 Stowie Adam P76 Stunkard Albert P35 Sullivan-‐Wilson Alexander S13.1 Sumova Alena S8.1 P115 Sutton Gregory S9.3

T Tachinardi Patricia P2, P49, P91 Tahara Yu S2.4, P107, P110 Takasu Nana P52 Talamantes Lopez Javier P25, P26 Tamai Katherine S10.1 Tamar Dayan S3.4 Teubner Brett S11.2 Timofeeva Elena S17.3 Tomioka Kenji S4.3 Tomotani Barbara P2, P49, P91 Tong Tina S1.3 Torre Aldo P62 Torres-‐Farfán Claudia S8.2 Tosatto Silvio C. E. S14.2 Trejo-‐Muñoz Lucero P112 Trres Iraci P41 Tsai Connie S18.2

251

Tule Candelaria P26

U Ueda Hiroki S11.3 Ugarte Araceli P20 Umezaki Yujiro S4.3

V Vaidya Nishtha P32 Valdez Diego S10.3, P117 Valdez Pablo P25, P26, P29, P33, P56, P57,

P58 Valencia Flores Matilde P62 Valentinuzzi Verónica P2, P49, P91 Valenzuela Guillermo S8.2 van Luijtelaar Gilles P40 Vanin Stefano S14.2 Vascincelos Denise P38 Vásquez Samuel P43 Vázquez Martínez Olivia P94, P130 Vega-‐González Arturo P46, P62 Velázquez-‐Amado Rosa P9 Venebra Muñoz Arturo P73 Verra Daniela S10.3 Vetter Céline S14.1 Vielma Jesús S11.4 Villalobos Leal Mónica P94 Viola Antoine S21.1, P30, P51 Vuillez Patrick P15

W Wada Naoya P119 Wakamura Tomoko P52 Walisewski Stefan P101 Waloch Maria P83 Waterhouse Jim P18 Webb Alexi S1.5 Weinert Dietmar P5, P15, P18 Welsh David S1.3 Wendt Sabrina S14.4 Whitmore David S10.1 Wiegand Mabel P34 Wilson Donald S13.1 Wirz-‐Justice Anna P30 Wright Jr. Kenneth S19.5 Wu Mingwei P80

X Xian Lijian P80

Y

Yagita Kazuhiro P87 Yagita Kazuhiro S1.4 Yamamoto Yoshiyuki S10.1 Yamanaka Yujiro S16.2 Yan Zhi P27, P28 Yan Zhuang P45, P59 Yasuyama Koouji S4.3 Yi Chun-‐Xia S9.4 Yoshii Taishi S14.2 Yoshikawa Tomoko S1.1 Yoshitane Hikari P119 Yu Wangjie S4.1

Z Zeng Jing P80 Zeng Zhaolei P80 Zepeda Rossana P101 Zhang Feng P28 Zhao Zi P27, P28 Zhou Qun Yong S7.4 Zoran Mark S18.1 Zuzewicz Krystyna P14

252

KEYWORD INDEX

A 5-‐HT S6 ABPM P32 ABSENCE EPILEPSY P40, P70 ACTIGRAPHY P62, P63, P66 ACTION SPECTRUM P4 ACTIVITY PATTERNS S3.4, P7 ACTIVITY-‐REST CYCLE P19, P21, P29 ADENOSINE S21.3 ADENOSINE TRIPHOSPHATE S18.1 ADIPOSE TISSUE S9.3, S11.2 ADRENAL S6.2 AGEING S21.1 P31, P66 ALCOHOL P77, P78 ALERTNESS P26 ALGAE S9.2 ALGORITHMS P46 ALZHEIMER'S DISEASE P39 AMBULATORY BP AMPLITUDE P8 ANALYSIS S20.3 ANTICIPATORY ACTIVITY S2.2, P96, P105 ARCUATE P86 ARRHYTHMICITY P92 ASTROCYTE S18.1, S18.2, S15.3 ASTYANAX S10.1 ATELES GEOFFROYI P21 ATTENTION P33, P56, P57 AUTISM P82 AUTONOMIC NERVOUS SYSTEM P51

B BASAL GANGLIA P88 BEHAVIOR S5.3, P21 BETA-‐OXIDATION P97 BIOLOGICAL CLOCK P45 BIOLUMINESCENCE S13.4 BLATTELLA GERMANICA S4.4 BLOOD GLUCOSE LEVEL P110 BMAL1 P119 BODY TEMPERATURE P91 BRAIN CLOCKS S13.2 BREAST CANCER P44

