The histaminergic neurotransmitter system : its

The histaminergic neurotransmitter system

– its development and role in neuronal plasticity in

zebrafish, Danio rerio

Jenny Maria Sundvik

Neuroscience Center and

Institute of Biomedicine, Anatomy

Faculty of Medicine

University of Helsinki

Finland

and

Helsinki Biomedical Graduate Program

ACADEMIC DISSERTATION

To be publicly discussed with the permission of the Faculty of Medicine, University

of Helsinki, in Lecture Hall 2, Haartman Institute, Haartmaninkatu 3,

on August 24th

2012, at 12 noon.

Helsinki 2012

Supervised by

Professor Pertti Panula

Neuroscience Center and Institute of Biomedicine, Anatomy

Faculty of Medicine


Finland

Thesis committee

Professor Esa Korpi

Institute of Biomedicine, Pharmacology

Faculty of Medicine


Finland

Dr. Tarja Stenberg

Institute of Biomedicine, Physiology

Faculty of Medicine


Finland

Reviewed by

Assistant Professor Sanna Lehtonen

Haartman Institute, Pathology

Faculty of Medicine


Finland

Assistant Professor Niclas Kolm

Department of Ecology and

Evolution, Animal Ecology

Evolutionary Biology Center

Uppsala University

Sweden

Opponent

Dr. Catherina Becker

Center for Neuroregeneration

The University of Edinburgh

United Kingdom

© Maria Sundvik 2012

ISBN 978-952-10-8136-1 (paperback)

ISBN 978-952-10-8137-8 (PDF)

http://ethesis.helsinki.fi

Unigrafia, Helsinki University Print

Helsinki 2012

To my family

Table of contents

List of original publications........................................................................................................... 7

Author contribution ......................................................................................................................... 7

Abstract .................................................................................................................................................. 8

Abbreviations ................................................................................................................................... 10

1 Introduction .................................................................................................................................. 12

2 Review of the literature ........................................................................................................... 14

2.1 Neuronal networks and plasticity ............................................................................... 14

2.1.1 Aminergic neurotransmitter systems............................................................... 15

2.1.2 Development of the histaminergic neurotransmitter system ............... 16

2.1.3 Synthesis, storage, release, and catabolism of the aminergic

neurotransmitters .................................................................................................... 17

2.1.4 Histaminergic receptor-signaling system ....................................................... 19

2.2 Neurotransmitter systems under normal conditions and disease ............... 21

2.2.1 Alertness and wakefulness .................................................................................... 21

2.2.2 Histamine and neurodegenerative diseases .................................................. 22

2.3 Zebrafish as a vertebrate model organism ............................................................. 25

2.3.1 Comparative neuroanatomy of fish and mammals ..................................... 26

2.3.2 Zebrafish behavior and motor functions ......................................................... 28

2.3.3 Behavioral pharmacology ...................................................................................... 30

2.3.4 Modification of gene expression with morpholino

oligonucleotides ........................................................................................................ 31

2.3.5 Modification of gene expression by inducing mutations into the

genome .......................................................................................................................... 31

2.3.6 Spatially and temporally targeted gene expression ................................... 32

2.3.7 Appropriateness of modeling human disease in a simple

vertebrate .................................................................................................................... 33

3 Aims of the study ........................................................................................................................ 34

4 Methods ........................................................................................................................................... 35

4.1 Animals ................................................................................................................................... 35

4.2 Manipulation of gene expression by morpholino oligonucleotides (III) ... 35

4.3 Pharmacological treatments ......................................................................................... 35

4.3.1 Treatment with histamine receptor ligands or α-

fluoromethylhistidine (III) ................................................................................... 36

4.3.2 Deprenyl, p-chlorophenylalanine, and fluvoxamine treatments (V) .. 36

4.3.3 γ-Secretase inhibitor DAPT treatments (IV).................................................. 36

4.4 Behavioral tests ................................................................................................................... 37

4.4.1 Locomotor activity of zebrafish (I, III–V) ........................................................ 37

4.4.2 Place preference (IV) ............................................................................................... 37

4.4.3 Place preference in a water column (V) .......................................................... 37

4.4.4 Locomotor activity of larval zebrafish over a 24-h period (III) ............ 38

4.4.5 Dark-induced flash responses in larval zebrafish (III, IV) ....................... 38

4.4.6 Optomotor response (III) ....................................................................................... 38

4.5 Lipophilic dye DiI tracing (III) ...................................................................................... 38

4.6 Real-time polymerase chain reaction ........................................................................ 39

4.6.1 Cloning of zebrafish hrh1, hrh2, and hrh3 receptors in adult

zebrafish (I) ................................................................................................................. 39

4.6.2 Expression of hrh1, hrh2, and hrh3 mRNA (I) .............................................. 39

4.6.3 Cloning full-length hdc cDNA and synthesis of capped

hdc mRNA (III) ........................................................................................................... 39

