A molecular and cellular analysis of the circadian system

A molecular and cellular analysis of the circadian system of the cockroach Rhyparobia (Leucophaea) maderae

The neuropeptide PDF and neurotransmitters involved in input pathways to the circadian clock

Dissertation

zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)

vorgelegt von

El‐Sayed Baz El‐Shabrawy El‐Sayed

Abteilung Tierphysiologie – Institüt fur Biologie Fachbereich 10/ Mathematik und Naturwissenschaften

Universität Kassel Kassel, Deutschland

Kassel, Februar 2015

„Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes“

A molecular and cellular analysis of the circadian system of the cockroach Rhyparobia (Leucophaea) maderae

The neuropeptide PDF and neurotransmitters involved in input pathways to the circadian clock

Dissertation

for obtaining the degree Doktor der Naturwissenschaften (Dr. rer. nat.)

Submitted by

El‐Sayed Baz El‐Shabrawy El‐Sayed

Animal Physiology ‐ Department of Biology Faculty of Mathematics and Natural Sciences

University of Kassel Kassel, Germany

Kassel, February 2015

i

Vom Fachbereich 10 / Mathematik und Naturwissenschaften der Universität Kassel, Kassel, Deutschland als Dissertation am 12.02.2015 angenommen.

Accepted as dissertation by the Faculty of Mathematics and Natural Sciences, University of Kassel, Kassel, Germany, 12.02.2015

Erstgutachter: Prof. Dr. Monika Stengl (First Referee)

Zweitgutachter: Prof. Dr. Charlotte Helfrich‐Förster (Second Referee)

Prüfungskommission: (Examination committee)

1‐ Prof. Dr. Monika Stengl (Abteilung Tierphysiologie, Universität Kassel) (Department of Biology, Animal Physiology, University of Kassel)

2‐ Prof. Dr. Charlotte Helfrich‐Förster (Lehrstuhl für Neurobiologie und Genetik, Universität Würzuburg) (Department of Neurobiology and Genetics, University of Würzburg)

3‐ Prof. Dr. Friedrich Herberg (Abteilung Biochemie, Universität Kassel) (Department of Biology, Biochemistry, University of Kassel)

4‐ Prof. Dr. Raffael Schaffrath (Abteilung Mikrobiologie, Universität Kassel) (Department of Biology, Microbiology, University of Kassel)

Tag der mündlichen Prüfung: 15.04.2015 (Date of oral defense)

ii

حيم حمن الر بسم الله الر

ھار مبصرة لتبتغوا ھار آيتين فمحونا آية الليل وجعلنا آية الن فضلا من وجعلنا الليل والن

نين والحساب وكل شيء كم ولتعلموا عدد الس ب .يلا ـلناه تفص ـفص ر

12 سراءالإسورة

Meanings:

In the name of Allah, the Entirely Merciful, the Especially Merciful.

“And We have made the night and day two signs, and We erased the sign of the night and made the sign of the day visible that you may seek bounty from your Lord and may know the number of years and the account [of time]. And everything We have set out in detail”

Holy Quran, Sura Al‐Israa Verse 12

Bedeutungen:

Im Namen Allahs, des Allerbarmers, des Barmherzigen.

“Und Wir haben die Nacht und den Tag zu zwei Zeichen gemacht.

Dann haben Wir das Zeichen der Nacht ausgelöscht und das Zeichen des Tages hell gemacht, damit ihr nach Huld von eurem Herrn trachtet und damit ihr die Zahl der Jahre und die (Zeit)rechnung wißt. Und alles haben Wir ganz ausführlich

dargelegt”

Holy Quran, Sura Al‐Israa Verse 12

iii

Eidesstattliche Erklärung

Hiermit versichere ich, dass ich die vorliegende Dissertation selbstständig, ohne

unerlaubte Hilfe Dritter angefertigt und andere als die in der Dissertation

angegebenen Hilfsmittel nicht benutzt habe. Alle Stellen, die wörtlich oder

sinngemäß aus veröffentlichten oder unveröffentlichten Schriften entnommen sind,

habe ich als solche kenntlich gemacht. Dritte waren an der inhaltlich materiellen

Erstellung der Dissertation nicht beteiligt; insbesondere habe ich hierfür nicht die

Hilfe eines Promotionsberaters in Anspruch genommen. Kein Teil dieser Arbeit ist in

einem anderen Promotions‐ oder Habilitationsverfahren verwendet worden.

Kassel, 11.02.2015 ________________ El‐Sayed Baz

iv

It is my honor to dedicate this work to my wife Norhan

v

vi

Contents

List of Figures .................................................................................................................................................. ix

List of Tables ................................................................................................................................................. xiii

Contribution statements ........................................................................................................................... xiv

Zusammenfassung.......................................................................................................................................... 1

Summary ............................................................................................................................................................ 2

1. Introduction ................................................................................................................................................ 3

1.1. Circadian rhythms ...................................................................................................................... 3

1.2. Cellular and molecular basis of the insect circadian clocks ....................................................... 5

1.2.1. The circadian clock system of the fruitfly Drosophila melanogaster ............................ 6

Cellular and molecular components of the circadian pacemakers in D. melanogaster .... 6

Molecular mechanisms of D. melanogaster clock system ................................................. 9

1.2.2. The circadian clock system in Rhyparobia maderae ................................................... 10

The accessory medulla (AME) .......................................................................................... 10

PDF‐immunoreactive neurons .......................................................................................... 12

Light input pathway to the accessory medulla ................................................................ 14

Molecular mechanisms of R. maderae clock system ....................................................... 16

1.3. The neuropeptide pigment‐dispersing factor (PDF) ................................................................ 18

1.4. Aims of this study ..................................................................................................................... 20

2. Materials and methods ......................................................................................................................... 23

2.1. Experimental animals ............................................................................................................... 23

2.2. Primary cell cultures ................................................................................................................. 23

2.3. Calcium imaging experiments .................................................................................................. 26

2.3.1. Loading cells with Ca2+ indicator dye .......................................................................... 26

2.3.2. Imaging setup and recording ...................................................................................... 26

2.3.3. Drugs and application.................................................................................................. 31

2.4. Förster Resonance Energy Transfer (FRET) Imaging experiments ........................................... 34

2.4.1. FRET–Sensor ................................................................................................................ 34

2.4.2. FRET‐Sensor Microinjection ........................................................................................ 34

2.4.3. cAMP imaging setup and recording ............................................................................ 36

2.5. Images analysis, measurements, and statistics ....................................................................... 38

Contents

vii

2.6. Behavioral experiments ........................................................................................................... 40

2.6.1. Running‐wheel assays ................................................................................................. 40

2.6.2. Injections ..................................................................................................................... 40

2.6.3. Data analysis ................................................................................................................ 41

2.7. Extracellular recordings ........................................................................................................... 43

2.7.1. Electrode implantations .............................................................................................. 43

2.7.2. Experiential setup and recordings ............................................................................... 44

2.7.3. Data analysis and measurements ............................................................................... 45

3. Results ......................................................................................................................................................... 49

3.1. Neurotransmitters‐induced Ca2+ responses in the cultured circadian pacemaker neurons of the cockroach Rhyparobia maderae ........................................................................................ 49

3.1.1. Responses to acetylcholine (ACh) ............................................................................... 49

4.1.1.1. Effects of ACh on intracellular Ca2+ levels ..................................................... 49

4.1.1.2. Responses to ACh is mediated by nicotinic receptors ................................. 51

4.1.1.3. ACh‐induced Ca2+ increase is mediated by voltage‐activated Ca2+ channels ... 52

3.1.2. Responses to histamine (HA) ...................................................................................... 53

3.1.2.1. Effects of histamine on intracellular Ca2+ levels ........................................... 53

3.1.2.2. Histamine‐receptors in AME neurons ........................................................... 54

3.1.3. Responses to GABA ..................................................................................................... 56

3.1.3.1. Effects of GABA on intracellular Ca2+ levels .................................................. 56

3.1.3.2. Different types of GABA receptors may mediate GABA‐dependent inhibitory responses ....................................................................................................... 57

3.1.4 Responses to other neurotransmitters ....................................................................... 59

3.2. Signaling mechanisms of the neuropeptide pigment‐dispersing factor (PDF) in the cultured circadian pacemaker neurons .................................................................................................. 61

3.2.1. Effects of PDF on the intracellular Ca2+ activity........................................................... 61

3.2.2. PDF signaling is not exclusively mediated via adenylyl cyclase (AC)/cAMP pathway . 63

3.3. Histamine phase‐shifts the circadian locomotor activity rhythm of the cockroach Rhyparobia maderae ................................................................................................................................... 68

3.4. Simultaneous electrophysiological analysis of circadian rhythms of the circadian pacemaker center, of the electroretinogram, and of leg muscle activity in the cockroach Rhyparobia maderae ................................................................................................................................... 71

3.4.1. Electrical activity of AME (EAA) recorded with the EAA‐electrode ............................. 71

3.4.2. Electroretinogram (ERG) ............................................................................................. 73

Contents

viii

3.4.3. Electromyogram (EMG) ............................................................................................... 75

3.4.4. Microinjections and correlation analysis .................................................................... 76

4. Discussion .................................................................................................................................................. 79

4.1. Possible roles of neurotransmitters in the circadian system of the Madeira cockroach ........ 79

4.1.1. Acetylcholine (ACh) is a key player in circadian clocks in many circuits ..................... 79

4.1.2. Histamine (HA) is involved in entrainment pathways to the circadian pacemaker neurons via cimetidine‐sensitive receptors ................................................................ 82

4.1.3. Other neurotransmitters involved in different functional circuits of the circadian clock ……………………………………………………………………………………………………………………..84

Functional role of GABA in the circadian clocks ............................................................... 84

Possible role of serotonin on the circadian clocks ........................................................... 86

Possible role of glutamate and octopamine on the circadian clocks ............................... 87

4.2. Pigment‐dispersing factor (PDF)‐dependent calcium‐ and cAMP –signaling pathways in circadian pacemaker neurons of the Madeira cockroach Rhyparobia maderae ..................... 89

PDF signals via adenylyl cyclase‐dependent and ‐independent pathways ........................... 91

4.3. Simultaneous electrophysiological analysis of circadian rhythms of the circadian pacemaker center, of the electroretinogram, and of leg muscle activity in the Madeira cockroach ........ 95

Suitability of the recording methods ..................................................................................... 96

References ....................................................................................................................................................... 98

Appendix ....................................................................................................................................................... 119

Dual FRET‐ and calcium‐imaging experiments .............................................................................. 119

Acknowledgements ................................................................................................................................... 123

ix

List of Figures

Page

1. Introduction

Fig. 1.1 Model of a circadian system 4

Fig. 1.2 Schematic outline of a phase response curve (PRC) of circadian rhythms in response to treatment with a stimulus (e.g. light, neurotransmitters, or neuropeptides

5

Fig. 1.3 Clock‐gene‐expressing neurons in the brain of Drosophila 7

Fig. 1.4 Morning (M) and evening (E) cells in the brain of Drosophila Melanogaster. 8

Fig. 1.5 The PER/TIM‐dependent circadian core feedback loop in D. melanogaster. 9

Fig. 1.6 Dorsal view of the adult male cockroach Rhyparobia maderae. 10

Fig. 1.7 3D‐reconstruction shows the accessory medulla (AME) and its associated neurons.

11

Fig 1.8 A 3D‐reconstruction of the Madeira cockroach optic lobe shows the PDF‐immunoreactive (PDF‐ir) somata and fibers.

13

Fig. 1.9 The contralaterally projections of the largest and medium‐sized aPDFMe neurons in the Madeira cockroach.

13

Fig. 1.10 A scheme of a possible peptidergic and GABAergic light entrainment pathway in the circadian clock (AME) of the Madeira cockroach.

15

Fig. 1.11

A Comparison between a light‐dependent PRC and the neuroactive‐substance‐dependent PRCs in R. maderae.

17

Fig. 1.12

Schematic view of the different methods employed for the analysis of neurotransmitter‐signaling and of the signaling of the neuropeptide PDF in the circadian pacemaker center of the Madeira cockroach Rhyparobia maderae

21

2. Materials and methods

Fig. 2.1 Schematic view of the method used to prepare the primary cell cultures of accessory medulla (AME) cells of the Madeira cockroaches to be processed for Ca2+‐imaging and FRET experiments.

25

List of figures

x

Fig. 2.2 Schematic drawing showing the location of different components inside of the fluorescence microscope and describing the light path for Polychrome V illumination.

28

Fig. 2.3 Typical configuration scheme of the calcium and FRET imaging system devices to be controlled by Live Acquisition software.

29

Fig. 2.4 Recording chamber used in calcium and FRET imaging experiments. 29

Fig. 2.5 Fluorescence image of the cultured AME cells loaded with Fura‐2. 29

Fig. 2.6 Stimulus solution application system designed for imaging experiments. 33

Fig. 2.7 Loading FRET sensors into an AME neuron via a microinjection pipette. 36

Fig. 2.8 Outline of Ca2+/FRET experimental steps. 38

Fig. 2.9 Schematic representation of running‐wheel assays 42

Fig. 2.10 Schematic view of the setup used for electrophysiological experiments 47

3. Results

Fig. 3.1 Acetylcholine (ACh) increased both the amplitude and the duration of calcium responses in the accessory medulla (AME) pacemaker neurons in primary cell culture of the Madeira cockroach.

50

Fig. 3.2 Acetylcholine (ACh) activates nicotinic, but not muscarinic cholinergic receptors in the cultured AME pacemaker neurons of Madeira cockroach.

51

Fig. 3.3 Acetylcholine (ACh) activates voltage‐dependent calcium channels in the cultured circadian pacemaker neurons of the Madeira cockroach.

52

Fig. 3.4 Effects of histamine (HA) application on the intracellular calcium concentrations in the cultured circadian pacemaker neurons of the Madeira cockroach.

54

Fig. 3.5 Histamine (HA)‐dependent response of type I is dose‐dependent. 55

Fig. 3.6 Histamine (HA)‐dependent decreases in the intracellular calcium concentrations of the cockroach circadian clock neurons are mediated via cimetidine‐sensitive HA receptors.

56

Fig. 3.7 Calcium responses of circadian pacemaker neurons of the Madeira cockroach Rhyparobia maderae to GABA.

57

List of figures

xi

Fig. 3.8 GABA‐dependent inhibitory responses were not mediated exclusively by GABAA receptors.

58

Fig. 3.9 Effects of GABAB receptor agonist baclofen on the intracellular calcium concentration in the cultured circadian pacemaker neurons of the Madeira cockroach.

59

Fig. 3.10 Glutamate (Glu) decreased the intracellular calcium levels in the cultured AME neurons of the Madeira cockroach Rhyparobia maderae.

59

Fig. 3.11 Calcium responses of circadian pacemaker neurons of the Madeira cockroach Rhyparobia maderae to octopamine

60

Fig. 3.12 Calcium responses of circadian pacemaker neurons of the Madeira cockroach Rhyparobia maderae to serotonin (5‐HT).

60

Fig. 3.13 Representative examples of different types of calcium responses induced by bath applications of PDF in cultured AME neurons of the Madeira cockroach Rhyparobia maderae.

62

Fig. 3.14 PDF‐sensitive types 1 and 2 AME neurons are dose‐dependently and reversibly.

63

Fig. 3.15 PDF increased the Ca2+ baseline and the frequency of oscillating Ca2+ transients of type 1 neurons via adenylyl cyclase (AC) activation.

65

Fig. 3.16 PDF‐induced responses in the AME neurons are not exclusively mediated by the cAMP/PKA pathway signaling.

66

Fig. 3.17 Application of 1 μM PDF and the effect of re‐perfusion with 10 μM PDF in the same cell.

67

Fig. 3.18 Running‐wheel recordings of the circadian locomotor activity of the cockroach Rhyparobia maderae show phase delays and phase advances after the injection of (2 x 10‐12 mol).

69

Fig. 3.19 Phase response curve (PRC) of histamine (HA) injections compared with saline injections for the locomotor activity rhythm of the Madeira cockroach.

70

Fig. 3.20 Injection of HA between CT 12 and CT 15 induced dose‐dependent phase delays.

70

Fig. 3.21 Position of the stainless steel microelectrode next to the AME. 72

Fig. 3.22 Extracellular electrical recordings of the AME (EEA‐recordings) can show regular activity.

73

List of figures

xii

Fig. 3.23 Distribution of the AME electrical activities over 24 circadian hours. 73

Fig. 3.24 Circadian rhythms of the electroretinograms (ERGs) amplitude level changes can be measured under LD and maintained under DD conditions.

74

Fig. 3.25 Representative results for the cockroach locomotor activity and electroretinogram (ERG) for 3 consecutive days.

75

Fig. 3.26 Microinjections of saline and neuropeptide PDF in an example cockroach. 76

Fig. 3.27 The average activity distributions of electrical activity of AME (EAA) and electromyogram (EMG) for seven consecutive days recorded from a cockroach maintained in DD conditions.

77

4. Discussion

Fig. 4.1 Proposed mechanisms of PDF signaling pathways in the AME neurons of the Madeira cockroach.

94

Appendix

Fig. A.1 Schematic representation of the system used for dual FRET and Ca2+ imaging. 114

Fig. A.2 Dual cAMP and Ca2+ imaging from AME neurons using the microinjected heterochromatic FRET‐Sensor (Fl‐Cα/DY560‐RIα) and loaded with fura‐2.

115

Fig. A.3 The regularly spontaneously active and the silent neurons in the primary cell cultures of the AME pacemaker of the Madeira cockroach R. maderae.

116

Fig. A.4 The silent AME neurons in the cultured AME pacemaker of the Madeira cockroach were converted into regularly spontaneously active state after PDF applications

116

xiii

List of Tables

Page

Material and methods

Table 2.1 Summary of the components and attached equipments of the experimental setups 27

Table 2.2 Timing for calcium imaging experiment and the EM‐CCD camera settings 30

Table 2.3 Summary of drugs and pharmacological agents with respective concentrations used in the experiments

32

Table 2.4 Summary of the headstages with their respective differential amplifiers used in the extracellular experiments

44

Results

Table 3.1 Effects of histamine (2 x 10‐12 mol) and saline (control experiments) injections through the compound eye on the phase of the circadian locomotor activity of the cockroach R. maderae.

69

Table 3.2 The period length (τ) of the locomotor activity of the cockroach R. maderae before (τ before) and after (τ after) histamine (HA) and saline injections

70

xiv

Contribution statements

Parts of this doctoral thesis have already been published by the author. Published materials in the original wording are marked with dark gray and are within quotation marks (e.g. "Calcium responses…"). Moreover, some text from the published work is reformatted and paraphrased here. The contributions of the author (El‐Sayed Baz) in each section are stated clearly as follows:

Section 3.1: Neurotransmitter responses of the cultured circadian pacemaker neurons.

• Acetylcholine (ACh) neurotransmitter

o Dose‐response experiments were initially designed, conducted, and analyzed by Dr. Hongying Wei together with Johannes Grosshans as appeared in: Baz E‐S, Wei HY, Grosshans J, Stengl M (2013). Calcium responses of circadian pacemaker neurons of the cockroach Rhyparobia maderae to acetylcholine and histamine. Journal of Comparative Physiology A 199:365–374. The example presented in this part (i.e. Fig. 3.1) was prepared by the author from similar experiments.

o Pharmacological experiments were designed, performed and analyzed by the author, together with Dr. Hongying Wei as appeared in Baz et al. (2013).

‐ Figures 3.2 and 3.3 present different examples, which are in principle similar to

those which appeared in Baz et al. (2013).

• Histamine (HA) neurotransmitter

o Calcium response types, dose‐responses, and pharmacological experiments were designed, conducted, and analyzed by the author. The results of this part were published in: Baz et al. (2013).

‐ Figures 3.4 and 3.5B in this thesis are as appeared in Baz et al. (2013), with some format modifications.

‐ Figures 3.5A and 3.6 present different examples, which are in principle similar to those which appeared in Baz et al. (2013).

• Glutamate, serotonin, octopamine, and GABA neurotransmitters

o The author has designed and conducted all relevant experiments presented here.

Contribution statements

xv

Section 3.2: Signaling mechanisms of the neuropeptide pigmentdispersing factor (PDF) in the circadian pacemaker neurons.

• Effects of PDF on calcium activity in cultured circadian pacemaker neurons.

o The author has designed, in collaboration with Dr. Hongying Wei, a number of experiments to investigate the signaling of PDF using calcium imaging. These experiments were published in: Wei H, Yasar H, Funk NW, Giese M, Baz E‐S, Stengl M (2014). Signaling of pigment‐dispersing factor (PDF) in the Madeira cockroach Rhyparobia maderae. PLoS ONE 9(9): e108757. The data and figures presented in this part were conducted by the author unless stated otherwise.

‐ Figures 3.13C and 3.14 are modified from Wei et al. (2014). The author has contributed to these experiments and provided some primary cell cultures during the time course of the study.

• Effects of PDF on cAMP activity in cultured circadian pacemaker neurons. o Förster resonance energy transfer (FRET) experiments were designed,

conducted, and analyzed by the author. All of the data and figures presented in this part were conducted by the author.

Section 3.3: Running‐wheel assays

• The author has designed, conducted, and analyzed all experiments in this section.

Section 3.4: Extracellular long‐term recordings of intact cockroaches

o The author has:

- established the methods, - designed the experiments and conducted some experiments, - designed the animal holder together with Marcel Heim and Marius

Bartholmai. o Investigation and analysis of recorded signals were performed together with

Marcel Heim & Ildefonso Atienza López.

o All of the presented figures in this section are adapted from recorded data that were obtained by Marcel Heim & Ildefonso Atienza López under the author’s guidance of their bachelor (Heim, 2014) and diploma (López, 2014) theses, respectively. Fig. 3.21: the brain staining performed by Marcel Heim, with

the help of Azar Massah and Andreas Arendt.

Unless otherwise stated, all figures and tables in this thesis were prepared by the author. All of the above‐mentioned work was supervised by Prof. Dr. Monika Stengl.

xvi

Abbreviations

5‐HT Serotonin ‐ 5‐hydroxytryptamine AC adenylyl cyclase ACh acetylcholine AME accessory medulla ANe anterior neuron AOC anterior optic commissure aPDFMe anterior PDF‐ immunoreactive neuron AT allatotropin cAMP cyclic adenosine monophosphate clk Clock CLK CLOCK CLK‐CYC CLOCK‐CYCLE cry cryptochrome CRY CRYPTOCHROME CT circadiane time cyc cycle CYC CYCLE DD constant darkness conditions DFVNe distal group of fronto‐ventral neurons DN dorsal neurons DN1, DN2, DN3 dorsal neuron group 1‐3 DN1a anterior dorsal neuron group1 DN1p posterior dorsal neuron group1 dPDFLa PDF‐immunoreactive neuron at the posterior dorsal edge of the lamina DT distal tract E –oscillators evening oscillators EAA electrical activity of the accessory medulla EMG electromyogram Epac exchange proteins activated by cAMP ERG electroretinogram FSK forskolin GABA gamma‐aminobutyric acid GABAA ionotropic GABA A receptors GABAB metabotropic GABAB receptors Glu Glutamate GTP guanosin triphosphate HA histamine H‐B eyelet Hofbauer‐Bucher eyelet HCN hyperpolarization‐activated, cyclic nucleotide‐gated cation channel IBMX 3‐isobutyl‐1‐methylxanthine ILP inferior lateral protocerebrum IP3 inositol trisphosphate ir immunoreactive La lamina LD light/dark cycle LL Constant light conditions

Abbreviations

xvii

l‐LNv large ventrolateral neuron l‐LNvs large ventro‐lateral neurons LNd dorsal lateral neuron LNds dorso‐lateral neurons LPN lateral posterior neuron LPNs posterior‐lateral neurons MALDI‐TOF matrix‐assisted laser desorption/ionization time of flight MB mushroom body Me medulla MFVNe medial group of fronto‐ventral neurons MIP myoinhibitory peptide MNe median neuron M‐oscillators Morning oscillators OA octopamine PDE phosphodiesterase PDF pigment dispersing factor PDFR PDF receptor PDH β‐pigment‐dispersing hormone per period PER PERIOD per /PER period / PERIOD PI pars intercerebralis PKA protein kinase A PKC protein kinase C PLC Phospholipase C POC posterior optic commissure pPDFMe PDF neurons located posteriorly to the medulla PRC phase response curve SCN suprachiasmatic nucleus s‐LNv small ventrolateral neuron sLNvs small ventro‐lateral neurons SLP superior lateral protocerebrum SMP superior medial protocerebrum tim Timeless TIM TIMELESS tim /TIM timeless / TIMELESS VACC voltage‐activated calcium channel VIP vasoactive intestinal peptide VLP ventrolateral protocerebrum VMNe ventromedian neuron VNe ventral neuron vPDFLa PDF‐immunoreactive neuron at the posterior ventral edge of the lamina VPNe ventro‐posterior neuron ZT Zeitgeber time Δφ phase shift τ (tau) period length

1

Zusammenfassung

Circadiane Schrittmacher koordinieren die täglichen Rhythmen in Physiologie und Verhalten in lebenden

Organismen. Die Madeira Schabe Rhyparobia maderae (Synonym: Leucophaea maderae) ist ein gut

etabliertes Modell, um die neuronalen Mechanismen der circadianen Rhythmen bei Insekten zu

studieren. Die akzessorische Medulla (AME) in den optischen Loben des Gehirns wurde als das circadiane

Schrittmacherzentrum der Madeira Schabe identifiziert, das circadiane Rhythmen in der Laufaktivität

steuert. Über die Neurotransmitter der Eingangswege in das circadiane System der Madeira Schabe ist

noch nicht viel bekannt. Das Hauptziel dieser Arbeit war es, mögliche Eingangssignale in die innere Uhr

der Madeira Schabe zu bestimmen. An primären Zellkulturen von AME‐Neuronen wurden Calcium‐

Imaging Experimente durchgeführt, um die Neurotransmitter‐abhängigen Veränderungen in der

intrazellulären Calcium‐Konzentration zu messen. Darüber hinaus wurde die Signalkaskade des

Neuropeptids Pigment Dispersing Factor (PDF), dem wichtigsten Kopplungsfaktor in circadianen

Schrittmachern von Insekten, in Calcium‐Imaging und Förster‐Resonanzenergietransfer (FRET)

Experimenten untersucht. Acetylcholin (ACh) erhöht die intrazelluläre Calcium‐Konzentration in der

Mehrzahl der circadianen Schrittmacherneurone der Madeiraschabe. Applikation von GABA, Serotonin

und Octopamin erhöhten oder reduzierten die intrazelluläre Calcium‐Konzentration in den AME‐

Neuronen, während Histamin und Glutamat die intrazelluläre Calcium‐Konzentration ausschließlich

reduzierten. Pharmakologische Experimente zeigten, dass die AME‐Neurone ACh über ionotrope

nikotinische ACh‐Rezeptoren detektierten, während GABA über ionotrope GABAA‐Rezeptoren und

metabotrope GABAB‐Rezeptoren detektiert wurde. Diese Ergebnisse deuten darauf hin, dass die

circadiane Aktivität der Schabe durch verschiedene Eingänge, einschließlich ACh, GABA, Glutamat,

Histamin, Octopamin und Serotonin, moduliert wird. Bei den FRET Studien wurde ein Proteinkinase A

(PKA)‐basierter FRET Sensor zur Detektion von cyclischem AMP (cAMP) verwendet. Es wurde gezeigt,

dass PDF über Adenylylcyclase‐abhängige und ‐unabhängige Signalwege wirken kann. Zusätzlich wurden

Laufrad‐Assays durchgeführt, um Phasenverschiebungen im Rhythmus der circadianen Laufaktivität zu

detektieren, nachdem der Neurotransmitter Histamin zu verschiedenen circadianen Zeiten injiziert

wurde. Histamin‐Injektionen durch die Komplexaugen der Schabe ergaben eine biphasische

Phasenantwortkurve (phase response curve) mit Phasenverzögerungen in der Laufaktivität am späten

subjektiven Tag und am Beginn der subjektiven Nacht und Phasenbeschleunigungen in der späten

subjektiven Nacht. Schließlich wurde eine extrazelluläre Ableittechnik an lebenden Schaben etabliert, die

gleichzeitige Langzeit‐Ableitungen von der AME, des Komplexauges (Elektroretinogramm = ERG), und der

Beinmuskulatur (Elektromyogramm = EMG) für mehrere Tage ermöglichte. Diese Methode bietet einen

Ausgangspunkt für weitere elektrophysiologische Untersuchungen des circadianen Systems der Schabe,

in denen Substanzen (z.B. Neurotransmitter und Neuropeptide) analysiert werden können, die einen

Einfluss auf den circadianen Rhythmus in der Laufaktivität haben.