C CAENORHABDITIS ELEGANS S14.3 CALCIUM IMAGING P128 CALCIUM P126, S7.2 CAPRINE S11.4 CARDIOVASCULAR S12.1 CAUDAL PHOTORECEPTOR P85 CELL CYCLE P75 CENTENARIAN P28

CEREBELLUM S13.2 C-‐FOS MRNA S17.3 CHERXAX QUADRICARINATUS P114 CHICKEN PINEAL GLAND P14, P83 CHILDREN P54, P33 CHRONOBIOLOGY P26 CHRONOPHARMACOLOGY P44 CHRONO-‐PREMETABOLIC CHRONOTERAPY P43 CHRONOTYPE P10, P34, P35, P38, P60 CIRCADIAN CLCOK S4.4, P93, P111 CIRCADIAN ENTRAINMENT S14.2 CIRCADIAN ORGANIZATION S2.3 CIRCADIAN REST-‐ACTIVITY CYCLE S16.5 CIRCADIAN RHYTHM S6.3, S7.2, S9.1, S13.1, P22,

P23. P24, P25, P32, P42, P95, P101, P106

CIRCADIAN SYSTEM S8.1, P18 CIRCADIAN S4.3, S5.3, S6.2, P20, P28,

P30, P51, P75, P77, P108 CIRRHOSIS P62 CLOCK GENES S9.3, S12.4, S13.1, S15.4,

S18.4, S21.1, P11, P39, P76, P101, P106, P114, P115 P120, P121

CLOCK PROTEINS S4.1 COCAINE S5.4, P76 COGNITION S21.3, P31 COHABITATION P92 COMPLEXITY S20.3 COMPUTER SIMULATIONS P49 CONSTANT ROUTINE P22 CORTICOSTERONE S6, S6.3, S8.4 CORTICO-‐STRIATAL CIRCUITS P23 COUPLING P4, P6 CRAYFISH P9, P114 CRUSTACEAN HYPERGLYCEMIC P122 CRYPTOCHROME P113

D DAILY MELATONIN RHYTHM P15 DANIO RERIO P120 DEIODINASES S11.1 DEPRESSION P41, P66 DESYNCHRONY S12.1 DESYNCRHONIZATION P43, P103 DEVELOPMENT S2.2, S8.3, P125 DIABETES S10.2, P61, P67 DISEASE S12.4 DISTURBED PHOTIC ENTRAINMENT P15 DIURNAL P2, P24 DJUNGARIAN HAMSTER P5, P15, P18 DMH S17.1 DOPAMINE S13.3, P65, P88 DROSOPHILA S3.1, S4.1, S4.3, S14.2 S18.3 DSPS P65

253

E EATING DISORDERS P35 EEG P74 ELDERLY P29, P30, P31, P41, P66 ELECTROPHYSIOLOGY S13.4 ENTERAL NUTRITION P42 ENTRAINMENT S3.2, S8.1, S8.3, S10.1,

S14.3, P3, P84 ENTRY TIME P16 EPILEPSY P40, P70, P81 EPWORTH SLEEPINESS SCALE P63 ESTROUS CYCLE P89 EVOLUTION S3.4 EXCESSIVE DAYTIME SLEEPINESS P63 EXECUTIVE FUNCTIONS P25 EXERCISE S5.4 EXIT TIME P16 EXTRA-‐SCN S13.4 EYA3 S11.3 EYESTALK P122

F FASCICULUS RETROFLEXUS S16.5 FEEDING S5.2, S13.2, S17.2 FEO S17.1, P98, P102, P110 FETAL S8.2 FIBROBLAST P30 FOOD ANTICIPATORY ACTIVITY P95, P111 FOOD ANTICIPATORY BEHAVIOR S17.1, S17.4 FOOD ENTRAINABLE OSCILLATOR S2.3, S17.3, P99 FOOD ENTRAINMENT S12.3, P95, P100, P103 FORCED DESYNCHRONIZATION S12.2, P48

G GABA S3.2, P94 GAP JUNCTIONS S18.2 GENE EXPRESSION S21.2 GENE MICROARRAY P80 GERBIL P7 GLAUCOMA P12 GLIA S18.2, S18.3 GLUCOCORTICOID S6.2, P105 GLUCONEOGENESIS P104 GLUCOSE S9.1, S9.4, P67 GLUTATHIONE BIOSYNTHESIS S3.1 GLYCOGEN P104 GUT PEPTIDES S17.4