4.6.4 Quantitative real-time polymerase chain reaction (III, IV) ..................... 40

4.6.5 Touchdown RT-PCR for identification of mutation in psen1-/- (IV) .. 40

4.7 Sequence comparisons and analysis of histamine receptors (I) ................... 41

4.8 [125I]iodoaminopotentidine binding to hrh2 (I) ................................................... 41

4.9 Whole-mount in situ hybridization (III, IV) ............................................................ 41

4.10 Immunohistochemistry (II–V) ................................................................................... 42

4.10.1 IHC with antibodies from different species on whole-mount larval

brains (II–V) ................................................................................................................ 42

4.10.2 IHC on whole-mount adult brains (II, IV) ..................................................... 42

4.10.3 IHC with two antibodies from the same species on cryosections

(II, III) ............................................................................................................................ 43

4.10.4 Enzyme histochemistry and activity assay (V) .......................................... 44

4.11 High-pressure liquid chromatography .................................................................. 44

4.11.1 Catecholamine and serotonin HPLC (V) ....................................................... 44

4.11.2 Histamine HPLC (III) ............................................................................................. 44

4.12 Microscopy ......................................................................................................................... 44

4.13 Image analysis ................................................................................................................... 45

4.14 Statistical methods .......................................................................................................... 46

5 Results ............................................................................................................................................. 47

5.1 Identification and characterization of the histamine receptors in

zebrafish and the behavioral effects of their ligands (I, III) ............................ 47

5.2 Cotransmitters of the histaminergic neurons (II, III) ......................................... 48

5.3 Histaminergic system in alertness/wakefulness (III) ........................................ 49

5.4 γ-Secretase regulates the development of histamine neurons (IV) ............. 50

5.5 Neuronal plasticity and the histamine system (III, IV) ...................................... 52

5.6 Monoamine oxidase affects locomotor activity via serotonin (V) ................ 54

6 Discussion ...................................................................................................................................... 56

6.1 The methods – drawbacks and strengths ................................................................ 56

6.2 Neuronal circuits underlying behavior (III, V) ...................................................... 57

6.3 The histaminergic system in zebrafish compared with that in

mammals (I–III) .................................................................................................................. 59

6.4 The potential role of histamine in neurodegenerative disease (III, IV) ..... 63

7 Conclusions .................................................................................................................................... 66

8 Future prospects ......................................................................................................................... 68

Acknowledgements ....................................................................................................................... 70

References ......................................................................................................................................... 72

APPENDIX Original publications

7

List of original publications

I Peitsaro N, Sundvik M, Anichtchik OV, Kaslin J, Panula P, 2007,

“Identification of zebrafish histamine H1, H2 and H3 receptors and effects of

histaminergic ligands on behavior”, Biochemical Pharmacology, vol. 73, no.

8, pp. 1205-1214.

II Sundvik M, Panula P, 2012, “The organization of the histaminergic system in

adult zebrafish (Danio rerio) brain: neuron number, location and co-

transmitters”, Journal of Comparative Neurology, accepted for publication.

III Sundvik M, Kudo H, Toivonen P, Rozov S, Chen YC, Panula P, 2011, “The

histaminergic system regulates wakefulness and orexin/hypocretin neuron

development via histamine H1 receptor in zebrafish”, The FASEB Journal,

vol. 25, vol. 12, pp. 4338-4347.

IV Sundvik M, Chen YC, Panula P, “Presenilin1 regulates histamine neuron

development and behavior in zebrafish, Danio rerio”, submitted.

V Sallinen V, Sundvik M, Reenilä I, Peitsaro N, Khrustalyov D, Anichtchik O,

Toleikyte G, Kaslin J, Panula P, 2009, “Hyperserotonergic phenotype after

monoamine oxidase inhibition in larval zebrafish”, Journal of

Neurochemistry, vol. 109, no. 2, pp. 403-415. This publication is included in

Dr. Ville Sallinen’s doctoral thesis.

Author contribution

I Sundvik set up a method for assessing behavior in larval zebrafish, designed

the behavioral experiment, conducted it, and analyzed the results.

II Sundvik designed the study partially, conducted all experiments, analyzed

the data, and wrote the manuscript.

III Sundvik set up behavioral methods for zebrafish larvae, conducted the

behavioral, molecular, and immunohistological studies, analyzed the results,

and wrote the manuscript.

IV Sundvik established the association between the histaminergic system and

psen1, designed the study, carried out the experiments, and wrote the

manuscript.

V Sundvik set up a method for assessing behavior in larval zebrafish, designed

the behavioral experiments, conducted them, analyzed the results, and took

part in writing the manuscript.

8

Abstract

The histaminergic neurotransmitter system is a modulatory system which

regulates many functions in the vertebrate brain. In the mammalian brain,

histaminergic neurons fire during waking and are involved in maintaining sleep-

wake homeostasis. Furthermore, histamine has been implicated in

neurodegenerative disorders as resulting from either up- or downregulation of the

neurotransmitter system and has been detected in patients suffering from

Alzheimer’s disease, Parkinson’s disease, and narcolepsy. Histamine is produced

from the amino acid ι-histidine by histidine decarboxylase (HDC). The aims of this

study were to characterize the histaminergic system in zebrafish, a nonmammalian

vertebrate, and to identify novel mechanisms that are mediated by the

histaminergic system.