2

Summary

Circadian pacemakers (clocks) coordinate the daily physiological and behavioral rhythms in almost all

living organisms. The Madeira cockroach Rhyparobia maderae (syn. Leucophaea maderae) is a well‐

established model to study the neural mechanisms underlying the circadian rhythms in insects. The

accessory medulla (AME) in the brain’s optic lobe of the Madeira cockroach has been identified as

the circadian pacemaker center that controls its circadian locomotor activity rhythms. Not much is

known about the neurotransmitters of input pathways to the cockroach circadian system. The main

aim of this thesis was to determine possible input signals into the circadian clock of the Madeira

cockroach. Ca2+‐imaging on the primary cell cultures of the AME neurons was performed to measure

the neurotransmitter‐dependent changes in the intracellular Ca2+ concentration. Moreover, the

signaling mechanisms of the neuropeptide pigment‐dispersing factor (PDF), the most important

coupling factor of the insect circadian pacemakers, were investigated using Ca2+‐imaging and

fluorescence resonance energy transfer (FRET) experiments. Acetylcholine (ACh) increased the

intracellular calcium concentration in the majority of the circadian pacemaker neurons of the

Madeira cockroach. Applications of GABA, serotonin and octopamine were observed to increase and

decrease the intracellular calcium levels in the AME neurons, while histamine and glutamate

decreased the intracellular calcium levels in the AME neurons. Pharmacological experiments showed

that the AME neurons were responsive to ACh via ionotropic nicotinic receptors, while GABA acts on

ionotropic GABAA receptors and metabotropic GABAB receptors. These results suggest that the

cockroach circadian activity is modulated by several different inputs, including acetylcholine, GABA,

glutamate, histamine, octopamine, and serotonin. FRET studies employed a protein kinase A (PKA)‐

FRET‐based sensor for cyclic AMP and showed that PDF signals via adenylyl cyclase‐dependent and

‐independent pathways. In addition, running‐wheel assays were performed to determine phase

shifts of the circadian locomotor activity rhythms following the injection of the neurotransmitter

histamine at different circadian times. Injections of histamine through the compound eyes of the

cockroach revealed a biphasic phase response curve with phase delays in the circadian locomotor

activity rhythm at the late subjective day/beginning of the subjective night and advances at the late

subjective night. Finally, an extracellular recording technique on living cockroaches was established

to allow simultaneous long‐term recordings from the AME, the eye (electroretinogram), and the leg

muscle (electromyogram) activity for several days. This method will provide a starting point for

further electrophysiological studies of the cockroach circadian system that aim to investigate the

substances (e.g. neurotransmitters and neuropeptides) that have an influence on the circadian

rhythm of the locomotor behavior.

3

1. Introduction

1.1. Circadian rhythms

Circadian (Latin, circa = approximately; dies = day) rhythms are any physiological

oscillations and behavioral patterns with a period close to 24‐hours. They have been

observed in most, if not all, living organisms from cyanobacteria to insects and

vertebrates. Circadian rhythms are regulated by a self‐sustained and autonomous clock

(= circadian pacemaker). Central circadian pacemakers that control the animal´s behavior

are located in insects in neuropils associated with the visual system (Nishiitsutsuji‐Uwo

and Pittendrigh, 1968a, Stengl and Homberg, 1994), in mollusks directly associated with

the eyes (Eskin, 1979), in birds in the pineal gland (Gaston and Menaker, 1968), and in

mammals in the suprachiasmatic nucleus (SCN) of the hypothalamus (Moore and Eichler,

1972). Circadian rhythms are characterized by specific properties. First, they can be

entrained/synchronized by rhythmic environmental external cues or inputs (e.g.

day/night cycles). This external time cues are called Zeitgebers (German for ‘time

giver’). Second, they cycle autonomously in absence of any external time with a period

length of approximately 24‐hours (endogenous free‐running period). Third, the period of

the rhythm remains constant over a wide range of temperatures, this propriety is known

as temperature‐compensation. The circadian clock system (Fig. 1.1) consists of the

endogenous pacemaker, which generates an oscillation of about 24 hours, entrainment

pathways, which are able to phase‐shift the pacemakers oscillation daytime‐

dependently), and output pathways (to effectors) (Helfrich‐Förster et al., 1998, Homberg

et al., 2003, Roenneberg and Merrow, 2005). Furthermore, the effectors feedback to the

pacemaker and/or entrainment pathways components (Golombek and Rosenstein,

2010).

The treatment with stimuli like light pulses or pulses of drugs (e.g. hormones

neurotransmitters, neuropeptides, etc) can accelerate ‘advance’ or slow down ‘delay’ the

circadian oscillator, causing phase shifts (Rusak and Bina, 1990). Phase response curves

(PRCs, Fig. 1.2), describe the relation between the circadian time and stimulus‐

Chapter 1. Introduction

4

dependent phase shifts. In PRC, the amplitude of the phase shifts in hours (Δφ) is plotted

on the Y‐axis against the circadian time on the X‐Axis. The circadian time (CT) is divided

into subjective day (from CT0 to CT12) and subjective night (from CT12 to CT24). Usually,

CT 12 is defined as the onset of the locomotor activity rhythm of a nocturnal animal

under free‐running conditions (Saunders et al., 2002, Golombek and Rosenstein, 2010).

The CT is measured with respect to a phase reference point (φ). The closest distance

between the same phases is the period (τ = tau). Therefore, one circadian hour equals to

τ/24‐hours (Golombek and Rosenstein, 2010). Depending on the CT, light pulses advance

or delay if the animals are kept in DD conditions (Saunders et al., 2002). The exposure of

an animal in a running wheel to light during the subjective day has no effect on the

circadian locomotor activity rhythms. However, light at the early night (dusk) delays the

locomotor activity rhythms, while light at the late night/early day (dawn) advances the

locomotor activity rhythms (Wiedenmann, 1977, Page and Barrett, 1989, Golombek and

Rosenstein, 2010). PRCs with both phase delays and phase advances, such as the light‐

dependent PRC are called “biphasic PRCs”, while PRCs with either only phase advances or

phase delays are called “monophasic PRCs” (Golombek and Rosenstein, 2010). Photic

PRCs are biphasic, while the non‐photic PRCs do not resemble a light‐dependent biphasic

PRC (Golombek and Rosenstein, 2010).

Fig. 1.1: Model of a circadian system. The circadian system consists of input pathways, rhythm generators (oscillator), and output pathways. The input signals are received via receptors (e.g. photoreceptors), and then delivered to a pacemaker that generates the circadian rhythm (oscillator). Input signals may also influence the effectors without the participation of the clock (masking). Solid line arrows indicate pathways between the components. Dotted line arrows show feedbacks from the effectors to the pacemaker and/or entrainment components. This scheme is a flowchart that is commonly used as a model of circadian systems among chronobiologists. Modified from Roenneberg and Merrow (2000), Friesen et al. (2001) & Golombek and Rosenstein (2010).

feedback

~Zeitgebers

Entrainment Pathway

Endogenous oscillator

Effectorpathway

input outputLight Temperature re

ceptors

Physiology/ Behaviore.g. body temperature

locomotor activity


5

Fig. 1.2: Schematic outline of a phase response curve (PRC) of circadian rhythms in response to treatment with a stimulus (e.g. light, neurotransmitters, or neuropeptides). (A) Representative actograms for the locomotor activity rhythm (black bars) of a nocturnal animal kept in constant darkness (DD). The free‐running period (τ) is shorter than 24‐hours. The beginning of the activity is the phase reference point (CT12) which determines the circadian time (CT). At different times of the day, the animal was injected with the same stimulus: 1: at CT 06, no change; 2: at CT 16, phase shift delay (‐Δφ); 3: at CT 21, phase shift delay (+Δφ). (B) A representative PRC shows the phase shifts against the CT after the injection of the stimulus (A 1‐3). The open bar at the bottom of the graph represents the subjective day while the black bar represents the subjective night. Modified from Refinetti (2006) & Golombek and Rosenstein (2010).

1.2. Cellular and molecular basis of the insect circadian clocks

As mentioned above, the circadian clocks drive the circadian rhythms with a period of

about 24 hours. The circadian pacemakers that control the physiology and behavior of

most insects are clusters or populations of neurons located in specific regions of the

brain (i.e. the optic and cerebral lobes) (reviews: Helfrich‐Förster et al., 1998, Tomioka

and Matsumoto, 2010). In addition to the brain clocks, there are circadian oscillators in

some peripheral organs such as the Malpighian tubules, the gut, the antennae, and the

eyes (Giebultowicz and Hege, 1997, Myers et al., 2003, Merlin et al., 2007, Ito et al.,

2008, Uryu and Tomioka, 2010). Lesion and transplantation experiments located the

circadian pacemaker, which controls the locomotor activity rhythms in the Madeira

days123456789101112131415

‐Δφ +Δφ

A1 2 3

subjective day subjective night

Advance +Δφ

Delay ‐Δφ

Circadian time (h)

Phase shift 1

2

3B


6

cockroach Rhyparobia maderae, in the accessory medulla (AME) with associated

pigment‐dispersing factor–immunoreactive (PDF‐ir) neurons, ventrally to the optic lobe’s

medulla (Stengl and Homberg, 1994, Reischig and Stengl, 2003b). In the fruitfly

Drosophila melanogaster, the PDF‐ir neurons, which express the circadian clock genes,

are also circadian pacemakers which arborize in the AME (Helfrich‐Förster, 2006). The

following sections will briefly summarize the cellular and molecular organization of two

insects’ circadian clocks: the pacemaker of the cockroach R. maderae, the animal species

model used in this thesis; and the pacemaker of the D. melanogaster, with the best‐

studied cellular and molecular circadian clock.

1.2.1. The circadian clock system of the fruitfly Drosophila melanogaster

Cellular and molecular components of the circadian pacemakers in D. melanogaster

There are about 150 circadian neurons in the brain of Drosophila, expressing the

circadian clock genes (reviews: Peschel and Helfrich‐Förster, 2011, Helfrich‐Förster,

2014). The circadian neurons are divided into several groups according to their

anatomical position and size (Fig. 1.3). The dorsal neurons (DN) consist of three groups:

DN1a,p, DN2, and DN3; the lateral neurons (LN) consist of four groups: dorso‐lateral

neurons (LNd), large ventrolateral neurons (l‐LNv), small ventrolateral neurons (s‐LNv),

and lateral posterior neurons (LPN) (Helfrich‐Förster, 2003, 2006, Tomioka and

Matsumoto, 2010, Peschel and Helfrich‐Förster, 2011). Four s‐LNv and four l‐LNv neurons

are pigment dispersing factor (PDF) positive (Helfrich‐Förster, 1995). Moreover, there is

one PDF negative s‐LNv (5th s‐LNv). The study of the circadian neuron projections reveals

that all known neurons with projections, except the I‐LNvs, send their fibers into the

dorsal protocerebrum (Helfrich‐Forster, 2005, Helfrich‐Förster et al., 2007) (Fig. 1.3A).

The AME receives arborizations from the s‐LNv and l‐LNv cells (PDF positive neurons), in

addition to few DN1 and DN3 cells (Helfrich‐Förster et al., 2007). Next to PDF, clock

neurons show immunoreactivity against several peptides/proteins (Fig. 1.3B); such as

neuropeptide F (NPF) (Hermann et al., 2012), cryptochrome (Cry), IPNamide (IPNa)

(Shafer et al., 2006), and short neuropeptide F (sNPF) (Johard et al., 2009). In addition,


7

the clock neurons utilize neurotransmitters such as acetylcholine and glutamate (Johard

et al., 2009, Collins et al., 2012).

Fig. 1.3: Clock‐gene‐expressing neurons in the brain of Drosophila. (A) A schematic diagram of the

brain shows the circadian clock neurons and their projections. Different groups of circadian neurons are labeled with different colors. The dorsal neurons consist of three groups named DN1a,p, DN2, and DN3, while the lateral neurons consist of four groups: dorsolateral neurons (LNd), large ventrolateral neurons (l‐LNv, four cells), small ventrolateral neurons (s‐LNv, four cells + a 5

th s‐LNv), and lateral posterior neurons (LPN). The accessory medulla (AME) receives aborizations from the s‐LNv and l‐LNv cells (PDF positive neurons). Also, few DN1 and DN3 cells send fibers to the AME. As shown on the right side of the brain, the light is transmitted from photoreceptor cells R1–6 and R7/8 of the compound eye and from the Hofbauer‐Bucher (H‐B) eyelet (from: Helfrich‐Förster et al. (2007)). (B) Neurochemical characterization of the circadian clock neurons in the brain and PDF aborization. The expressed peptides/proteins in the different circadian neurons are labeled in different colors. Modified from Peschel and Helfrich‐Förster (2011).

A

B

AME

Cryptochrome (Cry)

short Neuropeptide F (sNPF) Neuropeptide F (NPF)ion transport peptide (ITP)choline acetyltransferase (Cha)

IPN‐amideUnknown peptidergic content

Pigment dispersing factor (PDF)

POT


8

D. melanogaster expresses a bimodal locomotor activity pattern with morning (M)‐

and evening (E)‐activity (review: Helfrich‐Förster, 2014). The M‐activity peak is controlled

by the PDF expressing s‐LNv neurons, while the E‐activity peak is controlled by the fifth

sLNv and the three CRY‐positive LNd neurons (Fig1.4) (Grima et al., 2004, Stoleru et al.,

2004, Rieger et al., 2006, Tomioka and Matsumoto, 2010, Yoshii et al., 2012, Yao and

Shafer, 2014). However, E‐cells were capable of controlling both of the M‐activity and E‐

activity patterns depending on the environmental conditions. Some other neurons like

the DN can strongly contribute to drive M‐activity and E‐activity (Sheeba et al., 2010,

Zhang et al., 2010, Hermann‐Luibl and Helfrich‐Förster, 2015).

Fig. 1.4: Morning (M) and evening (E) oscillators in the brain of Drosophila Melanogaster. The PDF expressing s‐LNv neurons control the M peak of activity and therefore they identified as morning cells (M‐cells, red), while the E‐activity peak is controlled by the fifth sLNv (5th) and the three CRY‐positive LNd neurons, therefore they identified as evening cells (E‐cells, yellow). Also the DN neurons can contribute to the control of M‐activity and E‐activity peak. E‐oscillator appears to be dominant under constant light (LL) conditions. In constant darkness (DD) conditions, the M‐cells act as dominant master oscillator governing over the E‐slave oscillator. Abbreviations: see Figure 1.3. Modified from Hermann‐Luibl and Helfrich‐Förster,(2015).


9

Molecular mechanisms of D. melanogaster clock system

Following the discovery of the first clock gene, period (per), by Konopka and Benzer

(1971), several other clock genes were isolated and identified in Drosophila: e.g. timeless

(tim) (Sehgal et al., 1994), doubletime (dbt) (Kloss et al., 1998, Price et al., 1998), clock

(clk) (Allada et al., 1998), cycle (cyc) (Rutila et al., 1998), cryptochrome (cry) (Stanewsky et

al., 1998), vrille (Blau and Young, 1999), and shaggy (sgg) (Martinek et al., 2001). At least

two feedback loops in gene expression were identified in the circadian oscillator of

Drosophila that are interlocked to oscillate with a period of about 24 h; such as the

per/tim feedback loop and the Clk feedback loop (Emerson et al., 2009, Hardin, 2011).

The per/tim transcriptional feedback loop is presented in Figure 1.5. The circadian cycle

initiates around the mid‐day when per and tim mRNA levels rise. The transcription of the

per and tim genes start when the transcription factors CLOCK/CYCLE (CLK/CYC)

heterodimers bind to the respective E‐boxes (Emerson et al., 2009, Hardin, 2011). The

transcription of per and tim is repressed by the binding of a complex PERIOD (PER) and

TIMELESS (TIM) proteins to CLK and CYC (Hardin, 2005, 2011). The maximum levels of

PER and TIM proteins occur at the late night. Then, PER and TIM move into the nucleus

either as heterodimers or independently from each other and interfere with CLK/CYC‐

dependent activation (Stanewsky, 2002). Following the light exposure in the morning,

CRYPTOCHROME (CRY) is activated to reset the clock since CRY binds and degrades TIM

and, subsequently, leads to the degradation of PER (Tomioka and Matsumoto, 2010,

Hardin, 2011, Peschel and Helfrich‐Förster, 2011).

Fig. 1.5: The PER/TIM‐dependent circadian core feedback loop in D. melanogaster. CLOCK (CLK) and CYCLE (CYC) bind as a heterodimer to the E‐box of the timeless (tim) and period (per) promoter. per and tim mRNA are moved to the cytoplasm and translated into PERIOD (PER) and TIMELESS (TIM) proteins. PER and TIM accumulate in the cytoplasm and move into the nucleus to inhibit their own gene transcription. For details see text. Modified from Tomioka and Matsumoto (2010).

Nucleus Cytoplasm

TIM

CLKCYC

PERperiod

timelessTIM

TIMPER

degradation

CRY

CRYE-box

E-boxlight


10

1.2.2. The circadian clock system in Rhyparobia maderae

The Madeira cockroach Rhyparobia maderae (Syn.: Leucophaea maderae) (Fig. 1.6) is

also a well‐established model for understanding the circadian clock system in insects. The

Madeira cockroach was the first animal, where an endogenous circadian pacemaker was

localized (Nishiitsutsuji‐Uwo and Pittendrigh, 1968b). Lesion and transplantation studies,

in combination with immunocytochemistry, first located the circadian clock controlling

locomotor rhythms in the optic lobes (Nishiitsutsuji‐Uwo and Pittendrigh, 1968b, Page,

1982, Stengl and Homberg, 1994, Reischig and Stengl, 2003a). Further studies showed

that the clock is located in the accessory medulla (AME singular; accessory medullae,

AMAE, plural) in association with PDF‐immunoreactive (PDF‐ir) neurons (Stengl and

Homberg, 1994, Reischig and Stengl, 2003a).

Fig. 1.6: Dorsal view of the adult male cockroach Rhyparobia maderae.

The accessory medulla (AME)

The AME of the cockroach Rhyparobia maderae is a small, pear‐shaped, non‐

retinotopically organized neuropil, which is located at the inner ventromedial edge of the

medulla of both optic lobes (Petri et al., 1995, Reischig and Stengl, 1996, Homberg et al.,

2003). The AME consists of glomeruli (noduli) neuropile and interglomerular (interior

neuropile) region that is surrounded by a shell neuropil (Reischig and Stengl, 2003b). The

prominent input pathway into the AME is through the distal tract which connects the

noduli of the AME to the medulla, and possibly also the lamina (Reischig and Stengl,

1 cm


11

1996). About 250 neurons are associated with the AME neuropil (Fig. 1.7). They are

divided into 7 groups, named relative to their location to the AME and their morphologic

criteria: anterior neurons (ANe), distal and medial‐frontoventral neurons (DFVNe and

MFVNe), medial neurons (MNe), ventral neurons (VNe), ventromedial neurons (VMNe),

and ventroposterior (VPNe) neurons (Reischig and Stengl, 1996, 2003b, Söhler et al.,

2008). Several neuropeptides and neurotransmitters have been localized in the AME

associated neurons by using immunocytochemistry and mass‐spectrometry; such as PDF,

allatotropin (AT), baratin, corazonin, FMRFamid related peptides, leucomyosuppressin,

short neuropeptide F (sNPF), gastrin/cholecystokinin, leucokinin, myoinhibitory peptides

(MIP), orcokinin (ORC), histamine , γ‐aminobutyric acid (GABA), and serotonin (Homberg

et al., 1991, Stengl and Homberg, 1994, Petri et al., 1995, Reischig and Stengl, 1996,

Nässel, 2000, Petri et al., 2002, Reischig and Stengl, 2003b, Hofer and Homberg, 2006,

Söhler et al., 2007, Söhler et al., 2008, Schulze et al., 2012)

Fig. 1.7: 3D‐reconstruction shows the accessory medulla (AME) and its associated neurons. Abbreviations: DT: distal tract, DFVNe: distal‐frontoventral neurons, MFVNe: medial‐frontoventral neurons, MNe: median neurons, VMNe: ventromedian neurons, VNe: ventral neurons, VPNe: ventroposterior neurons. Anterior neurons are not shown. Scale bar = 50 µm. Modified from Reischig and Stengl (2003b).

AMEDT

MNe


12

PDF-immunoreactive neurons

The structure of PDF‐ir neurons has been extensively investigated. Antisera against the

neuropeptide PDF located four groups of PDF‐immunoreactive (PDF‐ir) neurons in the

optic lope of Madeira cockroach (Fig. 1.8): the anterior medulla neurons (aPDFMe) and

the posterior medulla neurons (pPDFMe) are located near to the AME, while the dorsal

lamina neurons (dPDFLa) and the ventral lamina neurons (vPDFLa) occur in the lamina

(Petri et al., 1995, Reischig and Stengl, 2003b). The characterization of aPDFMe was well‐

studied (review: Homberg et al., 2003). The aPDFMe neurons consist of about twelve

neurons with different sizes; which can be divided into four small‐, four medium‐, and

four large‐neurons (with one conspicuously largest neuron). The four large and medium

aPDFMes neurons belong to the VNes, while the four small aPDFMe neurons belong to

the DFVNes (Reischig and Stengl, 2003b). The aPDFMe neurons couple both the

contralateral AMAE with the optic lobe neuropils and the neuropils in the central brain as

circadian coupling pathways (Fig. 1.9) (Reischig and Stengl, 2002, Reischig et al., 2004,

Söhler et al., 2011). The largest aPDFMe neuron forms connections between the

bilaterally‐symmetric AMAE via the anterior and posterior optic commissure (AOC, POC),

and branches apparently in all PDF‐target areas, also in the medulla and lamina

(Homberg et al., 2003). In contrast, about three medium‐sized aPDFMes form a

connection to the contralateral AMAE, only via the AOC (Reischig et al., 2004, Söhler et

al., 2011). Branches of the small aPDFMe neurons appear to be limited to the ipsilateral

AME and possibly other ipsilateral optic lobe neuropils (Reischig and Stengl, 2003b).

Interestingly, the PDFMe neurons and their branches exhibit morphological plasticity

when the animal treated with different photoperiods. For example, the number of

medium‐sized aPDFMes decreases in animals reared in shorter Zeitgeber periods (e.g.

11:11 LD), while increases in animals raised in longer photoperiods (e.g. 13:13 LD) (Wei

and Stengl, 2011). This indicates that PDFMes appear to play important roles in the

photoperiodic adjustment to the annual changes, as in other insect species (Shiga, 2013).


13

Fig. 1.8: A 3D‐reconstruction of the Madeira cockroach optic lobe shows the PDF‐immunoreactive (PDF‐ir) somata and fibers. Two PDF‐ir groups are next to the medulla (Me): anterior medulla (aPDFMe) and posterior medulla (pPDFMe) neurons. Two PDF‐ir groups are next to the lamina (La): dorsal lamina (dPDFLa) and ventral lamina (vPDFLa) neurons. The aPDFMe neurons are subdivided into three sub‐groups according to their cells size: small (blue), medium (green), and large (yellow, with a largest neuron in red). Arrow shows that some fibers from the first optic chiasma (1. OC) enter the distal layer of the La. Scale bar = 50 µm. Modified from Wei et al. (2010).

Fig 1.9: The contralaterally projections of the largest and medium‐sized aPDFMe neurons in the Madeira cockroach. The largest aPDFMe neuron forms a connection between the contralateral AMAE via the anterior and posterior optic commissure (AOC, POC), and processes into the medulla (Me) and the lamina (La). This neuron is branching in all PDF‐target areas. The medium‐sized aPDFMe form a connection between the contralateral AMAE only via the AOC, branching in the dorsal lateral protocerebrum (dSLP) and other areas of the SLP and SMP; while the posterior PDFMe appears to terminate in the posterior optic tubercles (POTu). Abbreviations: AME: accessory medulla; ILP: the inferior lateral protocerebrum; SLP: superior lateral protocerebrum; SMP: superior median protocerebrum) (From: Söhler et al. (2011)).

AME

AME


14

Light input pathway to the accessory medulla

It was shown only that photoreceptors in or close to the compound eye are essential for

the photic entrainment with high intensity light:dark (L:D) cycles (Roberts, 1965,

Nishiitsutsuji‐Uwo and Pittendrigh, 1968b, Roberts, 1974). In addition, lesion

experiments showed that ipsi‐ and the contralateral compound eyes relay light input

information to the AME (Page et al., 1977, Page, 1978). Studies have shown that HA is

the neurotransmitter of insect photoreceptors (Hardie, 1987, 1988, 1989, Stuart, 1999).