H HAMSTERS S11.2 HATCHLINGS P84 HEALING P75 HEALTH P36 HIPPOCAMPAL DAMAGE P81

HOMEOSTASIS S21.3 HORMONES P102 HUMAN BRAIN P39 HUMAN PERFORMANCE P26 HUMANS S6.4, P43, P53, P115 HYPERTENSION S6.1, P27, P61, P79 HYPOCRETINS P100 HYPOPHYSIS P102 HYPOTHALAMUS P79, P100 HYPOXIA-‐INDUCIBLE FACTOR P9

I IMAGING S2.4 IMMUNE SYSTEM S15.1, S15.2, S15.4 IMMUNOMODULATION P44 IMPAIRED PHOTIC ENTRAINMENT P5 IN VIVO MUA S16.3 INDIRECT CALORIMETRY P42 INFLAMMATION P74, P83 INSECT S19.2 INSULIN P107 INTERVAL TIMING P23 INTRACELLULAR CALCIUM P127, P129 INTRINSIC CLOCK P88 INTRINSIC PHOTOSENSITIVITY S10.3 ION PUMPS S18.4

J JET-‐LAG S12.3, S15.2, P1, P47, P80 JNK P119

K KISSPEPTIN P90

L LACTATION S8.4 LATITUDE P11 LEARNING AND MEMORY S19.2 LESION S16.3 LIGHT S10.1, S10.4, P52 LIGHT-‐DARK CYCLE P49, P81, P121 LIGHTING DESIGN P36 LIPID METABOLISM P97 LIPOPOLYSACCHARIDE (LPS) S15.3 LIVER P94, P98, P99, P107 LOCOMOTOR ACTIVITY S18.3, P3 LUTEINIZING HORMONE SURGE P90

M MAGNETIC STORMS P13 MAMMAL P113

254

MANATEE P19 MATERNAL BEHAVIOR S3.3, P101 MATHEMATICAL MODELLING P47, P50, P48 MCTQ P34, P64 MEDICINE P45 MELANOPSIN S10.4, P12, P121 MELATONIN S6, S6.4, S8.2, S10.2, S10.3,

S12.3, P14, P41, P115 MENTAL DISORDERS P59 METABOLIC DAILY PROFILES P72 METABOLIC SYNDROME S12.4, P37 METABOLISM S9.1, S9.3, S12.1, P86 MICROARRAY P123 MINOR PSYCHIATRIC DISORDERS P38, P64 MONITORING P27 MORNINGNESS/EVENINGNESS P38 MOSQUITOES P93 MOTIVATION P96 MOTIVATIONAL ZEITGEBER P109 MOTOR SKILL P24 MOUSE P78 MPER2 P78 MRNA P83, P122, P124

N NARCOLEPSY P69 NATURAL CONDITIONS S14.2 NEUROGLOBIN P73 NEURONAL PLASTICITY S18.4 NEUROPEPTIDES S7.1, S7.2 NEWBORN P53 NIGHT EATING SYNDROME P35 NIH 3T3 P116 NOCTURNAL DIPPING PATTERN P32 NOCTURNAL P2 NON-‐HUMAN PRIMATES S8.2 NON-‐PHOTIC ENTRAINMENT S5.3, S8.4 NON-‐TRANSCRIPTIONAL S9.2 NTS P79 NULL P112 NURSING P108 NUTRIENTS S17.2

O OCTODON DEGUS P10 OLD P27 OLFACTORY S13.1, S19.2, P112, P106,

P125 ONTOGENESIS S8.1 OPN5 P117 OREXINS S7.3, S9.4, P69 ORNITHINE TRANSCARBAMYLASE P99 OUTPUT S7.4 OVULATION P89 OXIDATIVE STRESS P73

P PACEMAKER P126 PALATABLE FOOD S17.3 PALATABLE MEAL S17.4, P96, P109 PEPCK P104 PER1 P10, P98 PER1::EGFP S7.3 PER2 S6.3, S13.3, S14.4, P50 PER2::LUC S2.4 PERIOD VARIABILITY S20.2 PERIPHERAL CLOCK S10.4, P116 PEROXIREDOXIN S9.2 PHARMACOLOGY P41 PHASE ANGLE DIFFERENCE P16, P17 PHASE RESPONSE CURVE P8, P49, P112 PHASE WAVE P87 PHASE-‐SHIFT S4.4, P1, P40 PHOSPHOLIPID P116 PHOSPHORYLATION S4.1, P113, P119 PHOTOPERIOD S11.2, S11.3, S15.1, P1, P5,