The distribution analysis of HA‐immunoreactive (HA‐ir) neuron in the brain of the

Madeira cockroach showed that there are no direct connections between the compound

eye photoreceptors and the AME (Loesel and Homberg, 1999), but there is only one

centrifugal HA‐ir neuron appeared to connect between the AME and the medulla to

different regions in the lateral protocerebrum (Loesel and Homberg, 1999). Therefore, it

was hypothesized that HA may not be involved in the light entrainment, but as a non‐

photic input (Loesel and Homberg, 1999). At present, several candidates for the light

entrainment pathway of the clock of the Madeira cockroach were suggested (Schulze et

al., 2013, Schendzielorz and Stengl, 2014); however, no much is known about the cellular

and physiological nature of light entrainment pathways or other non‐photic inputs (Baz

et al., 2013). Ipsilateral light inputs are transmitted to the glomeruli of the AME via the γ‐

aminobutyric acid (GABA)‐immunoreactive distal tract since the distal tract connects

different layers of the medulla and possibly also the lamina with the glomeruli of the

AME (Reischig and Stengl, 1996, Petri et al., 2002, Schendzielorz and Stengl, 2014). The

contralateral light inputs are apparently mediated via the orcokinin (ORC)‐ir neurons of

the AME and are projected via the posterior optic commissure (POC) (Hofer and

Homberg, 2006). Allatotropin (AT)‐ir local neurons of the AME appear to receive the

information from the distal tract (Petri et al., 1995, Reischig and Stengl, 2003b). So far,

distal tract neurons do not appear to have either MIP‐, AT‐, or ORC‐ir processes, so it is

suggested that distal tract neurons signal only via GABA (Schendzielorz and Stengl, 2014).

Additionally, a possible role of myoinhibitory peptide (MIP) is suggested to transmit the

photic information to the AME (Schendzielorz and Stengl, 2014). The suggested

peptidergic and GABAergic light entrainment pathway in the AME of the Madeira

cockroach is shown in Figure 1.10.


15

Fig. 1.10: A scheme of a possible peptidergic and GABAergic light entrainment pathway in the

circadian clock (AME) of the Madeira cockroach. The expressed peptides/transmitters in

different neurons are labeled with different colors. Abbreviations: AT: allatotropin; GABA:

γ‐aminobutyric acid; MIP: myoinhibitory peptide; ORC: orcokinin. Ipsilateral light input

can be conveyed to the glomeruli (grey color) of the AME via the GABA‐immunoreactive

distal tract (DT). The distal tract combines different layers of the medulla (ME) with the

glomeruli of the AME. Moreover, the median AME neurons (MNes) can provide ipsilateral

light information via their connection between the glomeruli of the AME, the medulla,

the accessory laminae (ALae), and the proximal lamina (La). It is suggested that the local

distal‐frontoventral neurons of the AME (DFVNe) with either AL or colocalized GABA and

MIP or GABA and AT may be postsynaptic to distal tract neurons or median AME neurons

(MNes). Additionally, the contralateral light input can be provided by a MIP‐ and ORC‐

colabeled ventral AME neuron (VNe), which projects via the lobula valley tract (LoVT) and

the anterior optic commissure (AOC) to the contralateral optic lobe. ORC‐immunoreactive

and one orcokinin‐ and MIP‐colabeled ventromedian AME neuron (VMNe) are suggested

to transmit the contralateral photic input. These cells send fibers to the medulla and the

interglomerular region of the AME, and connect to the other AME via the LoVT and the

posterior optic commissure (POC). (slightly modified from: Schendzielorz and Stengl

(2014)).

Allatotropin (AT)

GABA

Myoinhibitory peptide (MIP)

Orcokinin (ORC)

AME


16

Phase response curves of different neuroactive substances suggest their role as clock inputs

Behavioral assays showed that light pulses at different circadian times reveal a PRC with

phase delays at the early subjective night (dusk) and phase advances at the late

subjective night (Wiedenmann, 1977, Page and Barrett, 1989). Injection studies,

combined with behavioral assays, were performed to search for light‐like phase‐shifting

inputs to the circadian clock; by comparing the light‐dependent PRCs to the obtained

PRCs after the treatment with different neuroactive substances such as: serotonin (Page,

1987), PDF (Petri and Stengl, 1997), GABA, AT (Petri et al., 2002), ORC (Hofer and

Homberg, 2006), Rhyparobia‐myoinhibitory peptide‐1 (MIP‐1) (Schulze et al., 2013),

acetylcholine (ACh) (Schendzielorz, 2013), 8‐br‐cAMP, 8‐br‐cGMP (Schendzielorz et al.,

2014), and Rhyparobia‐MIP‐2 (Schendzielorz and Stengl, 2014) (Fig. 1.11). Injection of

PDF showed a monophasic PRC with phase delays at dusk (Petri and Stengl, 1997).

Interestingly, some of these substances show biphasic PRCs, that share the same polarity

to light‐dependent phase shifts (e.g. AT, ORC, GABA and 8‐br‐cAMP), suggesting that they

take a part in the light‐entrainment pathways to the circadian clock. Moreover, other

substances (except MIP‐2: see below) show all‐delay monophasic PRCs at times similar to

the light‐dependent delay shifts, suggesting their possible inputs (not necessarily photic

inputs) to the clock to maintain the endogenous period length of the Madeira cockroach.

Only the injection with MIP‐2 is shown to result in an all‐advance monophasic PRC with

its maximal effect at late subjective night (Schendzielorz and Stengl, 2014).

Molecular mechanisms of R. maderae clock system

Not much is known about the clock genes in the circadian system of the Madeira

cockroach, although cockroaches are an established model in the circadian research. The

first molecular data of the R. maderae circadian clock has been recently published

(Werckenthin et al., 2012). It has been reported that period (per), timeless 1 (tim1), and

mammalian‐type cryptochrome (cry2) are most likely part of the Madeira cockroach

nuclear circadian clock. Moreover, it was noted that there are circadian expression levels

of these genes oscillated with peaks in the first half of the night. Short‐day animals

showed a lower daily mean of expression levels than the long‐day animals (Werckenthin

et al., 2012).


17

Fig. 1.11: A Comparison between a light‐dependent PRC and the neuroactive‐substance‐dependent PRCs in R. maderae. These PRCs were re‐drawn and modified after: Page and Barrett (1989) A; Page, (1987) B; Petri and Stengl (1997) C1; Schendzielorz et al. (2014) C2; Schulze et al. (2013) D; Schendzielorz and Stengl, (2014) E; Petri et al., (2002) F and G; Hofer and Homberg (2006) H; Schendzielorz, (2013) I. Abbreviations: 5‐HT: serotonin; PDF: pigment dispersing factor; MIP: myoinhibitory peptide; GABA: γ‐aminobutyric acid; Mas‐AT: Mas‐allatotropin; ORC: orcokinin; ACh: acetylcholine. Blue dotted‐line curves (B‐I) show PRC for light. For details see text.

Circadian time (h)0 06 12 18 24

4

2

0

‐2

‐4

Light

Rhyparobia‐MIP‐1


4

2

0

‐2

‐4

Phase shift (h)

Rhyparobia‐MIP‐2


4

2

0

‐2

‐4

Phase shift (h)

Subjective day Subjective night

GABA


4

2

0

‐2

‐4

Phase shift (h)

Mas‐AT


4

2

0

‐2

‐4

Phase shift (h)

ORC


4

2

0

‐2

‐4

Phase shift (h)

5‐HT


4

2

0

‐2

‐4

Phase shift (h)

ACh


4

2

0

‐2

‐4

Phase shift (h)

PDF


4

2

0

‐2

‐4

Phase shift (h)

A

B

C

D

E

F

G

H

I

1

2


18

1.3. The neuropeptide pigmentdispersing factor (PDF)

The insect neuropeptide PDF plays many roles in the circadian system network such as a

coupling pathway between pacemakers, an output pathway to the locomotor control

centers, and as possible part of the light entrainment pathway (Reischig et al., 2004, Lee

et al., 2009, Helfrich‐Förster, 2014, Shafer and Yao, 2014). Interestingly, the functional

role of PDF in insect’s circadian systems is similar to the role of neuropeptide vasoactive

intestinal peptide (VIP) in the mammalian circadian system (Homberg et al., 1991,

Helfrich‐Förster, 1995, Vosko et al., 2007, An et al., 2011, Wei et al., 2014). Despite of the

critical necessary of these neuropeptides, their signaling pathways are poorly understood

(Schneider and Stengl, 2005, Pakhotin et al., 2006, Taghert and Nitabach, 2012, Kudo et

al., 2013, Wei et al., 2014). The Identification of the receptors of PDF (PDFR) and VIP

(VPAC2R) revealed that they are G protein‐coupled receptors (GPCRs) (Aton et al., 2005,

Hyun et al., 2005, Mertens et al., 2005). Both signal through increases in the level of

intracellular cyclic adenosine monophosphate (cAMP) (Aton et al., 2005, Hyun et al.,

2005, Lear et al., 2005). GPCRs are composed of seven transmembrane α‐helices which

are connected to the extracellular domains (that serve as peptides‐binding sites) and the

intracellular domains (that bind the heterotrimeric GTP‐binding protein, which made up

of α, β and γ subunits) (Mains and Eipper, 2006). Genetic loss of PDF and VIP and their

respective receptors results in modifications in the expression of robust molecular and

behavioral circadian rhythms in insects and mammals, respectively; suggesting that they

are critical components in the neuronal clock network (Renn et al., 1999, Helfrich‐Förster

et al., 2000, Harmar et al., 2002, Colwell et al., 2003, Peng et al., 2003, Lin et al., 2004,

Hyun et al., 2005, Lear et al., 2005, Mertens et al., 2005, Maywood et al., 2006, Lear et

al., 2009, Shafer and Taghert, 2009, Im and Taghert, 2010, Hassaneen et al., 2011).

Although about 10% (16 of 150 cells) of the circadian pacemakers of Drosophila

express PDF, PDFR is expressed by 60% of all pacemakers (Shafer et al., 2008, Im and

Taghert, 2010). PDFR in the pacemaker neurons signals through the Gα‐subunit and

activates adenylyl cyclase (AC) in different PDF‐receptive clock neurons, resulting in

increasing the cAMP concentrations that activates protein kinase A (PKA) (Hyun et al.,

2005, Mertens et al., 2005, Shafer et al., 2008, Choi et al., 2009). The cAMP/PKA pathway

signaling leads to the stabilization of the PER and TIM proteins, and thereby, resetting of


19

the molecular clock within the target clock neurons (Helfrich‐Förster, 2014, Li et al., 2014,

Seluzicki et al., 2014). It was discovered that PDFR activates different ACs, e.g. PDF‐

signaling in the s‐LNvs is mediated through the AC3 activation, while in the LNds, it is

mediated through the activation of AC78C (an ortholog of the mammalian AC8) and at

least one additional unidentified AC (Duvall and Taghert, 2012, Duvall and Taghert, 2013).

Additionally, it is noted that PDF signaling in vitro causes Ca2+ increases (Mertens et al.,

2005). A recent study showed that the coupling of the PDFR to different G protein, Gq ,

activates the inositoltriphosphate (IP3)/Ca2+ signaling in flight control circuits of

Drosophila (Agrawal et al., 2013). Moreover, it has been discovered that PDF induces

depolarization and causes a small increase in the Ca2+ level in a subset of DN1p neurons

(Seluzicki et al., 2014).

In the Madeira cockroach R. maderae, a cellular mechanism of the PDF‐dependent

gating of the circadian locomotor activity rhythms via activation of downstream neurons

was suggested (Schneider and Stengl, 2005). Electrophysiological characterization of the

AME revealed that the large majority of the AME neurons spike spontaneously. They

display fast oscillations with ultradian periods in the gamma frequency range of 20‐70 Hz

superimposed on their circadian activity rhythms (Schneider and Stengl, 2005, 2006,

2007). Moreover, it was found that the oscillated neurons within the AME form different

ensembles, which can be rearranged into new ensembles of synchronized ultradian

oscillators by PDF application via both phase‐delays and phase‐advances (Schneider and

Stengl, 2005).


20

1.4. Aims of this study

The main aim of this thesis was to analyze whether circadian pacemaker neurons of the

accessory medulla (AME), the circadian pacemaker center of the cockroach Rhyparobia

maderae, respond to the neurotransmitters acetylcholine (ACh), histamine (HA), γ‐

aminobutyric acid (GABA), glutamate (Glu), serotonin (5‐HT), and octopamine (OA). In

addition, pigment‐dispersing factor (PDF) signaling was analyzed in AME neurons to

decipher the signal transduction cascade of this important circadian coupling signal. First,

Ca2+‐imaging combined with pharmacological experiments were performed to measure

neurotransmitter‐dependent changes in the intracellular Ca2+ levels of the dissociated

fura‐2 loaded AME neurons to examine whether these neurotransmitters relay inputs to

the Madeira cockroach circadian clock system. Next, the signaling of the neuropeptide

PDF were investigated using Ca2+ imaging and Förster resonance energy transfer (FRET)

experiments. In these experiments PDF was applied to the cultured AME neurons to

identify and distinguish between calcium‐ and cAMP‐ dependent changes in response to

PDF.

In addition to the imaging techniques, running‐wheel assays were performed to

determine phase shifts of the circadian locomotor activity rhythms following the injection

of the neurotransmitter HA at different circadian times. Finally, to obtain information

about neuronal connectivity and mechanisms underlying circadian rhythms in the

Madeira cockroach, a method using extracellular recordings was established to record

simultaneously electrophysiological parameters from live cockroaches over several days.

Using this system, the electrical activity of the AME was compared with electrical signals

of the compound eye (the electroretinogram = ERG) and with electrical activity of leg

muscles (electromyogram = EMG), to determine whether there are correlations between

the three simultaneously recorded signals. In addition, it should be examined whether

and how PDF might generate correlated phase shifts in the three simultaneously

recorded signals. Different experimental methods used for achieving the objectives of

this thesis are summarized in Figure 1.12.


21

Fig. 1.12: Schematic view of the different methods employed for the analysis of neurotransmitter‐signaling and of the signaling of the neuropeptide PDF in the circadian pacemaker center of the Madeira cockroach Rhyparobia maderae. The study employed calcium imaging & FRET experiments, behavioral‐ and injection experiments as well as in situ long‐term extracellular recordings to identify possible input signals employed in entrainment pathways to the circadian pacemaker center to further our understanding of the cellular network of the circadian system.

Extracellular RecordingsEMG- ERG-EAA

Cellular and Molecular Studies

Electrophysiological StudiesBehavioral Assays ~input output

Neurotransmitters &Neuropeptides

Pacemaker

Circadian locomotor activity rhythms

input output

‐Which neurotransmitters play a role in entrainment pathways to the circadian pacemaker center of the Madeira cockroach?

‐ How does the neuropeptide PDF signal in the clock?

22

23

2. Materials and methods

2.1. Experimental animals

Madeira cockroaches Rhyparobia maderae (Syn.: Leucophaea maderae) were housed at

the animal room of the Department of Animal Physiology, at the University of Kassel.

Colonies of cockroaches were maintained in plastic boxes (L 60 x W 40 x H 40 cm);

containing wood shavings as substrate and egg cartons for providing places to hide.

Average room temperature was 25 ± 2°C and relative humidity was 40–60%. Food (e.g.

dried dog food, apples, potatoes, and tomatoes) was provided twice a week and water

was available ad libitum. Animals were reared in different photoperiods of light/dark (LD)

cycles (either short‐day LD 06:18, normal‐day LD 12:12, or long‐day LD 18:06).

2.2. Primary cell cultures

The preparation of primary cell cultures of the accessory medulla (AME) for Ca2+‐imaging

and FRET‐imaging experiments is summarized in Figure 2.1 and described as the

following:

2–3 adult male cockroaches were cold‐anesthetized and sterilized with either 70%

ethanol or Barrycidal solution (Barrycidal 36, Biohit, Rosbach, Germany), between

Zeitgeber time (ZT) 1 and ZT 5. The head capsule was removed and fixed by thin pins into

a small tray filled with wax. Once the head capsule was opened (cuticle was removed

from the front), about 500 µL of culture medium were added to the opened windows and

then the trachea and fats that surround the brain were removed with a very fine pointed

forceps. The accessory medullae (AMAE) were carefully isolated from the optic lobes

using a glass capillary with a 200 µm tip diameter (GB150T‐10, Harvard Apparatus Ltd.,

Edenbridge, UK), pulled with a DMZ‐Universal‐Puller (Zeitz Instruments, Martinsried,

Germany). Following the isolation, the AMAE were directly transferred into a 35 mm

culture dish (Falcon) contained about 3 ml culture medium. For tissue dissociation, the

isolated AMEA were incubated in 500 µl collagenase/dispase‐solution (CD, enzyme

solution) for about 5 minutes in a water‐bath at 37°C. After the incubation, the CD

enzyme solution with the dissociated cells was gently mixed using a 100 µl micropipette‐

Chapter 2. Materials and methods

24

tip, and then transferred into 10 ml intermediate medium solution to stop dissociations.

The solution was then centrifuged at 500 rpm for 5 minutes at 8°C. The supernatant was

removed by a suction pump system. The cell suspension (about 150–200 µl) was

distributed onto 4–5 concanavalin–A coated 8–mm round glass coverslips (Thermo

Scientific, Braunschweig, Germany) placed in the center of sterile tissue culture dishes

(35–mm diameter, Falcon). The cells were allowed to settle for at least two hours before

adding 1 ml culture medium to each culture dish. The culture dishes were labeled with

date and time and kept at 20°C in a dark humidified incubator and were used for the

experiments in the following day.

Medium and solutions

Culture medium:

‐ 98 ml L–15 medium (Leibovitz’s L‐15 Medium, PAA Laboratories, Cölbe, Germany),

‐ 1 ml of the following mix (200 mg/ml glucose, 80 mg/ml fructose, 35 mg/ml L‐prolin and 6 mg/ml imidazol, all dissolved in L‐15),

‐ 1 ml glutamine,

‐ 100 µl gentamicin,

‐ 2.38 mg/ml Hanks’ balanced salt solution (HBSS, Gibco),

‐ pH 7.0, 360 mOsm/kg adjusted with NaOH and mannitol.

Enzyme solution (collagenase /dispase):

10 ml HBSS (Gibco) containing 1 mg/ml collagenase and 4 mg/ml dispase

Intermediate medium:

10 ml L–15 medium (PAA Laboratories, Cölbe, Germany) supplemented with 2.8

mg/ml yeastolate and 2.8 mg/ml lactalbumin

Hanks’ balanced salt solution (HBSS):

170 ml Aqua bidest, 30 ml 10xHBSS, 2 ml Penicillin/Streptavidin, 0.01 g

Phenylthiourea, 0.025 mg/ml Phenolrot, pH 7.0, 380 mOsm/kg adjusted with NaOH

and mannitol.

All chemicals were obtained from Sigma‐Aldrich unless otherwise declared.


25

Fig. 2.1: Schematic view of the method used to prepare the primary cell cultures of accessory medulla (AME) cells of the Madeira cockroaches to be processed for Ca2+‐imaging and FRET experiments. Enzymatic dissociation was used to get single cells. Cells were plated onto glass coated with concanavalin A. The cultured neurons were maintained in a humidified incubator for at least 24 hours before starting the experiments (for details see text).

137.0

2

45

3

6

87

20.0

The AME isolation Tissue dissociation

CentrifugationSupernatant removing

culture mediumadding Incubation

4‐5 min at 37°C

stop dissociation

Cells suspension distribution

500 rpm for 5 min at 8°C

After about 2 hrs at 20°C


26

2.3. Calcium imaging experiments

2.3.1. Loading cells with Ca2+ indicator dye

Before starting Ca2+ imaging experiment, the cultured AME neurons were examined

microscopically to make sure that they are healthy (e.g. deteriorated and contaminated

cells were considered as unhealthy), and then loaded with 4 µM calcium–sensitive dye

fura‐2 acetoxymethyl ester (Fura‐2 AM, Molecular Probes Inc., Eugene, OR, USA) for 40

minutes in darkness at room temperature. Fura‐2 AM stock solution (1 mM; dissolved in

dimethyl sulfoxide, DMSO) was diluted into 2 mL of standard saline solution. The

standard saline contained: NaCl 156 mM, KCl 4 mM, CaCl2 1 mM, HEPES 10 mM, and

glucose 5 mM (pH 7.1, osmolarity 380 mOsm).

2.3.2. Imaging setup and recording

Two imaging systems (TILL Photonic, Germany), functionally identical and featuring the

same design, were used for calcium imaging recordings. Some of the calcium‐imaging

experiments were performed using the first setup at the beginning of the study before

switching mainly to the second setup. The components of both setups are presented in

Table 2.1. The location of the components inside a fluorescence microscope and the

typical hardware configuration of an imaging system are shown in Figure 2.2 and Figure

2.3, respectively.

Following fura‐2 loading, the coverslip with cultured AME cells was transferred

carefully into the bath area of the recording chamber (Fig. 2.4) placed on the stage of

fluorescence microscope and thoroughly washed with standard saline using a perfusion

pump for about 10 minutes before starting to remove unloaded dye. All calcium imaging

experiments were performed at room temperature. The number of selected cells viewed

with a 20x objective per experiment (i.e. single cell culture) in an imaging area of 16 x 104

µm2 was approximately 20–25 cells (Fig. 2.5). The selected cells in the field of view were

manually identified using the imaging software (Region of Interest [ROI] for each single

cell).


27

Table 2.1: Summary of the components and attached equipments of the experimental setups.

Component First Imaging setup Second Imaging setup

Uses/Applications ‐ Calcium Imaging ‐ Calcium Imaging ‐ Fluorescence resonance energy

transfer (FRET) imaging

Microscope Examiner D1 microscope (Zeiss, Germany)

Leica DMI3000 B Inverted microscope (Leica Microsystems, Germany)

Camera Name Series Model Manufacturer

EM‐CCD cameraAndor 885 IXON DU885KCS‐VP Andor Technology Ltd, Northern Ireland

EM‐CCD camera Andor 897 IXON DU897_BV Andor Technology Ltd, Northern Ireland

Light source Polychrome V, Till‐Photonics, Gräfelfing, Germany as a monochromator: excitation wavelength from 320 nm – 680 nm

Imaging control Unit Imaging Control Unit (ICU), TILL Photonics

Imaging Software TILLvisION software(Version 4.5.60, Till Photonics)

Live Acquisition software (Version 2.2.0.9, Till Photonics)

CMount Adaptor 60 N‐C 2/3" 0.63x (Zeiss, Germany) 1X HC : 3/4" to 1" (Leica, Germany)

Used Objectives ‐ 20x objective: W N‐Achroplan, NA 1.0(Zeiss, Germany)

‐ 20x objective: HCX PL FLUOTAR L20x/0.40 CORR (Leica Germany)

‐ 40x objective: N PLAN L40x/0.55 CORR (Leica Germany)

Used filter set ‐ Fura 2 filter set (EX BP 340/30, EX BP 387/15, BS FT 409, EM BP 510/90)

(Zeiss, Germany)

‐ Fura 2 filter cube (EX 395 SP, DC LP 400, EM ET 510/80), (Leica Germany)

‐ FITC/RH filter cube (EX BP 490/15, EX BP 560/25, DCM 500, EM BP 525/20, EM BP 605/30) (Leica Germany)

Image splitter None Dual‐Channel Simultaneous‐Imaging System (DV2‐ Photometrics, Tucson, Arizona, USA)

Micromanublatores ‐ Two PatchStar micromanipulators (Scientifica Ltd, Uckfield, UK)

‐ One 3‐axes Mini 25 (Luigs&Neumann)‐ One PatchStar micromanipulator (Scientifica Ltd, Uckfield, UK)

Microinjection system

‐ Picospritzer II (General Valve Corporation, Fairfield, New Jersey, USA)

‐ Picospritzer II (General Valve Corporation, Fairfield, New Jersey, USA)

‐ Eppendorf Transjector 5246 (Eppendorf, Hamburg, Germany)

Perfusion Pump REGLO Digital MS‐2/6 (Ismatec, IDEX Health&Science, Germany)

LKB 2232 Microperpex S (LKB‐Produkter AB, Bromma, Sweden)


28

Fig. 2.2: Schematic drawing showing the location of different components inside of the fluorescence microscope and describing the light path for Polychrome V illumination. The coverslip with cells is mounted above the microscope objective, in which the excitation light passes through to the cells. The filter wheel contains up to five filters, specific for different fluorescence dyes. For FRET imaging, an image splitter (DV2) with specific filter settings is used to split the light from the microscope into two colors (fluorescence emissions Images at 520 nm and 580 nm) to be detected by the EM‐CCD camera. For Ca2+ imaging, a specific filter‐cube for fura‐2 was selected to detect fluorescence emission at 510 nm with two excitation wavelengths: 340 nm and 380 nm.

CCD camera

Dichroic FilterDV2 Filter Cube

Adjustable Mirror

Emission filters

C‐Mount adaptor

Camera Port Slider

Focus Wheel

Bypass mode/DV2 Slider

Condenser

Dichroic filter Wheel

Light from Polychrome V

Objective

Emission filter

Excitation filter

Dichroic mirror

Transmitted light lamphousing

Field diaphragm

cells

340 nm

380 nm

To camera

Fura‐2 filter set


29

Fig. 2.3: Typical configuration scheme of the calcium and FRET imaging system devices to be controlled by Live Acquisition software.

Fig. 2.4: Recording chamber used in calcium and FRET imaging experiments. The coverslip (⌀ 8 mm) with AME cells was transferred carefully to the bath area of the chamber with a fine forceps after loading with Fura‐2. The floor of the chamber was a 24 x 60 mm #1 glass coverslip.

Fig. 2.5: Fluorescence image of the cultured AME cells loaded with Fura‐2 (viewed with 20x Objective, 512 x 512 pixels ‐ excitation wavelength at 380 nm for 10 ms exposure time). Most of the cells were not in direct contact with each other. Scale bar = 50 µm.

TTL IN

TTL OUT 3

Manual Objective Changer

TTL OUT 2

TTL OUT 1

COM PORTCOM1

Camera Sync OUT

Trigger

DiDO1 DO3

Di

Cells

Perfusion output port

Fura‐2‐AM loading

40 minutes


30

For calcium imaging experiments, the filter wheel located in the microscope was

moved to an appropriate filter‐cube for Fura‐2 (340 and 380 nm excitation filters & 510

nm emission filter). The cultured AME neurons were illuminated at two excitation

wavelengths (340 nm for 30 ms followed by 380 nm for 10 ms) using a Polychrome V

monochromator (Till Photonics). The fluorescence emission from both excitation

wavelengths was detected at 510 nm and live image pairs were captured with an EM‐CCD

camera (Andor) at 500 ms intervals. In this case, intracellular Ca2+ levels were calculated

by the ratio of the emitted fura‐2 fluorescence at 340 nm and 380 nm excitations. Timing

for calcium imaging experiment and the EM‐CCD camera settings are provided in Table 2.2.

Table 2.2: Timing for calcium imaging experiment and the EM‐CCD camera settings

Parameter Specification/value

Channels count 2 channels 1st Channel 340/15 nm 2nd Channel 380/15 nm

Light Source Polychrome V

Exposure Time / Cycle (ms) 1st Channel 30 ms / 35 ms 2nd Channel 10 ms / 465 ms

Total cycle (ms) 500

Delay (ms) 0

Loop count e.g. 1200 for 10 min recording ‘Depending on the length of experiments’

Cooling On

Fan Full

Temperature (°C) ‐100

Output Amp Electron Multiply

AD channel 14 bit

Readout rate 35.0

Pre‐Amp. Gain 1.0

EM gain On

Binning Symmetrical Hor. (X) 1 Vert. (Y) 1

Clock (MHz) 10

VSS (µs), Vertical Clock Speed 3.3

VCVA, Variable Control Voice Actuator 0

Baseline clamp On

FT Mode On


31

2.3.3. Drugs and application

Drugs with respective concentrations employed for experiments are listed in Table 2.3.

Unless otherwise indicated, all drugs were dissolved and diluted in standard saline

solution (= different stimulus solutions) before their application to the cultured AME

neurons. Stocks of drugs dissolved in dimethyl sulfoxide (DMSO) were diluted (1/1000) in

standard saline solution. Stock solutions were diluted to their final concentration on the

day of the experiments. All treatments were performed at room temperature. The AME

cells were superfused continuously with normal standard saline. Drug solutions were

applied to the cells via a perfusion system, which could deliver and exchange of different

solutions to the recording chamber without interrupting the live‐cell imaging. The

configuration of the perfusion system is illustrated in Figure 2.6.

For the dose–response experiments, the neurotransmitters were bath applied at

different concentrations (i.e. application of the same neurotransmitter on the same

neurons with increasing concentration). For the pharmacological experiments, the

cultured AME neurons were pre‐incubated with antagonists/blockers for at least 5

minutes before treatment with their respective drugs. After the incubation, the

neurotransmitters or their agonists were applied in the presence of the same

agonists/blockers. In all experiments, the cultured AME neurons were washed out at

least 5 minutes after each drug application to prevent desensitization.

For single cell application, drugs were applied via a glass micropipette (tip diameter

of about 1 µm) using pressure pulses (40 ms, 40 psi) from the Picospritzer II (General

Valve Corporation, Fairfield, New Jersey, USA). Borosilicate glass capillaries (GC‐150F‐ 7.5,

Harvard Apparatus Ltd., Edenbridge, UK) were pulled with a DMZ‐Universal‐Puller (Zeitz

Instruments, Martinsried, Germany). The micropipette was filled with the stimulus

solution and positioned closely to the cell (distance about 100 ± 20 µm) using a 3‐axes

micromanipulator (Mini 25, Luigs & Neumann, Ratingen, Germany).


32

Table 2.3: Summary of drugs and pharmacological agents with respective concentrations used in the experiments

Drug Description, Manufacture Concentrations

Acetylcholine (ACh) Neurotransmitter, Sigma Different concentrations

Nicotine Nicotinic ACh receptor agonist, Sigma 50 µM

Pilocarpine Muscarinic ACh receptor agonist, Sigma 50 µM

Mecamylamine Nicotinic ACh receptor antagonist, Sigma 50 µM

Scopolamine Muscarinic ACh receptor antagonist, Sigma

50 µM

Mibefradil Voltage activated calcium channels (VACC) blocker, Sigma

10 µM

Histamine (HA) Neurotransmitter, Biogenic amine, Sigma Different concentrations

Cimetidine Vertebrates histamine H2‐receptor antagonist, Sigma

500 µM

Picrotoxin (PTX) Chloride channels blocker, Sigma 100 µM

Gamma‐aminobutyric acid (GABA)

Neurotransmitter , Sigma Different concentrations

Muscimol (MUS) GABAA receptor agonist, Biotrend 100 µM

Baclofen GABAA receptor antagonist 10 µM

Forskolin (FSK)* Adenylyl cyclase (AC) activator, Biotrend 10 µM & 20 µM

SQ 22536 AC inhibitor, Sigma 20 µM

Glutamate (Glu) Neurotransmitter, Sigma Different concentrations

Serotonin (5‐HT) 5‐hydroxytryptamine

Neurotransmitter, Sigma Different concentrations

Octopamine (5‐HT) Neurotransmitter, Sigma Different concentrations

3‐Isobutyl‐1‐methylxanthin (IBMX)*

Phosphodiesterase (PDE) inhibitor, Sigma

20 µM

Pigment dispersing factor (PDF)

Neuropeptide (NSEIINSLLGLPKVLNDA), Iris Biotech, Marktredwitz, Germany

Different concentrations

* Stock solutions were dissolved in dimethyl sulfoxide (DMSO) and diluted in standard saline solution. The rest was dissolved and diluted in standard saline solution.


33

Fig. 2.6: Stimulus solution application system designed for imaging experiments. Perfusion system with two peristaltic pumps allows liquid to flow through the bath area of a recording chamber (1 ml/min). Both pumps are controlled by computer. The first pump is connected to a manual switching system with six ports (named 1‐6); to manually alternate the flow of the solutions. The second pump is connected to an electronic 3‐way valve (manufactured by Parker) controlled by computer, to automatically alternate the flow between solutions (A & B). Solutions are conducted by Teflon tubing. Double pumps allow alternative perfusion assays and enable continuous flow of the normal standard saline with the possibility of controlling the solution volume and duration of application for a wide range of stimulus solutions over the cells without interrupting live imaging acquisition. The system is regularly disinfected with 70% ethanol and cleaned very well before and after the use with distilled water to avoid contaminations or clogging, and then kept dry.

1

2

3

4

5

6

1

2

34

5

6

654321

PC

BA

InOut

Waste “Used solution”

Standard saline and stimulus solutions

Pump I

Pump II

Waste

3‐wayvalve

Recording chamber

Cells

Solutions switch

Removal of solution “Drain”


34

2.4. Förster Resonance Energy Transfer (FRET) Imaging experiments

2.4.1. FRET–Sensor

The synthesis and characterisation of FRET–based cAMP biosensor have been extensively

addressed elsewhere (Stieger, 2011). Briefly, FRET studies employed the differently

fluorescence‐labelled two subunits of cAMP‐dependent protein kinase A (PKA) to

monitor cAMP changes in the AME neurons. The catalytic subunit (mCα, mouse, type α)

was labelled with fluorescein and the regulatory subunit (hRIα, humane, type Iα) was

labelled with DY560, rhodamine‐based label, to obtain the heterochromatic FRET‐sensor

(Fl–Cα/DY560–RIα). The regulatory subunit binds cAMP and, upon binding, the two

subunits of PKA dissociate and the catalytic subunit becomes active. Thus, the increased

distance between the two fluorophores suppresses energy transfer between both

fluorophores and the FRET‐signal decreases (Adams et al., 1991). Therefore, energy

transfer between the labelled‐subunits is a measure of the intracellular free cAMP

concentration.

Stock solutions of FRET‐Sensor was stored at ‐80 °C (in individual tubes, each

contains 10 µL with concentration of 50 µM). If stored well, the FRET‐Sensor should be

stable and responsive to cAMP for several months. At the day of the experiment, a tube

of FRET–Sensor stock solution was moved out to a box contained crushed ice to be

transferred from place to another. Directly after thawing, a portion of the stock solution

was diluted in standard saline in a nuclease‐free sterile protein low–binding microtube to

be used for injections when ready. The rest was stored at 4–6 °C to be used during the

following days. The concentration of FRET‐Sensor used for injection was 20 ± 5 µM. Then,

the sensor was kept into the ice during the course of the experiments and protected

from light.

2.4.2. FRETSensor Microinjection

Microinjection is the common and the only technique used so far to introduce such types

of FRET–sensors based upon cAMP‐dependent protein kinase (PKA) into cells.

Microinjections were performed by using either pulled glass micropipettes


35

manufactured with a needle puller (DMZ‐Universal puller; Zeitz‐instrumente,

Martinsried, Germany) from borosilicate glass capillary with filament (10 cm in length,

1.5 mm outside diameter, 1.17 mm inside diameter; Sutter Instruments, Novato, CA,

USA), or commercial glass micropipettes (Sterile Femtotips, Eppendorf, Hamburg,

Germany). The diameter of micropipette tip was approximately 0.5 ± 0.2 µm.

Microinjection pipettes were loaded with about 0.5 µL of the FRET‐Sensors by sterile

Microloader Pipette Tips (Eppendorf) or by standard polypropylene 0.1–2.5 µL sterile

microtips for back‐filling. The filament inside the pipette allows the sensor to be well‐

accumulated into the tip. After filling, the tip of the pipette was controlled under the

microscope to be sure that it has not been broken during the filling process and to note

the presence of air bubbles which prevent injections. Filled micropipettes with FRET‐

Sensor were inserted into the needle holder which is attached to

a PatchStar micromanipulator (Scientifica Ltd, Uckfield, UK), which can be moved in XYZ

and approach axes positions. The micromanipulator could be also controlled by the

computer with LinLab software (Version 1.678, Scientifica). The micropipette holder

connected to an Eppendorf Transjector 5246 (Eppendorf) used to introduce the FRET–

Sensor into the cytoplasm of each AME cells by pressure injection. The injection pressure

(Pi) was 100–140 hectopascals (hPa), time (ti) was 0.1 sec and the compensation pressure

(PC) was 55–75 hPa.

Microinjection was performed at 40x magnification using phase‐contrast of an

inverted microscope (Leica DMI3000 B Inverted microscope, Leica Microsystems,

Germany) with red light. Micropipette tip was moved into the recording chamber. Once

the tip was visible in the field of view, it moved up and down above the cell and finally

positioned few micrometers over the selected cell, and then the micromanipulator

moved along over the cell in the direction of the X‐axis. Immediately after the movement,

approach axis was activated and gently moved to touch and pierce the plasma

membrane, and then the above pre‐adjusted pressure was applied (Fig. 2.7). The total

volume of the injected sensor was estimated to be smaller than 10% of the cell volume.

The final intracellular concentrations were estimated to be 0.2–2 µM (Adams et al.,

1991). Successful injections were evaluated under the microscope in bright field. Also, an

image of the fluorescence emission of fluorescein was acquired at 520 nm when it


36

excited at 488 nm. Following microinjection, cells were washed with standard saline

solution. FRET experiment was performed at least 30 minutes after microinjection to

allow the cell to be recovered.

Fig. 2.7: Loading FRET sensors into an AME neuron via a microinjection pipette. (A) Schematic representation of the method of FRET‐sensor injection, showing how the pipette tip was moved towards the cell. (B) The proper positioning of the micropipette inside the cell to inject the sensor into the cytoplasm of an AME cell. A pressure pulse was delivered by using an Eppendorf Transjector 5246. Scale bar = 20 µm.

2.4.3. cAMP imaging setup and recording

Fluorescent resonance energy transfer (FRET) was used to detect the relative changes in

the intracellular cAMP level in response to the neuropeptide pigment‐dispersing factor

(PDF). FRET experiments were designed to identify whether cAMP responses to PDF

occurred or not. FRET imaging experiments were performed on an inverted microscope

(Leica DMI300B microscope, Leica, Germany) equipped with a Dual‐Channel

Simultaneous‐Imaging System (Dual View, DV2, Photometrics, Tucson, Arizona, USA)

attached to an EM‐CCD camera (IXON+DU_897BV, Andor) and a 40x objective (N PLAN

CellMovement steps Microinjection

40x objective

A

B


37

L40x/0.55 CORR, Leica Germany). Dual view was used to split the emission light in two

fluorescence channels to be simultaneously detected by the camera. The Imaging system

used for FRET studies is described in Table 2.1 and Figures 2.2 and 2.3.

After successful Ca2+ imaging experiments, the PDF‐responsive cells were selected to

be injected with the cAMP–dependent PKA FRET–sensor. Outline of the experimental

steps is summarized in Figure 2.8. The selected cells in the field of view were manually

identified using the imaging software (Live Acquisition, Version 2.2, TILL Photonics). The

PKA‐based sensor coupled to fluorescein (FITC as Donor) and rhodamine‐based (RH as

Acceptor) fluorescent dyes. The Fluorescence images for both fluorophores can be

obtained with the setup when: 1) The filter wheel, located in the microscope, was moved

to a filter‐cube for FITC/RH (excitation filter 490/15 & Dichromatic mirror 500, 580 &

suppression filter 525/20, 605/30, Leica); 2) The DV2 slider was moved to DV2 instead of

the Bypass Mode for acquiring simultaneous images; 3) DV2‐Cube filterset (HQ 525/50,

HQ 605/55 & DCM T565 LPXR, Photometrics) was completely inserted into the tube. The

DV2 channels were aligned in highly precise way (i.e. donor and acceptor channels were

adjusted pixel by pixel) followed the Photometrics DV2™ Alignment Procedure manual1,

and readjusted before each experiment. In practice, DV2 misalignment (e.g. the positions

of the two images are not identical) leads to inaccuracy in ratio images and subsequent

failure to detect FRET signal.

Cultured AME neurons were excited with light from a monochromator polychrome V

(Till Photonics) at 488/15 nm for 100 – 300 ms at 5 s intervals. Excitation of fluorescein

leads to emissions at 520 nm at 580 nm due to energy transfer between (donor)

fluorescein and (acceptor) rhodamine fluorescent dyes. FRET occurred when the distance

between both fluorophores is small (< 10 nm), for that reason the donor emission at 520

nm is decreased and the acceptor emission at 580 nm is increased. Binding of cAMP to

PKA leads to dissociation of its two subunits, resulting in an increased distance between

both fluorophores and subsequently the acceptor emission is reduced. Therefore,

changes in the intracellular cAMP levels were measured indirectly by an increase in the

ratio of 520 nm/ 580 nm emission. To reduce photobleaching and prolong recording

time, electron‐multiplying (EM) gain was increased (150–250) instead of increasing the 1 Available at: http://www.photometrics.com/support/pdfs/manuals/dv2_alignment.pdf (Accessed: 21 Nov. 2014)


38

exposure time since the EM gain allows minimal exposure of AME neurons to light at

excitation wavelengths.

Fig. 2.8: Outline of Ca2+/FRET experimental steps.

2.5. Images analysis, measurements, and statistics Sequence of images was acquired during the imaging experiments using the EM‐CCD

camera and its respective software (Table 2.1) and saved as image stacks. In addition to

online analysis, offline image analysis was performed using ImageJ software (version

1.46j, National Institutes of Health, USA). Fluorescence ratios for fura‐2 excitation

wavelengths at 340 nm and 380 nm was calculated (R = F340/F380) to measure the relative

changes in the intracellular Ca2+ concentration. Changes in the intracellular cAMP levels

were measured by calculation the ratios of the emitted fluorescence at 520 nm/ 580 nm

of the PKA‐based FRET‐fluorosensor (R = F520/F580). Fluorescence emission intensities

were background‐subtracted before the calculation of the ratios (i.e. one ROI with no

cells was specified to be selected as a background ROI for background subtraction from

Stimuli application

Fura‐2 Loading(40 min)

Calcium responses to stimulusYes

NO

Responses classification

FRET‐Sensor injection(Microinjection)

Data storage Offline analysis &

Quantitative analysis

Online analysis &

visualization

FiltersetFura‐2 filter cube

Filterset ‐ Filter cube: FI/RH

‐DV2 & its filter cube

The same stimuli application

StartCalcium Imaging

StartcAMP Imaging

(FRET)Stop


39

the emission intensities of fluorescent cells). Unless otherwise indicated, data are

expressed as fluorescence emission ratios. The ratios (F340/F380 & F520/F580) were

normalized to the baseline (R0) by dividing each ratio value by the average of 3‐5 basal

values before drug application to facilitate the comparison of the results between

independent experiments using different imaging setups and settings. For ratios images

presentation, the fluorescence is shown in pseudo-color and processed with Gaussian filter.

For the calculation of the intracellular calcium concentration ([Ca2+]i), the Gronkiewicz

equation (Grynkiewicz et al., 1985) was used as the following:

Kd: dissociation constant for Ca2+ fura‐2 binding to fura‐2 (assumed to be 2.66 µM,

according to Wei and Stengl (2012)).

ß: a factor which scales kd into the dissociation constant effective in the preparation.

R is Ratio of the fluorescence measured during the experiment (F340/F380).

Fmin and Fmax are the fluorescence intensities in the absence of Ca2+ or in the presence of a

saturating concentration of Ca2+, respectively.

Data are given as mean ± standard error (SE). Calcium responses amplitudes and

duration were measured with Spike‐2 software (version 7.02). Statistical comparisons

between different responses to neurotransmitters were performed with ANOVA test

with Tukey’s post hoc analysis with a significance level of P < 0.05. Since, Kolmogorov–

Smirnov was used as test for normality. Statistical comparisons of cAMP baseline levels

before and after the PDF application were performed using Mann‐Whitney U‐test. A

difference was considered statistically significant when p < 0.05.Data analysis and

graphics were performed using MS Excel 2007, SPSS (Statistical Package for Social

Science, Chicago, Illinois), and OriginPro 8 (version 8.754, OriginLab, Northampton, MA,

USA).

[ ]380380

max

min

max

min2

FF

RRRRKdßCa i ×−

−××=+


40

2.6. Behavioral experiments

2.6.1. Runningwheel assays

Phase shifts of the circadian locomotor activity in the Madeira cockroaches (R. Maderae)

were analysed in running‐wheel assays in constant darkness (DD) at various circadian

times (CTs) in response to the neurotransmitter histamine (HA) injection. HA‐dependent

phase shifts were assayed by obtaining phase response curves (PRCs). Only adult male

cockroaches were obtained from laboratory colonies under LD 12:12 photoperiod and

housed individually in running wheels (radius 3.5 cm, depth 3 cm) under DD conditions.

Running wheels were placed in wood cabinets/boxes with doors (16 individual running

wheels per cabinet). Cabinets were placed in a dark room under controlled temperature

(25°C). The room contained a dim red light lamp to be used when the cabinet door was

opened during care, maintenance, or obtained for injection. Cockroaches inside the

wheels have free access to water and sesame sticks food (Ad libitum). Running wheel

activity was recorded continuously, since the number of rotations per minute (1440

minutes = 24 hrs) was collected by custom‐programmed software developed by C. Lohrey

(University of Kassel). Each wheel was connected to a reed switch to monitor the wheel

activity. During the rotation, the switch was closed by two magnetic parts attached to the

wheel. At that stage, the data was saved to a text file format on a SD memory card

attached to the data collection system. The collected data were then copied to the

computer for analysis.

2.6.2. Injections

The injection method resembled methods developed by Schulze et al. (2013). On the

injection day, animals were removed from the running wheel and completely

anesthetized with a short, small CO2 dose and then positioned in a holder fabricated from

acrylic‐glass with metal base (made by the workshop of the University of Kassel), in which

the cockroach body was well‐fixed and its head capsule was kept outside to be viewed

under a stereomicroscope equipped with a dim red light source. Cockroaches were then

injected with a repetitive pipette (HandyStep, Brand, Wertheim, Germany). A disposable

syringe/injection needle was attached to a precision dispenser tip of the pipette filled

with the neurotransmitter. The needle tip was carefully inserted into the compound eye


41

to deliver the stimulus into the hemolymph. Each animal was injected with 2 µL HA and

was immediately returned to the running wheel. The HA concentration used for PRC was

10‐6 M (2 x 10‐12 mol), while for dose‐dependency responses analysis was 10‐4 M (2 x 10‐10

mol) and 10‐8 M (2 x 10‐14 mol). As a control, some cockroaches were injected with 2 µL

saline solution at different CTs. Cockroaches were kept to continuously free‐run in DD for

at least 10–15 days before the injection and left for about 15 days following the day of

stimulation. The injection was performed only for animals that showed a clear free‐

running rhythm. Arrhythmic animals were excluded from injections and further analyses.

The circadian time of the injection was determined before injection and was confirmed

during the phase shifts analysis. A schematic representation of running‐wheel assays is

illustrated in Figure 2.9.

2.6.3. Data analysis

Data were collected about 2 weeks after the application of the stimulus. Collected data

were exported as a text file (time series data, 1 day = 1440 data points) in a format

suitable to be used in ActogramJ software, ImageJ plugin (Schmid et al., 2011). The

software was used to draw double‐plotted aktograms and to determine the following

measurements: 1) free‐running period (τ, tau) prior to and after the injection using chi‐

square periodogram; 2) circadian time (CT) of the injection; 3) values of phase shifts (Δφ).

A regression line through the activity onsets (CT 12 was selected as a reference of the

activity onset and as phase point) was drawn before the injection to determine CT of

injection and τ. Following the injection, another regression line through the activity

onsets was drawn. Δφ was calculated by measuring the difference between eye‐fitted

lines. Results were presented as means ± SE. Phase shifts induced by HA and saline

injections were averaged in three hours bins at CT values of 1.5, 4.5, 7.5, 10.5, 13.5, 16.5,

19.5 and 22.5 and statistically analysed by the Kruskal‐Wallis test followed by Dunn’s post

hoc test with a significance level of P < 0.05. All statistical analyses and graphs were

performed by OriginPro 8 (version 8.754, OriginLab, Northampton, MA, USA) and

CorelDRAW Graphics Suite X3 (Ottawa, Canada).


42

Fig. 2.9: Schematic representation of running‐wheel assays. The cockroach is anesthetized with CO2 and then injected by a repetitive pipette into the compound eye. Then, the locomotor activity of the animals was recorded by wheel running assays. Each wheel was connected to a reed switch to monitor the wheel activity. During the rotation, the switch was closed by two magnetic parts attached to the wheel. The collected data was saved to a text file format on a SD memory card and then copied to the computer for analysis of the phase response curve. (For details see text).

Running‐wheel

run/stopSD

light

Injection

Anesthesia

Data collection

Data Analysis

Cockroach’s head

0 3 6 9 12 15 18 21 24Circadian Time (h)

Phase Shift (h

)

Delay

Advan

ce

An example for phase response curve

Results

CO2


43

2.7. Extracellular recordings

2.7.1. Electrode implantations

Stainless‐steel wire electrodes (30 µm diameter, Advent Research Materials Ltd.,

England, UK) were inserted into AME, compound eye, and leg muscles to search for

synchronization between AME outputs (electrical activity of AME = EAA) to the eye

(electroretinogram = ERG) and to leg muscles (electromyogram, EMG).This method was

established to get extracellular long‐term recordings (e.g. simultaneously neuronal

activity of AME, EMG, and ERG) for several days of intact cockroaches in LD cycles and/or

DD conditions.

Adult male cockroaches were anesthetized using CO2 and fixed in a custom‐made

holder made from acrylic‐glass, in which the animal was maintained in a horizontal

position. For inserting an electrode into the AME, a small window in the head capsule

cuticle above the optic lobe was opened under the dissecting microscope. Then, few

drops of the insect saline were added to the opened window. Through preparations, CO2

anesthesia was kept at a steady level via a chamber fitted to the holder in order to

prevent the animal movement and to reduce hemolymph pumping. With two fine

forceps the surrounded fat body was removed and the optic lobe was exposed. Then, the

electrode tip was carefully inserted into the area next to the AME. After the implantation

of the electrode into the AME, the window was completely covered by wax. A micro‐

dissecting needle was used to create a small hole in the compound eye and the tibia of

the third leg for inserting the tip of the electrodes using two fine forceps. Similar

procedures were applied for inserting the reference electrodes into the hemolymph (e.g.

middle of the head capsule and thorax). The inserted electrodes inside the holes were

fixed well with wax.

The electrode recording site in the optic lope was identified after the end of the

long‐term recording by applying Prussian blue reaction on the tissue to confirm its

contacts to the AME. Electrode marks were performed by passing a current pulse of 50

µA DC for 10 seconds using a custom built current source through the

measuring/reference stainless steel electrodes in the intact brain. The brain was removed


44

and treated with acidified solution of 2% potassium ferrocyanide which produces

Prussian blue plaques at the site of the recording electrode as an indication of iron

deposition in the tissue during the current application. So, the resulting blue can be

observed under the microscope. Brains were then stained with DAPI (4', 6‐diamidino‐2‐

phenylindole) for further confirmation of the exact location under the fluorescence

microscope.

2.7.2. Experiential setup and recordings

After finishing the animal preparation, the animal holder was transferred and placed to

its base, which was fixed within the faraday cage of the experimental setup. The male

PS/2 connecter within the cage was plugged into the holder via a female PS/2 connecter,

in which the measuring and reference electrodes were soldered. The other end of the

PS/2 connecter‐cable was connected to the headstages devices. Headstages were directly

connected to filter/amplifier units (npi electronic GmbH, Tamm, Germany). The signal

was processed with a high pass filter and a low pass filter. Headstages and differential

amplifiers, in which the recorded activities were fed through, are listed in Table 2.4. The

recorded signals are acquired and stored (e.g. sampling rate 500 Hz) on the computer

using analog/digital converter interface (Micro1401 mkII) and Spike2 software (Spike 2

V7.03, Cambridge Electronic Design Ltd., Cambridge, UK). Two light sources (i.e. LEDs)

were installed, the first was used in experiments under LD conditions to generate

day/night cycles within the Faraday cage, while the second was positioned near the front

of the compound eye and was used to generate light pulses (LP e.g. 10 ms at 30 minutes

intervals) via an optic fiber thread connected to a LED placed outside of the cage to

reduce ON/OFF effects. Lighting was controlled with Micro1401 mkII digital/analog

converter with Spike2 software. Figure 2.10 illustrates the experimental setup and the

location of the electrodes.

Table 2.4: Summary of the headstages with their respective differential amplifiers used in the extracellular experiments

Recordings/electrodes Headstage Filter/Amplifier unit

Signals from the AME, EAA EXT‐10/2F EXT‐01C & DPA‐2F

Signals from the eye, ERG ELC‐01MX 100M ELC 01‐MX

Signals from the leg, EMG EXT‐10/2F EXT 10‐2F


45

For comparison, the locomotor activities were recorded as EMGs and by a video

recording system. Multimedia files were automatically and simultaneously captured to

the computer with Spike2 video recorder equipped with an infrared (IR) camera (e.g.

Logitech Quickcam Pro 9000 Webcam, in which IR filter was removed manually) since the

holder base was supplemented with infrared LEDs to enable the camera to record the

cockroach movements in darkness. Moreover, the recording setup is equipped with a

microinjection system for automated series of neurotransmitter‐ and neuropeptide‐

microinjection experiments to analyze the circadian network. The head capsule was

viewed under a stereomicroscope supplied with a red light source (e.g. LED) to provide a

visual control of the injection procedures. The compound eye was penetrated by a pin

with a fine tip before inserting the microinjection capillary to avoid tip breaking during

the insertion. A borosilicate glass capillary with a diameter of 1 ± 0.2 µm (GC‐150F‐ 7.5,

Harvard Apparatus Ltd., Edenbridge, UK), pulled with a DMZ‐Universal puller (Zeitz‐

instrumente, Martinsried, Germany) was filled with the stimulus solution and positioned

directly into the pre‐opened hole in the compound eye using a

PatchStar micromanipulator (Scientifica Ltd, Uckfield, UK). As control injections, saline

solution was injected in the same way. Injected solutions were delivered into the

hemolymph using ejection pressure by Pneumatic PicoPump (PV 820, World Precision

Instruments, Inc., Sarasota, FL, USA). The injection time and its duration were controlled

via the recording software with the possibility to apply injections at different CTs.