P6, P7, P9, P19 PHOTOTRANSDUCTION P120 PINEAL GLAND P13 PLASTICITY S3.3, S11.1, P4, P123 POOR SLEEP AND SLEEP RHYTHM OB P59 POST-‐TRANSLATIONAL REGULATION P118 PREBÖTZINGER COMPLEX P129 PROCAMBARUS CLARKII P85 PROCESSING BODIES P118 PROINFLAMMATORY CYTOKINES S15.3 PROKINETICIN 2 S7.4 PROLACTIN P65 PROTEASOME ACTIVITY S3.1 PROTEIN S14.4, P127 PUBLIC TRANSPORT DRIVERS P37

Q QUALITY OF LIFE P28

R RABBIT S2.2, P108, P125 REPRODUCTION P103 REPRODUCTIVE SEASONALITY S11.4 RESETTING S14.4 RESPIRATORY RHYTHM P129 RESTRICTED FEEDING S2.4, P97, P107, P110 RETAIL STORES P36 RETINAL S10.2 S10.3 REWARD S5.2, S5.4 RGC-‐5 P117 RGCS P117 RHYTHMS P12 RODENTS S21.2 ROOSTING BEHAVIOR P17 RUNNING-‐WHEEL P91 RYANODINE RECEPTORS P124, P128

255

S15.1

S SCHEDULE CHANGE P33 SCN LESION P109, P111 SCN REORGANIZATION P6 SEASONAL REPRODUCTION P90 SEASONAL VARIATIONS P17 SEASONALITY P14 SERCA P126 SHIFT WORK S6.1, P48 SHIFTING CRITERIA TASK P25 SIGNAL ANALYSIS P46 SLEEP DEPRIVATION P56, P58 SLEEP QUALITY P55, P29, P55, P57, P53,

P54, P67, P72 SLEEP REGULATION S21.2 SLEEP S21.1, P37, P50, P54, P60,

P62, P66, P69, P70, P74 SLEEPINESS P52 SOCIAL BEHAVIOR S3.3, P123 SOCIAL RHYTHM P64, P92 SOCIO-‐SEXUAL RELATIONSHIPS S11.4 SOFTWARE P46 STATUS EPILEPTICUS P81 STRESS P20 STRESS GRANULES P118 STRIATUM S13.3 STUDENTS P60 SUBTERRANEAN P2 SUN COMPASS P84 SUNLIGTH P11 SUNSPOTS P13 SUPRACHIASMATIC NUCLEUS S2.3, S3.2, S5.2, S6.1, S7.1,

S7.3, S8.3, S12.2, S16.3, S18.1, P47, P73, P76, P86, P87, P124, P127, P128

SYNAPTIC TRANSMISSION S4.3 SYNCHRONISER S6.4 SYNCHRONIZATION P87 SYNDROME P61 SYRIAN HAMSTER S16.5 SYSTEM P94

T T CELLS S15.4 TEACHERS P55 TEMPERATURE CYCLES P93 TEMPERATURE P52 TEMPORAL PARTITIONING S3.4 TIME ESTIMATION P22, P31 TIME SERIES S20.2, S20.3 TSH S11.1, S11.3 TUCO-‐TUCO P91 TUMOR P80

U ULTRADIAN RHYTHM P85 UNMASKED BODY TEMPERATURE P18 UREA CYCLE P105

V VAGUS NERVE P89 VALIDATION P34 VALPROIC ACID P82 VASOACTIVE INTESTINAL PEPTIDE S7.1 VASOPRESSIN S7.4, S9.4 VISCERA OPERATION P59 VOCALIZATIONS P82 VOLCANO MOUSE P72

W WAG/RIJ RATS P40 WAKFULNESS P51 WAVEFORM P8 WAVELET S20.2 WITHDRAWAL P77 WORKING MEMORY P58

Y YAWNING P20

Z ZEITGEBER S14.3

256

Documents

THIRD&WORLD&CONGRESS&OF CHRONOBIOLOGY& ABSTRACTS.pdf · 2012. 3. 3. · 3" & & PRESIDENTMESSAGE& Chronobiology"has"become"awellZrecognized"discipline"in"contemporary"biomedical"research."The"