2.7.3. Data analysis and measurements

Recorded data were analyzed offline using Spike2 software (Version 7.03, Cambridge

Electronic Design Ltd., Cambridge, UK). Each single file was contained five channels (3

waveform channels for EAA, EMG, and ERG as well as 2 event channels for marking the

durations and times of LPs and microinjection). 24 hours files were created from the

combination of consecutively recorded files.

The recorded waveform channels of the neuronal activity of the AME were

converted to event channels. The mean frequency (number of events within a time

interval; expressed in Hz) and the instantaneous frequency (interevent‐interval,

distribution pattern of frequencies within time intervals; expressed in Hz) were then


46

calculated over the whole time of the experiment to visualize the electrical activity of

neurons. The data of the average number of events in the converted event channels per

minute were exported as a text file.

ERG‐amplitudes elicited by 10 ms LP every 30 minutes were calculated to examine

their level changes (subjective day and subjective night) during the course of the

experiments. The data of the animals with a very clear rhythmic ERG, based on the sine

curve obtained by OriginLab software, were imported as text file (time series data, 1 day

= 48 data points) to ImageJ program, plugin ActogramJ software to measure the circadian

time and free‐running period, using Chi‐Square periodogram.

The recorded EMGs waveform channels were converted to event channels. To

demonstrate the total animal activity and the real shape of locomotor activities, the

average number of events was calculated for 1 min intervals and imported as a text file

to ActogramJ software for plotting the aktograms. Moreover, the 1‐minute intervals are

grouped into 30 minutes intervals and plotted as histograms. Videos of recorded

movements were compared to the EMGs to evaluate the real locomotor activity patterns

of the animal. All values are reported as mean ± SE.

Data obtained from the locomotor activities and the AME neuronal activities

(average activity per 30 minutes) were directly compared to investigate the relationships

between AME and EMG. The correlation (Spearman's correlation coefficient) between

the two data sets was calculated by OriginLab software respective to the circadian time

of the animal, which calculated by the ERG rhythm.


47

Fig. 2.10: Schematic view of the setup used for electrophysiological experiments. The cockroach

holder was placed into a Faraday cage. Stainless‐steel electrodes were inserted into AME, compound eye, and leg muscles of an intact animal, allowing recording of its neural activity. Reference electrodes were placed in the hemolymph (e.g. head capsule). Simultaneous output signals from eye, leg and AME were passed through the headstages to the filter/amplifier units and then to the analog‐digital converter (ADC) and at the end to a computer to be collected by the software. All devices were connected to the ground. For obtaining ERGs, light pulses (LP, e.g. 10 ms) were given at 30 minutes intervals via a light emitting diode (LED) by digital‐analog converter (DAC). The LED was located outside of the cage and illuminated in front of the compound eye by a fiber optic thread. Another LED was used to simulate light/dark (LD e.g. 12:12) cycles with the ability to simulate light levels at dusk and dawn.The movements of the cockroach were acquired by an infrared (IR) camera placed below the animal holder. Image acquisitions were synchronized with the recorded signals. For details see text.

input

output

input

output

input

output

Headstages

Camera

Faraday cage

LP

ADC & DAC interface

Amplifier/Filter unit

<# P>+

CPU

Monitor

Signals from AME

Signals from compound eye

Signals from leg

Pictures captured by IR camera

LED con

trol

LED LD cycles control

48

49

3. Results

3.1. Neurotransmittersinduced Ca2+ responses in the cultured circadian pacemaker neurons of the cockroach Rhyparobia maderae

To identify possible inputs to the Madeira cockroach circadian clock system, Ca2+‐imaging

experiments with dissociated fura‐2 loaded AME neurons were performed to measure

neurotransmitter‐dependent changes in intracellular Ca2+ levels. Tested

neurotransmitters in this study were: acetylcholine (ACh), histamine (HA), γ‐aminobutyric

acid (GABBA), glutamate (Glu), serotonin (5‐HT), and octopamine (OA).

3.1.1. Responses to acetylcholine (ACh)

4.1.1.1. Effects of ACh on intracellular Ca2+ levels

Application of ACh increased the intracellular Ca2+ level in the majority of the tested cells

(n = 327 out of 347 cells). Both the amplitude and the duration of ACh‐induced calcium

responses in AME neurons increased in a dose‐dependent manner when applying

increasing concentrations of ACh, from 10 nM to 1 mM (Fig. 3.1). The threshold

concentration of ACh appeared to vary between 10 nM and 1 µM. The Ca2+ baseline

increased immediately after the initiation of ACh stimulation. The ACh‐dependent peak

amplitude was calculated from baseline to the maximum magnitude, while the response

duration was measured from the beginning of the intracellular Ca2+ increase until

recovery to the calcium baseline levels. The maximum peak of the response occurred

within few seconds after the ACh application. The increased Ca2+ level was associated

with small and rapid Ca2+ oscillations in about 80 % of the tested AME cells, which

disappeared after the recovery to the control level. With increasing ACh concentrations,

the intracellular Ca2+ level remained elevated for a short time after ACh washout from

the bath solution and then returned to control levels (i.e. the recovery was slower with

increasing concentrations indicating dose‐dependent recovery). A remarkable

desensitization was apparent at the higher concentration (e.g. 1 mM) in about 60 % of

the cells tested. In these neurons, the intracellular Ca2+ level was first increased but then

decayed quickly, since the normalized response of 1 mM ACh application = 89.31 % when

100 mM = 100 % (Fig. 3.1D).

Chapter 3. Results

50

Fig. 3.1: Acetylcholine (ACh) increased both the amplitude and the duration of calcium responses in the accessory medulla (AME) pacemaker neurons in primary cell culture of the Madeira cockroach. (A, C) Calcium response of an AME neuron loaded with fura‐2 to increasing concentrations of ACh (B) Pseudocolor images changes. The intracellular calcium level started to increase immediately after ACh application, accompanied by rapid Ca2+ oscillations. At higher concentrations of ACh, intracellular calcium levels increased only transiently, expressing fast desensitization. Ten minutes washes separated consecutive stimulus applications. Vertical arrow indicates the start of the stimulation. Black solid bars indicate the duration of the ACh application. (D) Normalized ACh dose‐response curve. Responses were normalized to the response at 100 µM ACh (=100 %). Data were plotted as mean ± SE.

0

1

2

3

4

-9 -8 -7 -6 -5 -4 -3

0

20

40

60

80

100

120

ACh

0.4

0.4R/R0

1 min

1 mM

100 µM

10 µM

1 µM

100 nM

10 nM

ACh 10 nM

B

100 nM 1 mM 100 µM 10 µM 1 µM R

atio

(340

/380

nm

A

low Ca2+

High Ca2+

Δt = 20 s 10 µm

D

30 s

R/R0

C 10 nM 100 nM 1 µM 10 µM 100 µM

Nor

mal

ized

Res

pons

e (%

)

Log [ACh] (M)

Response duration Response peak

Chapter 3. Results

51

4.1.1.2. Responses to ACh is mediated by nicotinic receptors

To determine whether the sustained Ca2+ increases induced by ACh were mediated either

via ionotropic nicotinic receptors (nAChRs) or via metabotropic muscarinic receptors

(mAChRs), nAChR and mAChR agonists and antagonists were applied (Fig. 3.2).

"Responses to the nAChR agonist nicotine (50 µM) resembled responses to 100 µM ACh

in all cells tested (n = 31 cells). In contrast, none of the tested cells responded to the

mAChR agonist pilocarpine (50 µM) (n = 31). Also, pre‐incubation with the mAChR

antagonist scopolamine (50 µM) did not block nicotine‐responses in any of the cells

tested (n = 11)" (Baz et al., 2013).

Fig. 3.2: Acetylcholine (ACh) activates nicotinic, but not muscarinic cholinergic receptors in the cultured AME pacemaker neurons of Madeira cockroach. (A). ACh (100 µM) responses were simulated via nicotine (Nic, 50 µM), the nicotinic ACh receptor agonist, while the muscarinic ACh receptor agonist pilocarpine (Pilo, 50 µM) was ineffective. Nicotine responses as well as ACh responses were blocked via pre‐incubation (5 min) with the nicotinic ACh receptor antagonist mecamylamine (50 µM), but not via preincubation (5 min) with the muscarinic ACh receptor antagonist scopolamine (50 µM). (B) No elevation of Ca2+ occurred in response to the ACh application in presence of mecamylamine (50 µM), while the response occurred in presence of scopolamine (50 µM). Black solid bars indicate the duration of the application. Open bars mark the presence of different bath solutions.

scopolamine

Pilo

0.05R/R

0

mec

amyl

amin

e

scop

olam

ine

Incu

batio

n

ACHPilo NicNicPiloNicsaline

ACHA

was

h

was

h

was

h

was

h

Incu

batio

n

1 min

mecamylamine

mec

amyl

amin

e

saline

was

h

scop

olam

ine

Incu

batio

n

was

h

Incu

batio

n

B AChmecamylamine

AChAChACh

0.1

R/R0

1 min

scopolamine

Chapter 3. Results

52

"After blocking the nAChRs with the nAChR antagonist mecamylamine (50 µM),

nicotine ceased to increase the intracellular Ca2+ level in all of the cells tested (n = 23).

Pilocarpine, nAChRs agonist, did not affect the intracellular Ca2+ level in presence of

scopolamine or mecamylamine. The mAChR antagonist scopolamine never affected the

ACh‐induced Ca2+ increase (n = 20), while the nAChR antagonist mecamylamine blocked

the ACh effect in all cells tested (n = 20) (Fig. 3.2B). Therefore, all ACh responses of the

AME cells appeared to be mediated via nAChRs, but not via mAChRs"(Baz et al., 2013).

4.1.1.3. AChinduced Ca2+ increase is mediated by voltageactivated Ca2+ channels

"In all AME cells tested, addition of 10 µM mibefradil (reversible voltage‐activated Ca2+

channel blocker) reduced the intracellular Ca2+ baseline level and blocked Ca2+ increases,

which were induced by 100 µM ACh or 50 µM nicotine (n = 22 cells) (Fig. 3.3). Therefore,

the ACh‐induced Ca2+ increase in the AME cells was mediated by voltage‐activated Ca2+

channels" (Baz et al., 2013).

Fig. 3.3: Acetylcholine (ACh) activates voltage‐dependent calcium channels in the cultured circadian pacemaker neurons of the Madeira cockroach. Pre‐incubation (5 min) with the voltage‐activated calcium channel blocker mibefradil blocks ACh (100 µM) and nicotine (Nic, 50 µM) dependent calcium increases in calcium imaging experiments of primary cell cultures of the accessory medulla. About 10 min washout of the blocker reversed ACh‐responses. Black solid bars indicate the duration of the application. Open bars mark the presence of different bath solutions.

saline

mib

efra

dil

mibefradil

mib

efra

dil

was

h

Incu

batio

n

Incu

batio

n

mibefradil

AChACh NicACh

0.11 min

R/R0

Chapter 3. Results

53

3.1.2. Responses to histamine (HA)

3.1.2.1. Effects of histamine on intracellular Ca2+ levels

Histamine (HA) solution (100 µM) evoked three different response types immediately

after the application (Fig. 3.4A). Response type I showed a rapid decrease in the Ca2+

baseline concentration following the HA application. This HA‐dependent decrease

remained for 325.84 ± 13.8 s (mean ± SE) after treatment and, thereafter, it recovered to

baseline levels. Response type II expressed an HA‐dependent sustained Ca2+ decrease

(long‐lasting response). Even after washout and in the absence of HA, it did not recover

to baseline levels. "Response type III included all cells which responded to HA application

with irregular changes in spontaneous calcium oscillations" (Baz et al., 2013).

"The percentages of each response type were calculated at three different

concentrations of HA (1, 10, and 100 µM) (Fig 3.4B). In 14.19 % of the tested cells (n =

191 cells), responses to 1 µM HA were observed (type I = 5.88 %, type II = 0 %, type III =

8.31 %). In 30.79 % of the tested cells (n = 311 cells), responses to 10 µM HA were

observed (type I = 14.78 %, type II = 4.15 %, type III = 11.86 %). In 48.65 % of the tested

cells (n = 266 cells), responses to 100 µM HA were observed (type I = 19.02 %, type II =

10.09 %, type III = 19.53 %)" (Baz et al., 2013).

"To examine the dose‐dependency of type I HA responses, amplitudes and durations

of the responses at increasing concentrations (100 nM to 100 µM) of HA were calculated.

The amplitude was measured from the control level to the maximum intracellular Ca2+

decrease. The duration of the responses was calculated from the beginning of the

intracellular Ca2+ decrease until recovery to control levels. While the durations of the

responses changed in a dose‐dependent manner (Fig. 3.5A), response amplitudes

appeared to be saturated since they did not show significant dose‐dependency (P > 0.05,

ANOVA). The threshold concentration for the HA effect varied between 100 nM and 1

µM. The responses were normalized to the response at 100 µM HA (=100 %). A dose–

response curve was constructed based on the normalized duration of the response (Fig

3.5B). The response duration increased with increasing concentrations of HA. Possibly

due to desensitization at higher concentrations of HA (1 mM) there was first an increase

Chapter 3. Results

54

in intracellular Ca2+ levels before Ca2+ levels decreased for longer time intervals" (Baz et

al., 2013).

Fig. 3.4: Effects of histamine (HA) application on the intracellular calcium concentrations in the cultured circadian pacemaker neurons of the Madeira cockroach. (A) Three different HA response types (named type I, II, III) can be distinguished according to response kinetics to 100 µM HA applications for 60 s (black solid bars). Type I HA‐response is transient, while type II HA‐response is longer lasting than the stimulus applications and do not recover to control levels after washout. Type III response includes all cells, which express irregular changes in spontaneous calcium oscillations after HA application. (B) At different concentrations of HA, 1, 10, and 100 µM the percentages of the histamine response types varied. Data presented as mean ± SE. Modified from Baz et al. (2013).

3.1.2.2. Histaminereceptors in AME neurons

Cimetidine (Vertebrates histamine H2 receptor antagonist) and picrotoxin (chloride

channel blocker) were employed to characterize the HA‐receptors or HA‐gated ion

channels (Gisselmann et al., 2002). Preincubation of AME neurons with 50 µM cimetidine

reduced or blocked HA‐dependent inhibitory response in about 40 % of the tested cells (n

= 13 out of 32), while almost all of the tested cells showed no HA‐responses with 500 µM

cimetidine. On the other hand, picrotoxin (100 µM) did not affect HA‐dependent

responses in all cells tested (n = 114 cells) (Fig. 3.6).

A

0.21 min

R/R0

HAType I

Type II

Type IIIHA

HA

I II III

0

5

10

15

20

25

B

Perc

enta

ge o

f res

pond

ed c

ells

(%)

HA Response Type

1 µM 10 µM 100µM

Chapter 3. Results

55

Fig. 3.5: Histamine (HA)‐dependent response of type I is dose‐dependent. (A) Response of an AME neuron to increasing concentrations of HA. The durations of the HA‐induced response increased dose‐dependently. As shown in the pseudocolor images changes and the recorded traces, the intracellular calcium level decreased immediately after HA applications and after the washout it remains for a few minutes and thereafter it recovered to the control level. Interestingly, at 1mM HA firstly increased the intracellular Ca2+ level, before it declined for a long time. Vertical arrow indicates the start of the application. The black solid bars indicate the duration of the application. (B) Normalized HA dose‐dependent response curve based on the response durations. Responses were normalized to the response at 100 µM HA (=100 %). Data were plotted as mean ± SE. Graph B is modified from Baz et al. (2013).

-7 -6 -5 -4

0

20

40

60

80

100

120

Δt = 20 s

1 mM

100 μM

10 μM

1 μM

HA 100 nM

R/R0

low Ca2+

0.21 min

HA 1 mM

HA 10 mM

High Ca2+100 nM

HA 1 mM

HA 100 mM

10 μm

A HA

Nor

mal

ized

Res

pons

e (%

)

Log [HA] (M)

B

Chapter 3. Results

56

Fig. 3.6: Histamine (HA)‐dependent decreases in the intracellular calcium concentrations of the

cockroach circadian clock neurons are mediated via cimetidine‐sensitive HA receptors. Preincubation of cultured AME neurons with histamine receptor antagonist cimetidine (500 µM) prevented 50 µM HA–dependent responses, while picrotoxin (100 µM) was ineffective. About 10 min washout of the blocker reversed HA‐responses. Black solid bars indicate the duration of the application. Open bars mark presence of different bath solutions.

3.1.3. Responses to GABA

3.1.3.1. Effects of GABA on intracellular Ca2+ levels

Application of GABA, the main inhibitory neurotransmitter of the insect nervous system,

showed mainly two response types (Fig. 3.7). The first response type was observed in

about 35 % of the tested cells (n = 37 of 105 cells). The response pattern could be

characterized by a decrease in the Ca2+ baseline following the application of GABA. This

GABA‐dependent decrease remained for some time after the treatment and, thereafter,

it recovered to baseline levels. Application of GABA in different concentrations (1 µM, 10

µM, 100 µM, and 1 mM) showed that the intracellular calcium levels were decreased in a

dose‐dependent manner (Fig. 3.7 A, B). The second response type was observed in about

9 % of the tested cells. The response pattern was characterized by a transient increase in

the Ca2+ baseline following the application of GABA. This GABA‐dependent increases

remained for some time after the treatment and, thereafter, it recovered to baseline

levels. Application of GABA at different concentrations (1 µM, 10 µM, 100 µM, and 1

mM) increased intracellular calcium levels dose‐dependently. Application of 1 mM GABA

evoked an unsteady, double‐peaked reaction with reduced amplitude, in comparison to

100 µM GABA (Fig. 3.7 C, D).

picr

otox

inIn

cuba

tion

was

h

was

h

Incu

batio

nci

met

idin

e

HApicrotoxincimetidine

HAHA HAHA

0.1

2 min

R/R0

saline

Chapter 3. Results

57

Fig. 3.7: Calcium responses of circadian pacemaker neurons of the Madeira cockroach Rhyparobia maderae to GABA. (A) GABA decreased the intracellular calcium levels dose‐dependently. (B) Normalized dose‐response curve of GABA‐dependent calcium decreases. Responses were normalized to the response at 1 mM GABA (=100 %). Data were plotted as mean ± SE. (C) GABA increased the intracellular calcium levels dose‐dependently. (D) Normalized dose‐response curve of GABA‐dependent calcium increases. Responses were normalized to the response at 100 µM GABA (=100 %). Data were plotted as mean ± SE. Black solid bars in A & B indicate the duration of drug application.

3.1.3.2. Different types of GABA receptors may mediate GABAdependent inhibitory responses

To characterize the receptor types involved in the GABA responses, neurons were

exposed to GABA or muscimol (GABAA receptor agonist) in the absence or presence of

picrotoxin (GABAA receptor antagonists). The response to muscimol resembled the

response to GABA (Fig. 3.8 A‐C) (n = 15 of 20 cells responded to GABA). Neither GABA‐

nor muscimol‐dependent decreases were completely blocked by the preincubation of the

AME neurons with 100 µM picrotoxin (n =10 cells). Thus, the inhibitory responses can be

mediated by GABAA receptors and may be GABAA receptors (Fig. 3.8A). In other neurons

tested, only muscimol‐but not GABA‐dependent Ca2+‐decreases were blocked by the

preincubation with picrotoxin (n = 4 cells), indicating that the inhibitory responses were

not mediated exclusively by GABAA receptors (Fig. 3.8B). In some AME neurons (n = 3),

picrotoxin was able to completely block GABA‐ and Muscimol‐dependent inhibitions,

indicating that the inhibitory responses were mediated only by GABAA receptors (Fig.

3.8C). Moreover, in few tested cells (n = 3 cells), the GABA response was not simulated

GABAGABAGABAGABA

0.52 min

R/R0

GABAGABAGABAGABA

0.22 min

R/R0

1 μM 10 μM 100 μM 1 mM

1 mM100 μM10 μM1 μM

125

100

75

50

25

01 µM 10 µM 100 µM 1 mM

[GABA]

Nor

mal

ized

resp

onse

(%)

1 µM 10 µM 100 µM 1 mM0

25

50

75

100

125

Nor

mal

ized

resp

onse

(%)

[GABA]

DC

BA

Chapter 3. Results

58

via the GABAA receptor agonist muscimol. In addition to the above experiments, few

experiments were performed to search for the presence of GABAB receptors in AME

neurons. To do so, the AME neurons were exposed to baclofen (GABAB receptor agonist)

after application of GABA. Only in a very limited number of neurons tested (n = 2 of 15),

baclofen resembled GABA‐responses, suggesting the presence of GABAB receptors in the

AME neurons (Fig. 3.9). More experiments have been performed (in: Giese, 2014), which

suggested the same results.

Fig. 3.8: GABA‐dependent inhibitory responses were not mediated exclusively by GABAA receptors. (A) GABA responses (100 µM) and muscimol responses were not completely blocked via preincubation (5 min) with the GABAA receptor antagonist picrotoxin. (B) Muscimol responses were blocked, while GABA responses were not abolished in presence of picrotoxin. (C) Both GABA and muscimol responses were blocked in presence of picrotoxin. Data obtained from different neurons. About 10 minute washout of the blocker reversed GABA‐ and muscimol‐responses. Black solid bars indicate the duration of the application. Open bars mark presence of different bath solutions

saline

was

h

picr

otox

inin

cuba

tion

GABAGABA

GABA GABA

GABAGABA Muscimol

Muscimol

MuscimolMuscimol

Muscimol

Muscimol

GABA

GABA

saline

saline

GABA

was

hw

ash

was

h

incu

batio

nPicrotoxin Picrotoxin

2 min

0.3R/R

0

2 min

2 min

0.2

0.2

R/R0

R/R0

picr

otox

inin

cuba

tion

was

hw

ash

picr

otox

in

Picrotoxin

C

B

Picrotoxin Picrotoxin

Picrotoxin

A

Chapter 3. Results

59

Fig.3.9: Effects of GABAB receptor agonist baclofen on the intracellular calcium concentration in the cultured circadian pacemaker neurons of the Madeira cockroach. Baclofen resembled GABA‐responses. Both (100 µM) decreased the intracellular calcium level, suggesting the presence of GABAB receptors in the AME neurons.

3.1.4 Responses to other neurotransmitters

Fewer calcium imaging experiments were carried out to investigate other

neurotransmitters that may change the intracellular calcium concentrations, such as

glutamate (Glu), octopamine (OA), and serotonin (5‐HT). In these experiments, only the

response types were distinguished as well as concentration‐dependence. Application of

Glu decreased the calcium concentrations of the AME cells dose‐dependently (n = 11 of

100 cells) (Fig. 3.10). Application of the OA either increased (Fig. 3.11A) (n = 29 of 60) or

decreased (Fig. 3.11B) (n = 7 cells) the intracellular calcium levels in the AME pacemaker

neurons in a dose‐dependent manner. Application of the 5‐HT either increased (Fig.

3.12A) (n = 17 of 60 cells) or decreased (Fig. 3.12B) (n = 14 cells) the intracellular calcium

levels in the AME pacemaker neurons in a dose‐dependent manner.

Fig. 3.10: Glutamate (Glu) decreased the intracellular calcium levels in the cultured AME neurons of the Madeira cockroach Rhyparobia maderae. (A) Application of Glu causes a decrease in the intracellular calcium levels in a dose‐dependent manner. Black solid bars indicate the duration of drug applications. (B) Normalized ACh dose‐response curve. Responses were normalized to the response at 1 mM ACh (=100 %). Data were plotted as mean ± SE.

GluGluGluGlu

0.11 min

R/R0

B1 μM 10 μM 100 μM 1 mM

A

125

100

75

50

25

01 µM 10 µM 100 µM 1 mM

[Glutamate]

Nor

mal

ized

resp

onse

(%)

R/R0

0.3

1 min

BaclofenGABA100 μM 100 μM

Chapter 3. Results

60

Fig. 3.11: Calcium responses of circadian pacemaker neurons of the Madeira cockroach Rhyparobia maderae to octopamine (OA). (A) Different concentrations of OA increase the intracellular calcium levels without clear dose‐dependency. (B) Application of OA can increase the intracellular calcium levels dose‐dependently, but starts to show desensitization at 100 µM and 1mM OA. Black solid bars indicate the duration of drug application.

Fig. 3.12: Calcium responses of circadian pacemaker neurons of the Madeira cockroach Rhyparobia maderae to serotonin (5‐HT). Application of 5‐HT either decreased (A) or increased (B) the intracellular calcium levels in fura‐2 loaded AME cells. The duration of the 5‐HT‐response expressed dose‐dependency more than the amplitude. Black solid bars indicate the duration of drug applications.

B5-HT5-HT5-HT

0.21 min

R/R0

5-HT5-HT5-HT

0.22 min

R/R0

A10 μM 100 μM 1 mM

1 mM100 μM10 μM

OAOAOAOA

0.2

1 min

R/R0

OAOAOAOA

0.2

2 min

R/R0

B

1 μM 10 μM 100 μM 1 mM

1 mM100 μM10 μM1 μM

A

Chapter 3. Results

61

3.2. Signaling mechanisms of the neuropeptide pigmentdispersing factor (PDF) in the cultured circadian pacemaker neurons

Using Ca2+ imaging and Förster resonance energy transfer (FRET) experiments, signaling

mechanisms of neuropeptide PDF were investigated. In these experiments, PDF was

applied to the cultured AME neurons to identify and distinguish between calcium‐ and

cAMP dependent changes in response to PDF.

3.2.1. Effects of PDF on the intracellular Ca2+ activity

Bath application of neuropeptide PDF to cultured AME neurons produced different types

of response pattern (Wei et al., 2014). The identified types were not characterized

according to their morphology, but only with respect to their responses to PDF (i.e.

changes in the intracellular calcium levels before and after PDF application). AME

neurons were classified into five types (named types 1‐5, Fig. 3.13).

"The type 1 AME neurons showed spontaneously occurring regular, large‐amplitude

Ca2+ transients (~3%; 51 of 1526 AME neurons; Wei et al., 2014). PDF increased the Ca2+

baseline concentration and also the frequency of oscillating Ca2+ transients in 58.8% of

type 1 AME neurons (n = 30 of 51) recorded (Fig. 3.13A). The threshold concentration for

the PDF effect varied between 100 nM and 250 nM. Besides regularly active type 1

pacemaker neurons, other AME neurons were less regularly active or not active at all.

The PDF‐sensitive type 2 neurons were silent, non‐spiking AME cells, with low

intracellular Ca2+ baseline levels, indicative of hyperpolarized membrane potentials. They

transiently increased intracellular Ca2+ baseline levels after PDF application (n = 29 of 792

silent recorded cells) (Fig. 3.13B). The PDF‐dependent Ca2+ increase occurred with

different kinetics in type 2 AME cells." PDF responses to different concentrations showed

that type 1 and type 2 AME pacemakers were dose‐dependent and reversible (Fig. 3.14)

(Wei et al., 2014). "The PDF‐sensitive type 3 cells were not silent as type 2 cells but

produced large Ca2+ transients which were less regular as compared to those of type 1

cells. In type 3 cells, PDF increased the Ca2+ baseline concentration only slightly or not at

all. The increase of the Ca2+ baseline was not dependent on the PDF concentration (Fig.

3.13C). In addition, PDF strongly decreased the amplitude of large Ca2+ transients in type

Chapter 3. Results

62

3 cells before blocking oscillating large Ca2+ transients altogether (n= 4 of 389 irregularly

active AME neurons; Wei et al., 2014). Type 4 cells had elevated intracellular Ca2+ levels

which rapidly decreased following PDF application (n=8 of 294 AME cells with elevated

intracellular Ca2+ baseline levels; Wei et al., 2014) (Fig. 3.13D)" (Wei et al., 2014). In

addition to the previously‐described types, it was interesting to find that few silent AME

neurons were modulated via PDF and produced spontaneously occurring regular, large‐

amplitude Ca2+ transients during the beginning of PDF washout (n = 3 cells) (Fig. 3.13E

and Fig. A.4). One of these cells showed a flip‐flop activity (Fig. 3.13E). One can consider

this type as a PDF response, since the silent AME cells were not observed to change their

calcium transient (i.e. convert into a spontaneous active) suddenly in all experiments

without an external stimulus even with long time recordings (Fig. A.3). Moreover, the

above‐described cell type was recorded from different cultures/experiments.

Fig. 3.13: Representative examples of different types of calcium responses induced by bath applications of PDF in cultured AME neurons of the Madeira cockroach Rhyparobia maderae. (A) Type 1 AME neurons are spontaneously active with regular Ca2+ transients. PDF increases the frequency of their Ca2+ transient and the Ca2+ baseline amplitude. (B) Type 2 AME neurons are silent cells with low baseline Ca2+ levels. The Ca2+ baseline levels increased in response to PDF. (C) "Type 3 cells are less regularly spontaneously active than type 1 cells. PDF application slightly increases baseline Ca2+ levels while suppressing high amplitude Ca2+ transients." This type was recorded and appeared as in Wei et al. (2014). (D) Type 4 AME neurons have a high Ca2+ baseline level, which is decreased by PDF application. (E) Type 5 AME neurons are silent AME neurons with low Ca2+ baseline levels, which exhibited no spontaneous activity. PDF converts the silent state into a spontaneously active state with large‐amplitude Ca2+ transients. A second PDF application returned the cell back to the silent state. Each graph contains a PDF‐responsive neuron from different experiments. Black solid bars indicate the duration of the PDF application.

A B C

D E

2 min

R/R00.2

1 min

[Ca2]i50nM

2 min

R/R00.5

1 min

R/R00.2

1 min

R/R00.2

PDF 1 µM PDF 250 nMPDF 1 µM

PDF 1 µM PDF 1 µM PDF 1 µM

Chapter 3. Results

63

Fig. 3.14: PDF‐sensitive types 1 and 2 AME neurons are dose‐dependently and reversibly. (A) PDF

application with different concentrations changes the frequency and amplitude of the spontaneous active neuron (type 1) in a dose‐dependent manner. (B) None spontaneous active neuron (type 2, silent cell) shows an increase in the intracellular calcium levels in dose‐dependent manner. Black solid bars indicate the duration of the PDF application. Modified from Wei et al. (2014).

3.2.2. PDF signaling is not exclusively mediated via adenylyl cyclase (AC)/cAMP pathway

With calcium imaging experiments, Wei et al. (2014) showed that PDF‐dependent

responses in type 1 AME cells were mediated via the activation of adenylyl cyclase (AC);

while in type 2 AME cells were not mediated by the PKA/cAMP signaling. Similar

experiment result is shown in Figure 3.15. Briefly, it was noted that forskolin (FSK), an AC

activator, caused an increase in the intracellular Ca2+ level in the tested AME neurons

that was similar to that observed with the PDF‐dependent responses for both AME types

(i.e. types 1 and 2). Preincubation with SQ22536, an AC inhibitor, reversibly blocked the

FSK‐dependent responses. PDF response in type 1 AME neurons was also blocked in the

presence of SQ22536. The PDF response was observed after washout of SQ22536,

confirming that PDF responses resulted from the activation of AC. In contrast, SQ22536

could not abolish the PDF responses of type 2 pacemakers, indicating that the response is

PDF 100 nM 250 nM 500 nM 1 µM

2 min

[Ca2]i50 nMw

ash

wash

wash

A

PDF 100 nM 250 nM 500 nM 1 µM

2 min

[Ca2]i10 nMw

ash

wash

wash

B

Chapter 3. Results

64

not mediated by AC activation (Wei et al, 2014). Therefore, the previous data support the

idea that cAMP seems to play a role in the PDF stimulation (i.e. cAMP is involved in the

signaling pathway). To verify this possibility, FRET experiments were carried out to

examine whether indeed the neuropeptide PDF signals via AC activation in Madeira

cockroach circadian pacemaker neurons. In these experiments, only PDF‐dependent

effects on the AME neurons type 1 and 2 were investigated. FRET studies employed the

differently fluorescence‐labeled two subunits of the cAMP‐dependent protein kinase A

(PKA) FRET sensor to monitor cAMP changes in the AME neurons.

First, with calcium imaging, types 1 and 2 of AME cells were identified in response to

PDF application. Afterward, the responded cells were injected by the FRET‐sensor to

image cAMP in neurons. Microinjection of the sensor was performed in 42 PDF‐

responsive cells of different sizes recorded from different cultures. Unfortunately, cell

survival rates achieved after the microinjection were just around 42%. Many factors

controlled the injection procedures (e.g. cell size, sensor volume and concentration,

photobleaching, etc.). Cells showed changes in the fluorescence (i.e. photobleaching) at

the time course after FRET‐sensor microinjection were excluded from the study.

To measure whether PDF has effects on cAMP levels in the individual type 1 AME

neurons, PDF was bath applied to the microinjected cells with FRET‐sensor. In 8 of 9

neurons successively microinjected, PDF (1 μM) caused an increase in the fluorescence

ratio which indicates an increase in the intracellular cAMP level. The maximal cAMP level

was achieved after 1‐2 min (Fig. 3.16A) and showed usually a long‐lasting response with

different amplitudes and kinetics. The fluorescence ratio changes were significantly

different from the baseline (Mann‐Whitney U‐test, p < 0.05) (Fig. 3.16C, D). Moreover,

co‐application of FSK (10 μM) and IBMX (20 μM) generated a response that was similar to

the response of PDF application (n = 2 cells) (Fig. 3.16E).

In contrast, almost all tested type 2 cells showed no cAMP response to bath

application of PDF (n = 7 of 8 cells) (Fig. 3.16B). Comparisons of the fluorescence ratio

changes before and after PDF application showed no significant change in the AMP levels

(Mann‐Whitney U‐test, p > 0.05) (Fig. 3.16C, D). Co‐applications of IBMX and FSK were

performed to the same cell that showed no PDF response to exclude the possibility that

Chapter 3. Results

65

these neurons were dead (Fig. 3.16F). The cAMP baseline of the tested cells was

increased only upon the AC activation and the phosphodiesterase (PDE) inhibition, PDEs

degrade cAMP. This may exclude any role of the AC in the response of this type of the

AME neurons to PDF.

Fig. 3.15: PDF increased the Ca2+ baseline and the frequency of oscillating Ca2+ transients of type 1 neurons via adenylyl cyclase (AC) activation. (A) Bath application of PDF (1 µM) in presence of SQ22536 (20 µM), adenylyl cyclase (AC) inhibitor, stops the PDF responses. The PDF‐response is re‐achieved after SQ22536 washout. Thus, the PDF‐ response in type 1 cells depends on the AC/cAMP signaling. (B) Since PDF application to type 2 cells is not affected by AC inhibition via SQ22536 (20 µM) PDF responses in type 2 cells are independent of AC. Black solid bars indicate the duration of the application. Open bars mark presence of different bath saline solutions.

PDF 1 µMsaline

SQ 22536

PDF 1 µM PDF 1 µM

1 min

R/R0

0.1

SQ 225

36incubatio

n

A

wash

PDF 1 µM

SQ 22536

PDF 1 µM PDF 1 µM

1 min

R/R0

0.1

SQ 225

36incubatio

n

B

wash

saline

Chapter 3. Results

66

Fig. 3.16: PDF‐induced responses in the AME neurons are not exclusively mediated by the cAMP/PKA pathway signaling. (A) Examples of PDF‐dependent cAMP increases in type 1 AME neurons show different response kinetics after the bath application of 1 µM PDF. (B) Examples of type 2 AME neurons show no cAMP increase in response to 1 µM PDF application. (C, D) Average plots of the PDF‐responses of the type 1 and 2 AME pacemaker neurons. Comparisons of cAMP baseline levels before and after PDF application in both AME types showed a significant PDF effect in type 1 after PDF application (Mann‐Whitney U‐test, p < 0.05); while no significant effect occurred in type 2 (Mann‐Whitney U‐test, p > 0.05). Also, there was a significant difference in the baseline changes between the types 1 and 2 AME, (Mann‐Whitney U‐test, p < 0.05), whereas the amplitude was larger in the type 1 response than in the type 2 response. Results are presented as means ± SE. (E) Effect of 1 µM PDF and re‐stimulation with 10 µM forskolin (FSK) and 20 µM IBMX. (F) The intracellular cAMP levels increased in response to 10 µM forskolin (FSK) and 20 µM IBMX in the AME cell which showed no cAMP response to PDF. The fluorescence ratio was normalized to the prestimulatory baseline level. Black solid horizontal bars indicate the duration of the application.

1 min

R/R00.2

PDF FSK + IBMXPDF FSK + IBMX

1 min

R/R00.2

A B

Type 1 AME neurons PDF response Type 2 AME neurons PDF response

1 min

R/R00.2 0.6

0.81

1.21.41.61.8

2

Type 1 Type 2 N

orm

aliz

ed ra

tio (F

520/F

580) Before PDF application

After PDF applicatiion

1 min

R/R00.2

1 min

R/R00.2

PDF

C D

PDF

E F

sig.

n.sig.

Chapter 3. Results

67

The dose‐response curve of the PDF application was hard to establish because of the

long‐lasting response of the majority of cells tested (i.e. the baseline was not recovered

to the control level after washing out). A second application of PDF at higher

concentrations (10 µM) (Fig. 3.17) further elevated the intracellular cAMP levels,

indicating a dose‐dependent increase (n = 2).

Fig. 3.17: Application of 1 μM PDF and the effect of re‐perfusion with 10 μM PDF in the same cell. Further increase in the cAMP concentrations was detected upon the application of a different concentration. Changes in the intracellular cAMP levels response pattern indicate that the response is concentration‐dependent. Black solid horizontal bars indicate the duration of the application.

Unfortunately, cAMP baseline elevations cannot be detected in responses to a single,

rapid PDF application (100 mM, 200 ms) via pressure ejection by a Picospritzer. The

cAMP‐dependent increases were only achieved with bath applied PDF. FRET was

combined with calcium imaging recording for the simultaneous measurement of cAMP

and calcium changes. Preliminary results of these attempts are shown in the Appendix.

1 min

R/R00.3

PDF 1 µM PDF 10 µM

Chapter 3. Results

68

3.3. Histamine phaseshifts the circadian locomotor activity rhythm of the cockroach Rhyparobia maderae Phase shifts of the circadian locomotor activity rhythm of the cockroach R. maderae in

response to histamine (HA) and saline, as control, were measured at different circadian

times (CTs). The phase shifts and CTs were calculated on the basis of the onsets of daily

activity of the animal (see: Material and Methods, section 2.6). Injection of HA (2 x 10‐12

mol; n = 59) at different CT induced phase delays at the late subjective day/early

subjective night and phase advances at the late subjective night. Examples of running‐

wheel recording of HA‐dependent phase shifts are presented in Figure 3.18. The phase

shifts in the activity onsets were averaged to show the mean ± SE for 3‐hours bins of CTs

of injections. Saline injections (n = 39) at different CTs induced non‐significant phase

shifts (Kruskal‐Wallis test; p > 0.05). Comparing HA‐dependent phase shifts with controls

after the saline injections showed significant differences (Kruskal‐Wallis test, p < 0.05) at

CT 10.5, CT13.5 and CT 19.5 (Dunn’s post hoc test; p < 0.05); while the other points

showed little or no phase difference. Figure 3.19 shows phase response curves (PRCs) for

the locomotor activity rhythm of the cockroach, injected with HA and saline. The

outcomes of the PRC are summarized in Table 3.1, which gives the CTs, respective phase

shifts and the number of animals. Applying HA at different doses (2 x 10‐14 mol and 2 x 10‐

10 mol) between CT12 and CT 15 induced dose‐dependent phase shifts. 2 x 10‐14 mol HA

induced phase delays of ‐0.89 ± 0.31 hours (n = 4) while 2 x 10‐10 mol HA revealed phase

delays of ‐2.26 ± 0.42 hours (n = 5). In comparison with saline injections, 2 x 10‐14 mol HA

showed no significant difference while significant differences were detected when

applying 2 x 10‐10 mol HA (Dunn’s post hoc test, p < 0.01, Fig. 3.20).

No significant differences in the circadian period (τ) length of the locomotor activity

were found at all CTs tested when comparing the period before and after saline and HA

injections (Mann‐Whitney U‐test, p > 0.05; Table 3.2). With the periodogram analysis

(Chi‐Square), the period length before the saline injections (n =39) was 23.53 ± 0.068

hours and 23.44 h ± 0.074 hours after injections. For HA injections, the period length

before injections (n =59) was 23.52 ± 0.034 hours and after injections was 23.50 ± 0.038

hours.

Chapter 3. Results

69

Fig. 3.18: Running‐wheel recordings of the circadian locomotor activity of the cockroach Rhyparobia maderae show phase delays and phase advances after the injection of (2 x 10‐12 mol). (A, B) HA given at CT 13.45 (arrow) induced a phase delay (‐Δφ in hours) of‐2.44 as resulted from the regression analysis through activity onsets. (C, D) HA given at CT 18.53 (arrow) induced a phase advance (+Δφ in hours) of 2.67 as resulted from the regression analysis through the activity onsets. Animals were kept in constant darkness condition. No significant differences in the period length (τ) were observed before and after the HA injection (Mann‐Whitney U‐test, p > 0.05). The horizontal axis is the time of the day, in hours; the vertical axis is the consecutive days.

Table 3.1: Effects of histamine (2 x 10‐12 mol) and saline (control experiments) injections through the compound eye on the phase of the circadian locomotor activity of the cockroach R. maderae.

Stimulus CT (hours)

HA Saline Δφ (mean ± SE) n Δφ (mean ± SE) n

00:00 ‐ 03:00 0.53 ± 0.27 5 ‐0.29 ± 0.28 4 03:00 ‐ 06:00 ‐0.48 ± 0.41 5 ‐0.11 ± 0.47 4 06:00 ‐ 09:00 ‐1.02 ± 0.30 6 0.02 ± 0.36 6 09:00 ‐ 12:00 ‐2.58 ± 0.32* 7 0.35 ± 0.44 6 12:00 ‐ 15:00 ‐2.47 ± 0.33* 10 ‐0.27 ± 0.30 6 15:00 ‐ 18:00 ‐0.75 ± 0.48 10 0.29 ± 0.41 5 18:00 ‐ 21:00 2.16 ± 0.34* 9 ‐0.12 ± 0.27 4 21:00 ‐ 24:00 0.52 ± 0.19 7 ‐0.31 ± 0.29 4

* Significant differences (Dunn’s post hoc test, p < 0.05) between HA‐ and saline induced phase‐shifts

1

5

10

15

25

20

1

5

10

15

25

20

1

5

10

15

25

20

24 24246 12 18 18126

A B

Δφ = ‐2.44 hCT

24 24246 12 18 18126

τ after = 23.75

τ before = 23.68

24 24246 12 18 18126

C D

Δφ = 2.67 hCT

τ after = 23.37

τ before = 23.4

24 24246 12 18 18126

1

5

10

15

25

20

Chapter 3. Results

70

-4

-3

-2

-1

0

Pha

se s

hifts

(circ

adia

n ho

urs)

Saline

Histamine (mol)

**

Fig. 3.19: Phase response curve (PRC) of histamine (HA) injections compared with saline injections for the locomotor activity rhythm of the Madeira cockroach. (A) HA injection (2 x 10‐12 mol) through the compound eye caused a biphasic PRC with delays (‐Δφ) at the beginning of the subjective night and advances (+Δφ) at late subjective night. PRCs were obtained by plotting those shifts (Δφ in circadian hours) against the circadian time of the injections. Black line is HA PRC. The curve is the results of 3 hours average of individual data points from different animals, which are plotted in the middle of each 3‐hours bin (open circle, mean ± SE) and presented as b‐spline curves. Grey dotted line is the saline PRC. Saline injections at different CTs induced non‐significant phase shifts. The HA injection caused significant phase delays, compared to the saline injections as control, (Dunn’s post hoc test; p < 0.05; asterisk: significant) at CT 10.5 (‐2.58 ± 0.32), CT 13.5 (‐2.47 ± 0.33), and CT 19.5 (2.16 ± 0.34). (B) Scatter plot of phase shifts after the HA injections with B‐spline curve.

.

Fig. 3.20: Injection of HA between CT 12 and CT 15 induced dose‐dependent phase delays. Data represent the mean phase shift (mean ± SE) resulted from the injections of saline as control (n = 6), 2 x 10‐10 mol HA (n = 5), 2 x 10‐12 mol HA (n = 10), and 2 x 10‐14 mol HA (n = 4). Significant differences were detected between saline injection and 2 x 10‐10 mol HA as well as 2 x 10‐12 mol HA (asterisk: significant; Dunn’s post hoc test; p < 0.01).

Table 3.2: The period length (τ) of the locomotor activity of the cockroach R. maderae before (τ before) and after (τ after) histamine (HA) and saline injections.

τ before (hours)

(mean ± SE)

τ after (hours)

(mean ± SE)

HA 23.52 ± 0.034 23.50 ± 0.038

Saline 23.53 ± 0.068 23.44 h ± 0.074

0 3 6 9 12 15 18 21 24-6

-4

-2

0

2

4A

Pha

se s

hift

(CT

hour

s)

Circadian Time

**

*

0 3 6 9 12 15 18 21 24-6

-4

-2

0

2

4B

Pha

se s

hift

(CT

hour

s)

Circadian Time

Chapter 3. Results

71

3.4. Simultaneous electrophysiological analysis of circadian rhythms of the circadian pacemaker center, of the electroretinogram, and of leg muscle activity in the cockroach Rhyparobia maderae To obtain information about neuronal connectivity and mechanisms underlying circadian

rhythms in cockroach, a method using extracellular long‐term recordings at three

locations in situ was established. Stainless steel microelectrodes were inserted into AME,

compound eye, and leg muscles to search for synchronization between AME outputs

(electrical activity of AME = EAA) to the eye (electroretinogram = ERG) and to leg muscles

(electromyogram, EMG). Two sets of exploratory experiments under LD and DD

conditions have been performed to assay the relationships between the three

simultaneous signals (i.e. EEA, ERG, and EMG), and to improve the effectiveness of the

established method. The first set of experiments was performed with about 7

cockroaches1. The second set of experiments was performed with 8 animals2. Length of

the recording was not uniform and can be changed from one animal to another; ranging

from 2 to 10 days depending on the animal´s survival and the purpose of the experiment.

During this study, many changes have been undertaken to improve the quality of the

measurements and to reduce the recording artifacts. The records presented here are

examples of different experiments.

3.4.1. Electrical activity of AME (EAA) recorded with the EAAelectrode

The position of the EAA‐electrode in the optic lobe was examined directly after the

recording (see: Material and Method, section 2.7) and before the analysis (Fig. 3.21).

Most of the EAA‐recordings showed no regular activity. Some extracellular recordings (n

= 2) from the AME showed a regular activity. An example of this regular activity of EAA‐

recordings is shown in Figure 3.22, with an inter‐burst interval of about 250 s. Moreover

it can be seen that between two bursts, there were two instantaneous frequency bands,

indicative of two synchronized AME neuron populations which fired at the same

frequency but different phase. During the burst they merged into one band, indicative of

burst‐dependent phase‐synchronization (delta phi = 0) (Fig. 3.22B). To analyze the

1 The experiments and analysis were carried together with Ildefonso Atienza López as a part of his diploma practical work.

(López, 2014) 2 The experiments and analysis were carried together with Michael‐Marcel Heim during his bachelor practical work (Heim,

2014).

Chapter 3. Results

72

distribution of the electrical activity of the AME over 24 hours, the EAA signals of 30‐

minutes bins were investigated. The maximum activities were recorded during the

subjective night (Fig. 3.23).

Fig 3.21: Position of the stainless steel microelectrode next to the AME. (A) Histological section of the left lobe stained by DAPI. The black arrow shows the electrode placement. White arrow shows the estimated area of the AME tissue. In this example, the recording site is about 50 µm away from the left AME. (B) Iron deposits “Prussian blue” visualized and magnified by a transmitted light microscope. Current through a stainless steel microelectrode was given to produce Iron deposits in the recording site and followed by potassium ferrocyanide reaction after the brain isolation to produce Prussian blue reaction. (C) Schematic view of the optic lobe showing its anatomical structure in relation to A.

lateral

ventral

AME

200 µm

Prussian blue

A

CB

Chapter 3. Results

73

Fig. 3.22: Extracellular electrical recordings of the AME (EEA‐recordings) can show regular activity. (A) An example of EEA‐recordings for about 7200 s with regular bursts of spikes. (B) Instantaneous frequency plot shows that two synchronized populations of neurons with the same AP frequency and stable phase difference (two parallel bands) synchronized their phase during the bursts (two bands merged into one band).

Fig. 3.23: Distribution of the AME electrical activities over 24 circadian hours. This plot shows the average activity distributions (30‐minutes bins) of all recorded days from a cockroach. The maximum activities are recorded during the subjective night. Three peaks of activity can be identified. The first peak is at the beginning of the subjective night (CT 12‐13). The next is in the middle of the subjective night (CT 18.5 ‐19.5). The last one is a short half‐hour peak occurs in the last third of the subjective night of CT 21.5‐22.

3.4.2. Electroretinogram (ERG)

The electroretinogram (ERG = extracellular recording of photoreceptor neurons)

amplitude levels varied during the subjective day and night in response to a short light

pulse (e.g. LP = 10 ms, generated every 30 min). The amplitude of ERG during the

subjective night was higher than the amplitude of ERG during the subjective day. These

EAA (m

V)

‐0.10

‐0.05

0.00

0.05

0.01

0.15

Time (sec)

0

2

1

3

4

5

Instantane

ous Freq

uency

(Hz)

A

B

0 500 1000 1500 2000 2500 3000 3500 50004000 4500 5500 6000 6500 7000

0

100

200

300

400

500

600

700

800

06 12 18 24 06

No of events/ 30 minutes

Circadian time (hours)

Chapter 3. Results

74

changes were observed under both, LD and DD conditions. Thus, the ERGs offered a good

indicator to measure the internal circadian clock of the tested animals. Many factors (e.g.

LP duration, electrode position, and light intensity) were tested to identify the ERG wave

patterns and to observe the changes during the day and the night time. Figure 3.24

shows an example of rhythmic ERG obtained under LD 12:12 conditions and another

example under DD conditions. Further analysis for the ERG amplitude values was

performed with chi‐square periodogram to calculate the period length and the circadian

time. Only animals with clear ERG and sustained for a long period could be analyzed,

these animals showed period length closed to 24 hours.

Fig. 3.24: Circadian rhythms of the electroretinograms (ERGs) amplitude level changes can be measured under LD and maintained under DD conditions. (A) Circadian ERG rhythm was obtained at 12:12 LD conditions (LP = 100 ms/30 min for 2 days). (B) Circadian ERG rhythm was obtained at DD conditions (LP = 10 ms/30 min for 3 days). It is clear that the amplitude level is increases throughout the subjective night and decreases during the subjective day. Light phases are indicated in white bars, while dark phases are indicated in black bars.

06 09 12 15 18 21 24 06 09 12 15 18 21 24 03 0603

0.01 mV

Time (hours

06 09 12 15 18 21 24 06 09 12 15 18 21 24 03 0603 09 12 15 18 21 24 0603

0.02 mV

Time (hours)

A

B

Chapter 3. Results

75

3.4.3. Electromyogram (EMG)

Electrical recordings of tibia muscles (EMG) were used to measure the

cockroach locomotor activity rhythms. Recorded waveform of EMGs over several days in

LD and DD conditions were converted to movement events to assay the animal

locomotor activity in a quantitative way. When locomotor activities were synchronized

with the ERGs signal, the ERG amplitudes peaked during the subjective night in

correlation with a high locomotor activity. Unfortunately, most of the analyzed EMGs

signals showed no rhythmic changes by using chi‐square periodogram. On the first day

after the preparation, the animals showed high levels of movements. An example of

animal locomotor activity is presented in Figure 3.25.

Fig. 3.25: Representative results for the cockroach locomotor activity and electroretinogram (ERG) for 3 consecutive days. (A) The locomotor activity of a cockroach under 12:12 LD cycles. (B) Circadian rhythm of ERG amplitudes levels, compared to the locomotor activity of the same animal on graph A. (C) The locomotor activity for a different cockroach maintained under DD conditions. (D) Circadian rhythm of ERG amplitudes levels, compared to the locomotor activity of the same animal on graph C. The locomotor activities on the first day after the preparation showed a high number of movements (=events). Events were measured in 1‐minute bins. ERGs amplitudes were evoked as a response to a light pulse of 10 ms generated every 30 minutes. The ERGs amplitudes were normalized to the maximum recorded value (Max. = 100 %). Light periods are indicated in white bars, while dark periods are indicated in black bars.

25

50

75

100

24 12 24 12 24 12 24

Norm. ERG

amplitu

de (%

)

Time (hours)

0

100

200

300

400

500

No. of Events /m

in

ZT

0

100

200

300

400

500

24 12 24 12 24 12 24

No. Of events /m

in

CT

25

50

75

100

Norm. ER

G amplitu

de (%

)

Time (hours)

24 24 24 2412 12 12 24 24 24 2412 12 12

A

B

C

D

Chapter 3. Results

76

3.4.4. Microinjections and correlation analysis

Using the microinjection system, saline and PDF solutions were injected directly into the

hemolymph through the compound eye of only one animal (Fig. 3.26). The application of

saline as control showed no effect on the EAA, since no changes were observed in the

original recording, the mean frequency, and the instantaneous frequency (Fig. 3.26A).

Application of PDF may show changes in the neuronal activity of the AME, the mean

frequency, and the instantaneous frequency directly after the application (Fig. 3.26B).

Fig. 3.26: Microinjections of saline and neuropeptide PDF in an example cockroach. (A) Microinjection of saline solution (0.1 µl) as a control has no effect on the EAA. No changes were observed in the original recording, the mean frequency, and the instantaneous frequency (Inst. Frequency). (B) Microinjection of PDF (0.1 µl of 0.2 mmol) shows changes in the neuronal activity phase of AME, the mean frequency, and the instantaneous frequency directly after application. The injected solutions were delivered directly to the hemolymph via a glass micropipette inserted into the compound eye. The injections were performed around CT 6 on the sixth day after the starting of the recordings. Mean frequency and instantaneous frequency were calculated with a threshold of ‐0.23 mV (grey line in the original recording)

Finally, recorded EAA and EMG for seven consecutive days from an animal were

compared to find the relationships between the two datasets. 30 minutes bins of EAAs

and EMGs were used for such comparisons. Only on the first day, there was no higher

correlation (Spearman’s correlation coefficient = 0.16), possibly due to the continuous

movement of the animal. From the second day onward, values of the correlation

coefficients between the two datasets were increased significantly (Fig. 3.27).

EAA (m

V)Inst. Frequ

ency (H

z)

A B

Mean Frequ

ency (H

z)

EAA (m

V)Inst. Frequ

ency (H

z)Mean Frequ

ency (H

z)

Saline injection PDF injection

10 sec 10 sec

Chapter 3. Results

77

Fig. 3.27: The average activity distributions of electrical activity of AME (EAA) and electromyogram

(EMG) for seven consecutive days recorded from a cockroach maintained in DD conditions. The recoded EAA and EMG data are averaged in 30 min intervals. The values of the Spearman’s correlation coefficients (CC) between the two datasets were presented on the right side for each day. The locomotor activities on the first day after the preparation show a high number of movement events, which make the correlation difficult to detect. Starting from the second recording day, high correlations can be detected. Changes in the correlation ratios cannot be determined after the injection of 0.2 mmol PDF at the beginning of the sixth recording day (black arrow).

0

1 5 0

3 0 0

4 5 0

6 0 0

7 5 0

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

02 0 04 0 06 0 08 0 0

1 0 0 01 2 0 01 4 0 01 6 0 0

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

05 0 0

1 0 0 01 5 0 02 0 0 02 5 0 03 0 0 0

02 0 04 0 06 0 08 0 01 0 0 01 2 0 01 4 0 01 6 0 0

05 0 0

1 0 0 01 5 0 02 0 0 02 5 0 03 0 0 03 5 0 0

01 0 02 0 03 0 04 0 05 0 06 0 07 0 08 0 0

02 0 04 0 06 0 08 0 0

1 0 0 01 2 0 0

0

5 0

1 0 0

1 5 0

2 0 0

02 0 04 0 06 0 08 0 0

1 0 0 01 2 0 0

0

5 0

1 0 0

1 5 0

2 0 0

01 0 02 0 03 0 04 0 05 0 06 0 07 0 0

0

2 0

4 0

6 0

8 0

1 0 0

06 12 18 24 06

No of EAA events/ 30 minutes

Circadian time (hours)

No of EMG events/ 30 minutes

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

EMGEAACC = 0.16

CC = 0.73

CC = 0.69

CC = 0.71

CC = 0.82

CC = 0.75

CC = 0.76

78

79

4. Discussion

4.1. Possible roles of neurotransmitters in the circadian system of the Madeira cockroach

In this thesis the neurotransmitter and neuropeptide inputs to the circadian pacemaker

center of the Madeira cockroach, the accessory medulla (AME) in the brain’s optic lobes,

were characterized. To determine whether the neurotransmitters acetylcholine (ACh),

histamine (HA), γ‐aminobutyric acid (GABA), glutamate (Glu), serotonin (5‐HT), and

octopamine (OA), as well as the neuropeptide pigment‐dispersing factor (PDF) relay

inputs to the Madeira cockroach circadian clock system, Ca2+‐imaging experiments with

dissociated AME neurons were performed to measure the neurotransmitter‐dependent

changes in the intracellular Ca2+ levels. Application of ACh increased baseline Ca2+ levels

in the majority of the tested pacemaker neurons. Application of GABA, 5‐HT, and OA

either decreased or increased intracellular calcium concentrations in the AME neurons.

HA and Glu appeared to decrease the intracellular calcium concentrations in AME

neurons. These results suggest that the cockroach circadian activity is modulated by

several different inputs, including ACh, HA, GABA, 5‐HT, Glu, and OA.

4.1.1. Acetylcholine (ACh) is a key player in circadian clocks in many circuits

Using Ca2+ imaging experiments, ACh increased the intracellular calcium in the majority

of the circadian pacemaker neurons of the Madeira cockroach (~95% of cells tested, Fig.

3.1). Thus, it can be assumed that ACh appears to play a very prominent role in different

circuits of the AME (Baz et al., 2013). Since preliminary anti‐PER‐immunocytochemistry

stained the majority, or even possibly all AME neurons of the Madeira cockroach and

since circadian clock gene expression cycles in the AME (Werckenthin et al., 2012), it is

likely that the majority of AME neurons are circadian pacemaker cells. However, whether

only subgroups of the AME neurons are independent endogenous circadian clocks cannot

be resolved with existing data, since specific antibodies against other circadian clock

Chapter 4. Discussion

80

proteins were not obtained yet and since recording from isolated AME neurons over the

course of several days was not performed yet.

It was found that the AME neurons were responsive to ACh via ionotropic nicotinic

receptors (Fig. 3.2). Since the ACh‐induced increases in the intercellular Ca2+ were

completely blocked in the presence of the nicotinic ACh‐receptors (nAChRs) antagonist,

mecamylamine, but not the muscarinic ACh‐receptors (mAChRs) antagonist,

scopolamine; this suggested that the ACh signaling is mediated via nAChRs. Further

support for presence of nAChRs was that the nAChRs agonist, nicotine, mimicked the

response to ACh. In contrast, none of the tested cells responded to the mAChR agonist

pilocarpine. However, it cannot be excluded that mAChRs may also be present in the

AME pacemaker neurons. Furthermore, the ACh‐dependent depolarizations appeared to

open voltage‐dependent Ca2+ channels (Fig.3.3) since mibefradil, which blocks voltage

dependent low‐voltage‐activated (LVA) and to a lesser extent high‐voltage‐activated

(HVA)‐type Ca2+ channels (Wei and Stengl, 2012), decreased the ACh responses (Baz et

al., 2013).

In the cockroach Rhyparobia maderae, acetylcholinesterase (AChE, the degrading

enzyme of ACh) staining was detected in all optic lobe neuropils. While the lamina was

more weakly labeled, strong staining was observed in seven layers of the medulla, in the

lobula and also in the glomeruli of the AME (Schendzielorz, 2013). However, no AChE

activity was detected in the GABA‐immunoreactive distal tract (Schendzielorz, 2013),

which was suggested to transmit ipsilateral light information to the glomeruli of the AME

(Reischig and Stengl, 1996, Loesel and Homberg, 2001, Petri et al., 2002). Thus, ACh can

be possibly released via medulla tangential cells connecting the medulla and possibly also

the proximal lamina to the glomeruli of the AME, as candidates for a light entrainment

pathway. Since ACh‐injections produced a monophasic all‐delay phase response curve

(PRC) similar to that the delays of the light PRC, ACh can play a role in relaying light‐

dependent phase delays to the clock at dusk (Schendzielorz, 2013). Combined with the

behavioral data, ACh is most likely not responsible for the excitatory photic inputs

recorded in the AME neurons (Loesel and Homberg, 2001), which apparently phase‐


81

advance the clock at dawn, but possibly it can modulate the excitatory photic

information reaching the AME.

In the cockroach Periplaneta americana, the ACh content and the activity of its AChE

also showed inverse circadian rhythms, indicating that also in insects ACh is released

from circadian pacemaker neurons (Cymborowski et al., 1970, Vijayalakshmi et al., 1977,

Subramanyam et al., 1981). In Drosophila, ACh appears to be involved in photic

entrainment of circadian clock cells, since ACh released from the larval photoreceptor

organ (Bolwig's organ) increased the activity of the light‐sensitive large pigment‐

dispersing factor‐immunoreactive ventrolateral pacemaker neurons (l‐LNvs) (Wegener et

al., 2004, Helfrich‐Förster et al., 2007, McCarthy et al., 2011). Also, a subset of the lateral

neurons contains choline acetyltransferase, the key enzyme in the biosynthesis of ACh,

indicating that they may be cholinergic (Yasuyama et al., 1995, Johard et al., 2009).

"Several studies have provided strong evidence for ACh as a key player in the

circadian pacemaker center both in mammals and in insects (Vijayalakshmi et al., 1977,

Lewandowski, 1983, Wegener et al., 2004, Johard et al., 2009, Hut and Van der Zee,

2011, Keene et al., 2011, McCarthy et al., 2011, Lelito and Shafer, 2012). In mammals,

circadian fluctuation in the cholinergic system such as ACh content, cholinergic enzyme

activity, cholinergic receptor, and vesicular acetylcholine transporter were reported,

indicating that ACh releasing as well as perceiving cells are circadian clock neurons (Hanin

et al., 1970, Schiebeler and von Mayersbach, 1974, Nordberg and Wahlström, 1980,

Mash et al., 1985, Morley and Garner, 1990). Indeed, immunocytochemistry located

enzymes characteristic of cholinergic neurons in ‘‘simple bipolar’’ cells in the

suprachiasmatic nucleus (SCN), which are suggested to transfer light information (Hut

and Van der Zee, 2011). While in the SCN, postsynaptic cells contained only few nAChRs

(van der Zee et al., 1991) apparently all SCN neurons were immunopositive for mAChR

(van der Zee and Luiten, 1999, Van der Zee et al., 2004, Yang et al., 2010). Accordingly, in

vitro, the majority of SCN cells were inhibited by mAChR agonists, whereas only some

SCN cells were excited or non‐responsive (Yang et al., 2010). In contrast, in vivo studies

reported mostly excitatory effects of ACh and nicotine in the SCN (Miller et al., 1987,

Nashmi and Lester, 2006). Thus, ACh does play a very prominent role in the SCN not only

in the light entrainment pathway, but apparently also in other circuits which might differ


82

depending on different Zeitgeber times or different behavioral or physiological contexts

(Golombek and Rosenstein, 2010, Gritton et al., 2013). In contrast to mammalian clock

neurons in the fruit fly, as in the cockroach, ACh activated mostly nAChRs, thus being

excitatory (Wegener et al., 2004, Lelito and Shafer, 2012). Since ACh has such wide‐

spread functions in apparently all circuits of the mammalian and the insect circadian

pacemaker system this neurotransmitter is well suited to control the general response

range of circadian pacemaker neurons (Baz et al., 2013)."

4.1.2. Histamine (HA) is involved in entrainment pathways to the circadian pacemaker neurons via cimetidinesensitive receptors

HA is an important neurotransmitter in the nervous systems of a wide range of organisms

(Schwartz et al., 1991, Fleck et al., 2012). HA is involved in the regulation of different

functions in the insect brain, such as biological rhythms, thermoregulation, energy

metabolism, feeding rhythms, and learning and memory (Nässel et al., 1988, Pirvola et

al., 1988, Homberg and Hildebrand, 1991, Pollack and Hofbauer, 1991, Nässel and Elekes,

1992, Buchner et al., 1993, Bornhauser and Meyer, 1997, Loesel and Homberg, 1999,

Monastirioti, 1999, Ignell, 2001, Hong et al., 2006, Huang et al., 2011). In addition to

these HA function in higher order brain centers, HA is the neurotransmitter of insect

photoreceptors (Hardie, 1987, 1988, 1989, Buchner et al., 1993, Stuart, 1999).

Photoreceptors in or close to the compound eye of insects are essential for photic

entrainment (Roberts, 1965, Nishiitsutsuji‐Uwo and Pittendrigh, 1968b, Roberts, 1974).

HA was found to mimic the physiological response to light in many insect species (Elias

and Evans, 1983, Hardie, 1987, 1988).

Using Ca2+ imaging experiments, HA appeared to inhibit a subset of the AME neurons

via chloride gated channel receptors (Figs. 3.5 and 3.6) (Baz et al., 2013). HA injections in

running wheel‐assays revealed a biphasic PRC which resembled a light‐dependent PRC

with phase delays in the circadian locomotor activity rhythm at the late subjective

day/beginning of the subjective night and phase advances at the late subjective night

(Fig. 3.18). These results highlight the possibility that HA is well‐involved/mediated in the

entrainment pathways of the Madeira cockroach. Previous HA Immunostaining studies in


83

the Madeira cockroach brain revealed that the photoreceptor cells of the compound eye

innervate in the lamina and the medulla of the optic lobe, but no direct inputs of

photoreceptors to the cockroach circadian clock were known. Therefore, it was assumed

that the entrainment signals reach the AME indirectly (Loesel and Homberg, 1999). There

is only one known centrifugal HA‐immunoreactive (HA‐ir) neuron with arborizations in

the AME that connects the AME to the medulla and to higher‐order central brain regions

(such as the anterior optic tubercle, the inferior lateral protocerebrum, and the lateral

horn of the lateral protocerebrum) (Loesel and Homberg, 1999). Thus, it is likely that this

neuron receives its input from the anterior optic tubercle or the medulla and

receives/relates information from other modalities (and possibly also from the behavioral

state in higher order integration centers in the protocerebrum). It can be hypothesized

that the HA‐ir neuron integrates multimodal inputs before it phase‐shifts the AME

neurons. This assumption is in agreement with the function of HA in the mammalian

circadian system. "In the mammalian clock, HA appears to be involved in circadian

entrainment (Stephan et al., 1981, Moore, 1983, Tuomisto, 1991). Previous behavioral

studies showed that HA release in rats is under circadian control with maximum release

during the dark period (Mochizuki et al., 1992). Jacobs et al. (2000) suggested that HA

receptors are the final gate at which both photic and non‐photic entrainment

mechanisms converge before sending a resetting signal to the intracellular biological

clock of mammals (Jacobs et al., 2000). Furthermore, HA is a clock input signal, which

shifts the circadian activity phase dependently (Itowi et al., 1991, Stehle, 1991)."

"In the SCN, HA‐dependent activation is mediated via histamine H1 receptors while

inhibitory effects were H2 receptor‐dependent (Yuh Liou et al., 1983, Stehle, 1991).

Furthermore, in vivo studies suggested that HA is a neuromodulator of glutamatergic

transmission via its effects on the NMDA receptor (Eaton et al., 1995, Eaton et al., 1996).

In the insect optic lobes, HA serves as the neurotransmitter of photoreceptors by directly

activating a picrotoxin‐sensitive chloride channel in the postsynaptic cells (Hardie, 1987,

1988, 1989, Stuart et al., 2007).” In contrast, the HA‐dependent‐decrease in the

intercellular Ca2+ concentrations of the AME neurons is mediated via the cimetidine‐

sensitive receptors chloride gated channel (Fig. 3.6) (Baz et al., 2013). In Drosophila,

cimetidine (antagonists of vertebrates H2 receptors) can effectively block the histamine


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receptors (Gisselmann et al., 2002, Hong et al., 2006). Two histamine‐gated chloride

channels (HisCl1 and HisCl2) have been identified at D. melanogaster photoreceptor

synapse (Gengs et al., 2002, Gisselmann et al., 2002, Zheng et al., 2002, Roeder, 2003,

Pantazis et al., 2008). Immunostaining experiments showed that one of these receptors

(HisCl1) expressed in l‐LNv neurons (Hong et al., 2006), suggesting that PDF neurons can

receive histaminergic signals. It was reported that histamine HisCl1 receptor and its

signaling is important for regulation of the sleep/wake and temperature

preferences (Hong et al., 2006, Oh et al., 2013). Drosophila mutants with a deficiency in

histamine synthesis display no histamine immunoreactivity in the photoreceptors; and

being blind and they have defects in the electroretinogram phenotype (Burg et al., 1993,

Melzig et al., 1996). Apparently histamine‐gated chloride channel receptor also is in the

AME neurons of the Madeira cockroach (Fig. 3.6). This finding suggests that HA indeed

target the AME neurons and acts on intracellular calcium via a HA‐receptor. Possibly, HA

and its receptors in the AME clock neurons is required also for the temperature choices

and sleep/wake regulation as suggested for Drosophila (Hong et al., 2006, Oh et al.,

2013). Further experiments are required to test these hypotheses.

4.1.3. Other neurotransmitters involved in different functional circuits of the circadian clock

Other neurotransmitters that affected the isolated neurons of the circadian clock in the

Madeira cockroach were GABA, Glu, 5‐HT, and OA. The results suggest that the circadian

clock system receives both inhibitory and excitatory GABAergic inputs, serotonergic

inputs, as well as octopaminergic inputs, because these three neurotransmitters can

either decrease or increase the intracellular calcium concentration in AME neurons. In

contrast, in preliminary experiments, it was found that Glu relays only inhibitory inputs

into the clock since it always decreased the intracellular calcium levels in the AME

neurons tested.

Functional role of GABA in the circadian clocks

GABA was reported to be an important neurotransmitter in the circadian clock of the

Madeira cockroach. Immunostaining experiments described many different GABA‐ir


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neuronal systems which innervated the AME. Next to GABA‐ir local interneurons of the

AME, GABA‐ir median neurons as well as the prominent GABAergic distal tract connects

the AME to the lamina and the ipsilateral medulla as possible light entrainment pathways

(Reischig and Stengl, 1996, Loesel and Homberg, 2001, Petri et al., 2002, Schendzielorz

and Stengl, 2014). Consistent with the hypothesis that GABA is involved in light

entrainment pathways, injections of GABA revealed biphasic light‐like PRCs with phase

delays at the late subjective day/early subjective night and phase advances at the middle

of the subjective night (Petri et al., 2002). Thus, it can be assumed that there are two

different neuronal circuits in the AME, called morning (M) or evening (E) oscillator circuits

which either are phase advanced at dawn (M‐cells) or delayed by light at dusk (E‐cells) to

couple the clock to changing photoperiods (Helfrich‐Förster, 2009, Yoshii et al., 2012).

Possibly, GABA‐ergic interneurons of the AME play a role in the formation of these two

M‐ and E‐oscillator circuits. This assumption is supported by extracellular recordings of

the AME which showed that GABA forms synchronized assemblies of regularly spiking

AME neurons (Schneider and Stengl, 2005). Moreover, with enzyme‐linked

immunosorbent assay (ELISA), it was found that cAMP levels of AME and optic lobe tissue

under light‐dark (LD) and constant conditions produce a peak at dusk and a peak at

dawn, reminiscent of E‐ and M‐oscillator circuits (Schendzielorz, 2013).

The data presented here suggest the presence of both, ionotropic GABAA receptors

and metabotropic GABAB receptors in the circadian clock neurons of the Madeira

cockroach (Fig. 3.8 and 3.9). However, future studies are required to further

characterize, in details, the receptors that mediated the GABA‐dependent effects in the

AME neurons. GABA and its receptors are expressed in circadian neurons of mammals

and Drosophila (Hill, 1985, Knipper and Breer, 1988, Mezler et al., 2001, Moore et al.,

2002, Wang et al., 2003, Hamasaka et al., 2005, Kahsai et al., 2012, Gmeiner et al., 2013).

They have been characterized to play an important role in maintaining sleep in mammals

and Drosophila (Parisky et al., 2008, Chung et al., 2009, Gmeiner et al., 2013). In

Drosophila circadian clock neurons, GABA acts as a slow inhibitory transmitter via the

metabotropic GABAB receptors but not by the ionotropic GABAA receptors (Hamasaka et

al., 2005). In the SCN, in addition to GABA inhibitory Ca2+ transients, GABA was shown to

also act as an excitatory transmitter (Albus et al., 2005, Choi et al., 2008, Farajnia et al.,


86

2014). Similar excitatory and inhibitory results were also obtained in the Madeira

cockroach circadian neurons (Fig. 3.7). In mammals, it is suggested that the balance

between GABAergic excitation and inhibition in the SCN network can be modulated

according to the photoperiods (Farajnia et al., 2014). Maybe, GABA and its receptors in

the circadian system are sensitive to changes in photoperiods, especially when it is

assumed that GABA is involved in the light input pathway. Future experiments are

needed under different photoperiods to test this hypothesis. As noted above, AME and

optic lobe tissue of the Madeira cockroach showed daily changes in cAMP levels with a

peak at dusk and a peak at dawn (Schendzielorz, 2013). It is still unknown whether the

metabotropic GABAB receptors on AME neurons of the Madeira cockroach are

responsible for daytime‐dependent rhythms in cyclic nucleotide levels. Future

experiments are required to test theses hypotheses.

Possible role of serotonin on the circadian clocks

5‐HT is known to be an important neurotransmitter in the circadian system of insects

(Page, 1987, Nässel et al., 1991, Petri et al., 1995, Wurden and Homberg, 1995, Pyza and

Meinertzhagen, 1996, Cymborowski, 1998, Helfrich‐Förster et al., 1998, Tomioka, 1999,

Saifullah and Tomioka, 2002, Yuan et al., 2005, Nall and Sehgal, 2014) and mammals (Rea

et al., 1994, Bobrzynska et al., 1996, Sanggaard et al., 2003, Horikawa and Shibata, 2004,

Smith et al., 2015). In Madeira cockroach, running wheel experiments accompanied with

5‐HT injections suggested that it induces only phase delays at dusk (Page, 1987).

Immunohistochemical labeling with anti‐serotonin antibodies revealed that 5‐HT is

concentrated in the coarse neuropil and appears to omit the glomeruli (Petri et al., 1995).

Here, application of 5‐HT were observed to increase and decrease the intracellular

calcium levels in the AME neurons (Fig. 3.12). However, the function of 5‐HTand its

possible role in the entrainment and the coupling of the circadian pacemakers is largely

unknown. In crickets, it was suggested that 5‐HT is involved in the photic entrainment

pathway and involved in the coupling mechanism between the optic lobe pacemakers

(Tomioka, 1999, Saifullah and Tomioka, 2002, Tomioka, 2014). In blowflies, a modulatory

effect of 5‐HT on the light response of the circadian system was reported, suggesting a

possible role for 5‐HT as mediator of the circadian rhythms in the insect’s visual system


87

(Nässel et al., 1991, Cymborowski, 1998). Also in blowflies, injection of 5‐HT caused

phase shift and reduced the locomotor activity level (Cymborowski, 2003). In Drosophila,

molecular connections between serotonin 5‐HT1B receptor signaling and the central clock

component TIM were identified (Yuan et al., 2005). Moreover, three of the seven

mammalian receptor families (5‐HT1A/B Dro, 5‐HT2 Dro, and 5‐HT7 Dro) were characterized

in the brain of Drosophila (Nichols and Nichols, 2008, Becnel et al., 2011). In Drosophila

circadian clock neurons, 5‐HT acts as an inhibitory transmitter since it decreased the

intracellular Ca2+ level and caused inhibition of the spontaneous oscillations in Ca2+ levels

(Hamasaka and Nässel, 2006). In mammals, circadian variations in the extracellular 5‐HT

and its receptors were detected (Dudley et al., 1998, Meng et al., 2015). It was

documented that 5‐HT input is one of the projections between the midbrain raphe nuclei

and the SCN, in which 5‐HT provides both photic and non‐photic signals (Hay‐Schmidt et

al., 2003, Paulus and Mintz, 2013, Smith et al., 2015). Recently, it was shown that 5‐HT

signaling in rodents is regulating some of the circadian properties through the 5‐HT1A

receptor since the deletion of such receptor decreased the running‐wheel activity in LD

and constant light conditions (Smith et al., 2015, Westrich et al., 2015). Beside its general

role in the circadian system, 5‐HT is implicated in the regulation of different physiological

functions in CNS, such as aggression/impulse control, sleep control, cognitive function,

food regulation, motor activity, pain, reproduction, and sensory functions (Murphy et al.,

1998, Westrich et al., 2015).

Possible role of glutamate and octopamine on the circadian clocks

Several studies indicated that Glu can act on the circadian clock system (Hamasaka et al.,

2007, Raghu and Borst, 2011, Duffield et al., 2012, Ehnert et al., 2012, Kahsai et al.,

2012). Circadian variations in the extracellular Glu levels were detected in rats (Meng et

al., 2015). The role of Glu in the Madeira cockroach circadian system is still unknown, as

it is not defined yet which neurons of the AME express Glu. Glu may be involved in the

adaptation to changing photoperiods, since it might mediate processes of plasticity and

learning (Giurfa and Menzel, 2013). In Drosophila, application of Glu induced a decrease

in the intracellular calcium in the circadian pacemaker cells (Hamasaka et al., 2007).

Furthermore, Glu‐induced responses is mediated metabotropic glutamate receptors


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(Hamasaka et al., 2007). This is in accordance to the results of preliminary experiments of

this study, which showed that the application of Glu decreased the intracellular calcium

levels in about 10 % of the cells tested in a dose‐dependent manner (Fig. 3.10). Future

immunocytochemical and functional studies are needed to examine the function of Glu

in the circadian pacemaker of the Madeira cockroach. Finally, OA seems to play a role in

the Madeira cockroach circadian clock since OA application induced both excitatory and

inhibitory responses in AME neurons (Fig 3.11). It is reported that OA, beside other

neurotransmitters like GABA and 5‐HT, affects Drosophila sleep (Yuan et al., 2006, Agosto

et al., 2008, Crocker et al., 2010). Moreover, the effect of OA on the sleep/wake cycle

was mediated by cAMP‐dependent pathways (Crocker et al., 2010). Further studies are

required to investigate the possible mechanisms underlying the changes in the

intracellular calcium of the cultured AME pacemaker neurons in response to Glu, 5‐HT,

and OA; and also to characterize the receptors on which theses neurotransmitters act.


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4.2. Pigmentdispersing factor (PDF)dependent calcium and cAMP –signaling pathways in circadian pacemaker neurons of the Madeira cockroach Rhyparobia maderae

Next to neurotransmitters, many neuropeptides are employed in the information

processing in the network of the circadian clock (Nässel, 1991, Reghunandanan and

Reghunandanan, 2006, Taghert and Nitabach, 2012, Helfrich‐Förster, 2014). Several

extensive studies performed on the circadian network systems of insects have shown

that the PDF is the most important circadian coupling signal, a circadian input signal to

the clock, and forms an important clock output since it controls circadian locomotor

activity rhythms (Helfrich‐Förster et al., 2000, Homberg et al., 2003), comparably to the

peptide vasoactive intestinal polypeptide (VIP) in the mammalian circadian clock (Vosko

et al., 2007, Taghert and Nitabach, 2012, Helfrich‐Förster, 2014). Previous extracellular

recordings from the isolated AME suggested that PDF can synchronize, activate, or

inactivate the pacemaker neurons of the Madeira cockroach (Schneider and Stengl,

2005). Previous behavioral experiments suggested that PDF injections at dusk induced

phase delays while at dawn induced narrow phase advances in the locomotor activity of

the Madeira cockroach (Petri and Stengl, 1997, Schendzielorz et al., 2014). Interestingly,

the PDF‐dependent PRC is a quit similar in shape and amplitude as the VIP‐dependent

PRC (An et al., 2011). In Madeira cockroach, the same biphasic, light‐like PRC was

obtained when cAMP was injected, with delays at dusk and advances at dawn. This

suggested that maybe PDF signals via cAMP and is possibly involved in photic

entrainment (Schendzielorz et al., 2014). In the mammalian circadian clock, daily changes

in cAMP were reported with maxima around dusk and dawn (Prosser and Gillette, 1989,

Prosser and Gillette, 1991, Witte and Lemmer, 1991, Ferreyra and Golombek, 2000).

Also, AME and optic lobe tissue of the Madeira cockroach showed daily changes in cAMP

concentrations peaked at dusk and dawn (Schendzielorz, 2013). Thus, PDF as most

prominent coupling signal may be released at dusk and dawn to recruit an E oscillator

circuit at dusk and an M‐oscillator circuit at dawn which respond differently to light

(Schendzielorz et al., 2014). However, it is poorly understood how PDF signals in the

Madeira cockroach circadian clock. Here, the signaling mechanisms of PDF were further


90

investigated by using Ca2+ imaging and Förster resonance energy transfer (FRET)

experiments (cAMP‐imaging) on cultured AME neurons of the Madeira cockroach.

With standard calcium imaging methods used in this study, the AME neurons can be

classified into five response types (Fig. 3.13), according to their calcium concentration

changes in the primary culture and their responses to the neuropeptide PDF (Wei et al.,

2014). The most distinctive two types of AME pacemaker neurons either expressed

spontaneous calcium transients (type 1; Fig. 3.13A) or were silent neurons without

calcium transients (type 2; Fig. 3.13B). Other cells had relatively few or irregular calcium

transients with interval variations between transients (types 3 and 4; Fig. 3.13C,D).

Application of PDF increased the frequency of Ca2+ transients in type 1 and baseline Ca2+

levels (Wei et al., 2014). The same results were obtained from two imaging setups, which

indicate that the spontaneous activity and PDF‐dependent responses were not due to the

imaging procedure (i.e. the responses are not setup‐dependent). PDF‐elicited oscillations

of types 1 and 3 AME cells are similar to those observed previously in extracellular

recordings (Schneider and Stengl, 2005, Wei et al., 2014). Wei et al. (2014) found that

there were two different PDF‐response groups among type 1 cells. They assumed that

type 1 AME neurons are candidates for regularly bursting bright‐ and dark‐rhythm

neurons: "Type 1 cells are reminiscent of two types of regularly bursting AME neurons

identified previously in intracellular recordings which occurred at a similar low frequency

as type 1 cells (Loesel and Homberg, 1998). Illumination of the ipsilateral compound eye

activated one type (bright‐rhythm cell) and inhibited the other (dark‐rhythm cell)

independent of light intensity. Both types generated regular membrane potential

oscillations causing regular bursts of action potentials of around 40 Hz. They never

responded to the motion or the polarized light. These AME pacemakers had ramifications

in the ipsilateral AME and medulla and projected to the contralateral optic lobes, sending

processes to the ventral protocerebrum (Loesel and Homberg, 1998). It can be

hypothesized that these regularly bursting bright‐ and dark‐rhythm neurons correspond

to the two groups of regularly bursting type 1 pacemakers which were synchronized

differentially by PDF, thereby maintaining stable phase‐differences. These bright‐ and

dark‐rhythm neurons are ideally suited to allow for adaptation to different photoperiods

via differential synchronization with PDF‐sensitive pacemaker neurons controlling


91

locomotor activity either at the beginning (evening cells = E oscillators) or the end of the

night (morning cells = M oscillators) (Helfrich‐Förster, 2014)" (Wei et al., 2014).

Interestingly, application of PDF converted silent neurons into active ones (Fig.

3.13E, Fig. A.4). Possibly the switching from non‐active state to active state is important

for permitting a more effective synchronization and coupling between the AME cells in

vivo via changes of the pacemakers´ electrical activity through the membrane excitability.

This is in consistent with PDF functions in the Madeira cockroach, which assumed to

inhibit, activate, and synchronize action potential rhythms of circadian pacemaker

neurons (Schneider and Stengl, 2005). In Drosophila, Focal PDF application has been

found to induce depolarization and increase action potential firing rates which are

directly correlated with an increase in intracellular calcium levels in DN1P neurons

(Seluzicki et al., 2014). The transition from a silent neuron to a spontaneously active state

is important to control some specific animal behaviors and to maintain homeostasis

(Kaczmarek and Levitan, 1987, Cohen and Miles, 2000, Kononenko and Dudek, 2004).

Furthermore, electrical changes of pacemaker cell membrane potentials are crucial clock

components (Nitabach et al., 2002, Wu et al., 2008, Choi et al., 2009, Depetris‐Chauvin et

al., 2011, Choi et al., 2012, Mizrak et al., 2012, Ruben et al., 2012, Brancaccio et al., 2013,

Wei et al., 2014).

PDF signals via adenylyl cyclasedependent and independent pathways

Pharmacological studies with calcium imaging suggested that PDF changed the Ca2+

baseline and the frequency of oscillating Ca2+ transients via the cAMP elevations only in

type 1 but not in type 2 AME neurons (Wei et al., 2014). This could be confirmed with

preliminary FRET experiments, which showed that PDF signals via AC activation only in

type 1 but not in type 2 pacemaker cells. PDF‐dependent Ca2+ and cAMP responses were

long‐lasting response. Possibly the long lasting response is required for the long‐range

mechanisms, such as the long‐term changes in gene expression (Li et al., 2014, Seluzicki

et al., 2014). Long‐lasting PDF and VIP responses were reported in Drosophila and

mammals circadian pacemakers, respectively (Gurantz et al., 1994, Kononenko and

Dudek, 2004, Shafer et al., 2008, Kudo et al., 2013). However, it is unknown whether the


92

long‐lasting PDF responses play a role in the temporal integration of multimodal inputs

(Wei et al., 2014).

In this study, FRET employed the differently fluorescence‐labeled subunits of cAMP‐

dependent protein kinase A (PKA) to monitor the cAMP changes in the AME neurons. The

used FRET–based cAMP biosensor was synthesised by Kai Stieger, modifying established

methods (Stieger, 2011) based on the work of Adams et al. (1991). After the

development of the first cAMP reporter (named: FlCRhR), based on dissociation PKA in

the early 1990s (Adams et al., 1991), it was widely used to monitor cAMP changes in

several studies including invertebrates neurons (Bacskai et al., 1993, Hempel et al., 1996)

and mammalian neurons (Liu et al., 1999, Vincent and Brusciano, 2001, Goaillard and

Vincent, 2002, Gorbunova and Spitzer, 2002). However, it is reported that these

chemically labeled PKA‐FRET sensors may change the real cAMP kinetics and have a lot of

difficulties during the microinjection, especially with different cells sizes (Schwartz, 2001,

Rich and Karpen, 2002, Nikolaev and Lohse, 2006, Willoughby and Cooper, 2007). An

alternative approach was established using some genetically encoded indicators, based

on the activation of PKA and exchange protein activated by cAMP (Epac) (Zaccolo et al.,

2000, DiPilato et al., 2004, Ponsioen et al., 2004, Lissandron et al., 2005, Zaccolo et al.,

2005). Here, despite some difficulties, the modified PKA‐dependent FRET sensor was

shown to be adequate to report cAMP changes in a limited number of the dissociated

AME neurons. In Drosophila, a genetically encoded cAMP FRET sensor, Epac1‐camps, was

used to determine the possible effects of PDF on the cAMP concentration in the circadian

pacemaker neurons (Shafer et al., 2008, Duvall and Taghert, 2013). Unfortunately,

genetically encoded techniques are not possible so far for the Madeira cockroach. Future

experiments will employ also other FRET sensors to further characterize the cAMP

signaling in circadian pacemaker neurons in response to different neuropeptides and

neurotransmitters.

The findings presented here regarding the AC‐dependent PDF‐signaling in the

Madeira cockroach (Fig. 3.16) are consistent with PDF signaling in the fruitfly and with

VIP signaling via AC/cAMP‐dependent pathways in the circadian pacemakers of mammals

(Gurantz et al., 1994, Vosko et al., 2007, Atkinson et al., 2011, Shafer and Yao, 2014, Wei


93

et al., 2014). Accordingly, the PDF receptor (PDFR) of Drosophila looks likes the VIP

receptor (VPAC2), since they are secretin‐like G protein‐coupled receptors (GPCRs) that

activate AC which leads to increases in intracellular cAMP (Nielsen et al., 2002, Meyer‐

Spasche and Piggins, 2004, Hyun et al., 2005, Lear et al., 2005, Mertens et al., 2005,

Dickson and Finlayson, 2009, An et al., 2011, Wei et al., 2014). cAMP‐dependent signaling

is a crucial component in the mammalian and Drosophila circadian clock (Gerhold et al.,

2005, Hao et al., 2006, O'Neill et al., 2008, An et al., 2011, Taghert and Nitabach, 2012,

Helfrich‐Förster, 2014). In the Madeira cockroach PDF signals via adenylyl cyclase‐

dependent and ‐independent pathways. In SCN, VIP activates both cAMP/PKA and

phospholipase Cβ (PLCβ) pathways to convey phase information (An et al., 2011). In

Drosophila pacemaker neurons, PDFR signals through the Gα‐subunit and activates AC

resulting in increasing the cAMP concentrations that activates protein kinase A PKA

(DeHaven and Cuevas, 2004, Shafer et al., 2008, Duvall and Taghert, 2012, Duvall and

Taghert, 2013). Furthermore, In Drosophila, the coupling of the PDFR to different G

protein, Gq , activates the inositoltriphosphate (IP3)/Ca2+ signaling in flight control circuits

of Drosophila (Agrawal et al., 2013). On the basis of the above findings, it can be

proposed that in the Madeira cockroach PDFR acts via Gαs/AC to increase intracellular

cAMP and Ca2+ level in type 1 AME neurons, while it acts via Gq/PLCβ pathway to induce

Ca2+ release from intracellular store in type 2 AME neurons, as was also suggested

previously (Wei et al., 2014). The possibility of cross talk between both pathways cannot

be excluded. The possible PdfR signaling pathways in AME neurons are summarized in

Figure 4.1. Further evidence for cAMP‐dependent calcium rises in the AME neurons were

obtained by using dual imaging of Ca2+ and cAMP. Unfortunately application of PDF was

not possible due to some technical reasons and difficulties in the microinjection of the

PDF‐responsive neurons. Instead it was able to determine that the activation of AC

increased both cAMP and Ca2+ baseline levels in the AME neurons (Fig A.2). The cAMP

baseline increased immediately after application of the AC activator forskolin. These

increases were also followed by rises of the Ca2+ baseline. This suggested that the calcium

channels might be regulated through cAMP/PKA pathways since activation of adenylyl

cyclase AC leads to increased cAMP, which binds to PKA regulatory unit, and then further

signaling cascades will regulate the influx of Ca2+ ions through the calcium channels,

which resulted in a marked increase in calcium concentrations (i.e. Ca2+ increases are


94

mediated by a cAMP/PKA pathway). Future imaging experiments of Ca2+ and cAMP are

required to test the effect of PDF.

Fig. 4.1: Proposed mechanisms of PDF signaling pathways in the AME neurons of the Madeira cockroach. (A) PDFR signals via adenylyl cyclase (AC)‐dependent pathway in type 1 pacemaker neurons. PDFR acts via Gαs/AC to increase intracellular cAMP and Ca2+ level. Activation of AC leads an increase in cAMP, which leads to an increase in intracellular Ca2+

concentration. When cAMP levels rise, further signaling cascades will regulate the influx of Ca2+ ions through the calcium channels. (B) PDFR signals via AC‐independent pathway in type 2 pacemaker neurons. PDFR acts via Gq/ phospholipase Cβ (PLCβ) pathway to activates the inositoltriphosphate (IP3)/Ca

2+ signaling. The IP3 induces Ca2+ release from intracellular

store, probably endoplasmic reticulum. IP3 binds to ion channel which opens and allows Ca2+ to move into the cytosol.

.

PDFR

ATP

cAMP

Regulatorysubunits

Inactivecatalyticsubunits

PKA

activecatalyticsubunits

cAMPcAMP

cAMPcAMP

PDF

Ca2+

Ca2+

P

P

Gs

Nucleus

Cytosol

Type 2 AME neuronType 1 AME neuron

PDF

PDFR

Gq

Cytosol

A B

??

?


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4.3. Simultaneous electrophysiological analysis of circadian rhythms of the circadian pacemaker center, of the electroretinogram, and of leg muscle activity in the Madeira cockroach

Although the activity of circadian clock neurons has been well‐studied

electrophysiologically (Loesel and Homberg, 2001, Schneider and Stengl, 2005, 2006,

2007), the relationship between the AME electrical activity (EEA) and behavioral activity

(i.e. the effect of the behavior on the AME and how the circadian network controls the

behavioral rhythms) is still not well‐understood. Consequently, extracellular long‐term

recordings for several days of intact cockroaches were established to search for

synchronization between AME outputs to the eye (electroretinogram, ERG) and to leg

muscles (electromyogram, EMG) in light‐dark cycles and/or constant conditions (In:

Heim, 2014, López, 2014). Recordings of EMG signals were used to measure the

cockroach locomotor activity, which is controlled by the AME (Helfrich‐Förster et al.,

1998, Homberg et al., 2003). Recording of ERG has shown to be of great value, since

changes in the visual sensitivity indicate the endogenous circadian time of the animal

(Wills et al., 1985, Colwell and Page, 1989, Chang and Lee, 2001, Baz et al., 2009). In the

Madeira cockroach, the AME controls both ERG amplitude and the locomotor activity

rhythms (Page, 1982, Wills et al., 1985, Page, 1990, 2001). It is known that the ERG

amplitude levels for nocturnal animals are high during the subjective night and low

during the subjective days (Colwell and Page, 1989, Pyza and Meinertzhagen, 1997, Baz

et al., 2009). The shape of the ERG in response to short light pulse shares some similarity

to those for other insects, consisting of a peak or trough region which returned to the

baseline (Yinon, 1970, Wills et al., 1985, Colwell and Page, 1989, Leboulle et al., 2013).

The ERG shape or kinetics can be also modified in response to the background

illumination and the electrode position (Colwell and Page, 1989). Decreasing the light

pulse duration from 100 ms to 10 ms was shown to provide better ERGs‐waves

facilitating the measurements of their amplitude changes during the subjective day and

subjective night and. Comparing the obtained ERG wave data with those obtained from

Colwell and Page (1989), for the same cockroach species, show some similarity in the

waveform since both contain an on‐transient and a sustained component. Moreover,

light‐dependent changes in the electrical activity were not only observed in the visual


96

sensitivity via ERG recordings, but also observed in the AME electrical activity. Such

results prove that the AME received the given photic inputs which spread over from cell

to cell. This is in consistence with the intracellular recording data, which showed that

some of the AME neurons responded to the light stimuli (Loesel and Homberg, 2001).

Using the technique described here, the characterizations of the electrical activity of the

intact AME were similar to those obtained by glass microelectrodes from isolated AME

tissue as described by Schneider and Stengl (2005). They also described irregular and

regular patterns of the electrical activity of the isolated AME.

Suitability of the recording methods

The techniques described here have some prominent advantages and disadvantages,

which will be discussed below with giving some notes to improve the recording methods.

Here, first attempts were made to obtain simultaneous extracellular recordings of the

neuronal activity of the AME (EEA), visual sensitivity (ERG), and locomotor activity (EMG)

in the cockroach R. maderae in situ with stainless‐steel wire electrodes. While it can be

confirmed that the EEA electrode was positioned next to the AME, by the Prussian blue

reaction (Tsuruoka et al., 2003), it should be noted that the extracellular electrodes

receive the neuronal activity from many cells located in the area of the recording site

(Segev et al., 2004). Staining of the recording site can be used in further researches to

find out the relationships between the electrode placement and the EEA electrical

activity patterns. Previously, all of the electrophysiological recordings were performed

with a glass electrode (Loesel and Homberg, 2001, Schneider and Stengl, 2005, 2006,

2007). The combination of the use of both glass and stainless‐steel electrodes is possible

in further studies since the glass electrodes can be used to monitor the electrical activity

of the AME in extracellular as well as intracellular recordings. Dye‐filled glass

microelectrode may be used to mark the recording sites (Loesel and Homberg, 2001, Park

and Griffith, 2006). Also using bundle of four electrodes (tetrode) can be used in future

experiments (Bender et al., 2010, Zill, 2010). Insertion of tetrodes can be used to monitor

different regions in the brain that supervise walking behavior (Strausfeld, 1999, Bender et

al., 2010). For comparison, locomotor activities were recorded as EMGs and with a video

recording system to discriminate between different movement types. During an


97

experiment, it was only possible to get data from one animal since the setup was only

equipped with three amplifiers for recording from three locations. Several animal holders

and multi ‐amplifiers and ‐filter units can be constructed in order to record from different

animals side by side to accelerate data acquisition. The animal holder used seems to be

reasonable to fix the animal with minimum stress. Throughout the experiments, the

cockroach was initially fixed to different animal holders, in which its body was positioned

vertically between two half shells. Moreover, some animals were placed horizontally into

plastic Petri dish as a holder, in which a hole was drilled to keep the head capsule

outside. Some animals showed arrhythmic ERG amplitudes and arrhythmic locomotor

activity. Possible reasons are that the optic lobe might be damaged during the insertion

of the electrode into the AME area and/or that the ERG electrode was not in the correct

position. Some other animals showed clear rhythmic ERGs, but arrhythmic locomotor

activity, probably because these animals have no rhythmic locomotor activity as shown

for some animals in running‐wheels assays (personal observation). The developed

method used in this study provides means to monitor the circadian rhythm of the

locomotor activity in an insect species by analyzing the EMGs in comparison to AME

activity and visual sensitivity. In view of the above, further improvements in the

recording setup are necessary to be established in further applications.

Current methods can be used as a suitable examination system for checking the

effects of different neurotransmitters and neuropeptides on the circadian system and

behavior, by applying drugs directly into the hemolymph through the compound eyes.

On the basis of the method described above, The AME neuronal activities and locomotor

activities via EMGs can be easily monitored and compared over 24‐hours, by monitoring

the endogenous circadian time via changes in the ERG amplitudes during the subjective

day and night. In this study, only a few successful injections were performed e.g. saline,

ACh, and PDF applications. Here, the goal of this study was primarily to establish a

recording system that will provide a starting point for a series of further experiments to

investigate how chemical stimuli modulates the circadian rhythm of the locomotor

behavior.

98

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Appendix

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Appendix

Dual FRET and calciumimaging experiments

Simultaneous imaging of Ca2+ and cAMP is a powerful technique to analyze the

relationship between Ca2+ and cAMP/PKA signaling pathways in the cultured neurons

(DeBernardi and Brooker, 1996, Goaillard et al., 2001, Harbeck et al., 2006, Willoughby

and Cooper, 2006, Dunn et al., 2009, Niino et al., 2009). The measurements of cAMP and

Ca2+ in the same time frame with the described imaging setup were not possible due to

the filter position (see sections 2.3.2 and 2.4.3). The filter wheel of the microscope was

switched manually after finishing a calcium imaging experiment from the position of

FURA‐2‐set filter to FRET‐ set filter to start a FRET experiment (Fig. 2.2). In this study

some modifications were performed to the setup in an attempt to measure parallel FRET

and calcium imaging. For this purpose, a motorized automated filter switcher was

developed, in a way that the filter wheel can be automatically moved to an appropriate

filter position at the exposure time of the selected excitation wavelength (Fig. A.1). Using

Multi Channel recordings (Ca2+ imaging: 340 nM and 380 nM & FRET: 488 nM), the

excitation wavelengths were switched and fluorescence emission images were acquired

one directly after another for 340 nm and 380 nm excitation channels followed by two

simultaneously images of the fluorescence emissions (at 520 nm and 580 nm) of the

donor and acceptor channels. The filter position is changed and controlled by the

computer and the imaging software without affecting the imaging experiments. The

motor circuit was designed to be stopped directly after changing to the filter position,

since sudden reversal of the voltage of the motor during spinning can burn out the motor

and its circuit controller. For this reason the filter movement and its related images

acquisition were performed every 5 seconds. Preliminary results showed the possibility

of imaging cAMP and Ca2+ at the same time in AME neurons (n = 3) (Fig. A.2). these

attempts provided a new tool as start‐point for further experiments. Application of FSK

and IBMX increased both cAMP and Ca2+ baseline levels. It can be noted that the cAMP

baseline increased immediately after application of FSK+IBMX associated with rises of the

Ca2+ baseline. This suggested that the calcium channels might be regulated through

cAMP/PKA pathways since activation of adenylyl cyclase AC leads to increased cAMP,

Appendix

120

which binds to PKA regulatory unit, and then further signaling cascades will regulate the

influx of Ca2+ ions through the calcium channels, which resulted in a marked increase

in calcium concentrations (i.e. Ca2+ increases are mediated by a cAMP/PKA pathway).

Fig. A.1: Schematic representation of the system used for dual FRET and Ca2+ imaging. A motorized automated filter switcher was used to change the position of the filter cube. The motor was moved in forward and reverse direction by trigger circuit. The motor was designed in the way that it controlled the movement of the proper filter before exposure to its wavelength. Using Multi Channel recordings (Ca2+ imaging: 340 nM and 380 nM & FRET: 488 nM), the excitation wavelengths were switched and fluorescence emission images were acquired one directly after another for 340 nm and 380 nm excitation channels followed by two simultaneously images of the fluorescence emissions (at 520 nm and 580 nm) of the donor and acceptor channels.

Filter cube for Calcium Imaging

Filter cube for FRET Imaging

Second

s

Wavelength

340 380 488

09

Exposure Time30 ms

Exp. 10 ms

Exp. 100 ms

Fura‐2 Filter‐set

Ca+2

Imaging

F 340/F

380

FRET Filter‐set

cAMP Im

aging

F 520/F

580

00.0100.0200.0300.0400.050

055.0205.0405.0605.0805.100

10

Appendix

121

Fig. A.2: Dual cAMP and Ca2+ imaging from an AME neuron using the microinjected heterochromatic FRET‐Sensor (Fl‐Cα/DY560‐RIα) and loaded with fura‐2 Ca

2+ indicator. Co‐application of forskolin (FSK; 20 µM) and IBMX (20 µM) increases intracellular cAMP‐ and Ca2+ levels in the AME neurons (n = 3).

0 50 100 150 200 2500.5

1.0

1.5

2.0

0 50 100 150 200 2500.5

1.0

1.5

2.0

Rat

io (F

520/F

580)

Time (sec)

Ca2+

Rat

io (F

340/F

380)

Time (sec)

cAMP

FSK+IBMX

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122

Supplementary Figures:

Fig. A.3: The regularly spontaneously active and the silent neurons in the primary cell cultures of the AME pacemaker of the Madeira cockroach R. maderae. (A) Type 1 AME neurons are spontaneously active with regular Ca2+ transients. (B) Type 2 AME neurons are silent cells with low baseline Ca2+ levels.

Fig. A.4: The silent AME neurons in the cultured AME pacemaker of the Madeira cockroach were converted into regularly spontaneously active state after PDF applications. Silent AME neurons with low Ca2+ baseline levels and without spontaneous activity were switched into active ones with regular and large‐amplitude Ca2+ transients. Black solid bars indicate the duration of the drug application.

0.2

2 min

R/R0

0.2

2 min

R/R0

A

2 min

R/R00.3

PDF 1 µM

BPDF 1 µM PDF 1 µM PDF 1 µM

2 min

R/R00.2

123

Acknowledgements

I am thankful to almighty ALLAH who has given me the power to complete this thesis. I

would like to thank people who helped and supported me from the first day until finishing

my thesis. I owe deepest gratitude to my supervisor Prof. Dr. Monika Stengl, head of the

Animal Physiology Dept., Kassel University, who gave me the opportunity to be a member in

her laboratory and her continuous guidance as well as encouragement and interesting

helpful suggestions during my work. My appreciation and special thanks to Prof. Dr.

Charlotte Helfrich‐Förster for reviewing this thesis as well as Prof. Dr. Friedrich Herberg and

Prof. Dr. Raffael Schaffrat for their participation in the examination committee. I really

express my heartfelt thanks to the Egyptian Ministry of Higher Education (MoHE) and the

German Academic Exchange Service (DAAD) for the financial support of my PhD‐scholarship.

I extend my gratitude to Dr. Hongying Wei for the nice teamwork and sharing her

technical experience. I am very grateful to Dr. Achim Werckenthin for helpful comments. My

special thank is addressed to Christin Sender, Christina Wollenhaupt, Gisela Kaschlaw, Karin

Große‐Mohr, and Ursula Reichert. Special thanks to the spirit of Christa Uthof.

My thanks also go to the all members of the animal Physiology Dept. Institute of

Biology, Kassel University: Andreas Arendt, Andreas Nolte, Azar Massah, Hanzy Yasar,

Ildefonso Atienza López, Julia Gestrich, Dr. Julia Schendzielorz, Kai Stieger, Marcel Heim,

Maria Giese, Marius Bartholmai, Nico Funk, Petra Gawalek, Dr. Thomas Schendzielorz, Robin

Schumann, Dr. Wolfgang W. Schwippert, and all of the other members for productive

suggestions and interesting discussions. Also I would like to thank all members of the

Biochemistry Dept. Institute of Biology, Kassel University for collaboration.

Further, I want to thank my friend Ahmed M. El‐Gabbas for his time and for his critical

reading of the thesis and helpful suggestions. Also, many thanks extend to Dr. Wael R.

Abdel Fattah for his effective comments. I am really thankful for my family in Egypt for

constant encouragement and support.

Finally, and most importantly, I would like to thank my wife for her unwavering love,

understanding, tolerance, persistent confidence in me, and taking the load off my shoulder

during the past few years.

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