We found that histamine receptor ligands altered the behavior in larval zebrafish

in a dose-dependent manner. Cloning and sequence analysis of the histaminergic

receptors indicated that the zebrafish histamine receptors were similar to the

corresponding mammalian receptors. We also studied the neurotransmitter

phenotype of histaminergic neurons by immunohistochemistry and confocal

microscopy and found that these neurons resemble those of rats.

Immunohistochemical comparison of the histaminergic system in male and female

adult brain revealed no significant differences in location, distribution, or neuron

number, and thereby no differences between the sexes were observed. We

studied the functioning of the histaminergic system in hdc knockdown animals

(morpholino oligonucleotide approach), and found that the neurotransmitter was

important for mediating rapid behavioral responses to environmental cues, and

that these behavioral responses were driven via the histamine receptor, hrh1. We

studied the interaction of histamine with a wakefulness associated neuropeptide

system, hypocretin/orexin (hcrt), which is implicated in narcolepsy. We reported a

novel mechanism for histamine that regulates development of hcrt neurons.

Transient knockdown of hdc with morpholino oligonucleotides and

pharmacological inactivation of hrh1 with pyrilamine reduced the number of hcrt

mRNA-positive neurons in zebrafish larvae. This effect was rescued by

overexpression of hdc mRNA. Since histaminergic neurons play important roles in

the central nervous system, we were interested in identifying a factor that

regulates the development of the histaminergic neurons themselves. We found

that in the Alzheimer’s disease-associated gene, presenilin1 (psen1), mutant

zebrafish, the histaminergic system was altered. During the developmental and

9

larval stages, the histamine neuron number was significantly reduced in psen1

mutants, whereas at the young adult age there was a significant increase in

histamine neuron number in psen1 mutants in comparison to wild-type fish. These

results showed us that psen1 was essential for the development and normal

functioning of the histaminergic neurotransmitter system. Histamine is

metabolized by histamine N-methyltransferase (HNMT), followed by monoamine

oxidase (MAO). We studied the effect that pharmacological inhibition of MAO has

on the locomotor behavior of fish. We found that pharmacological inhibition of the

MAO enzyme mainly affects the serotonin system in zebrafish, with characteristic

behavioral consequences that could be rescued.

Taken together, the results compiled in this thesis provide evidence that the

histaminergic neurotransmitter system in zebrafish is similar to that in mammals.

These results show that the zebrafish is an excellent tool in studies that provide

novel insights into neurotransmitter system functions in the vertebrate central

nervous system on both the mechanistic and behavioral levels. We found that the

histaminergic system is important for rapid behavioral responses, affects the

development of the hcrt neurotransmitter system, and is itself regulated in a

temporal manner by psen1.

10

Abbreviations 3D Three-dimentional

5-HIAA 5-Hydroxyindoleacetic acid

AADC Aromatic ι-amino acid decarboxylase

AAS Ascending arousal system

AC Adenylate cyclase

AD Alzheimer’s disease

Akt/GSK3 Serine/threonine protein kinase/ Glycogen synthase kinase 3

ALDH Aldehyde dehydrogrnase

AMPK Adenosine monophosphate-activated protein kinase

ANOVA Analysis of variance

aph-1 Anterior pharynx-defective 1

APP Amyloid precursor protein

Ca2+

Calcium

cAMP Cyclic adenosine monophosphate

cDNA Complementary DNA

CEL-I Plant endonuclease

CNS Central nervous system

COMT Catechol-O-methyl transferase

CREB cAMP response element-binding

DAG Diacylglycerol

DAO Diamine oxidase

DAPT N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethylester

Dc Central area of dorsal telencephalon

Dd Dorsal area of dorsal telencephalon

DβH Dopamine β-hydroxylase

DiI Lipophilic tracer dye

Dl Lateral area of dorsal telencephalon

Dm Medial area of dorsal telencephalon

DMSO Dimethyl sulfoxide

ι-DOPA ι-Dihydroxyphenylalanine

DOPAC 3,4-Dihydroxyphenylacetic acid

Dp Posterior area of dorsal telencephalon

dpf Days postfertilization

E3 Embryo medium

EDAC 1-Ethyl-3,3(dimethyl-aminopropyl) carbodiimide

ENU N-ethyl-N-nitrosurea

α-FMH α-Fluoromethylhistidine

GABA γ-aminobutyric acid

gad1 Glutamate decarboxylase 1, or gad67

gad2 Glutamate decarboxylase 2, or gad65

Gal4 Transcriptional activator, galactose utilization

HA Histamine

Hc Caudal zone of periventricular hypothalamus

hcrt Hypocretin/orexin

hcrt1 Hypocretin receptor subtype 1

hcrt2 Hypocretin receptor subtype 2

HDC Histidine decarboxylase

HNMT Histamine N-methyltransferase

hpf Hours postfertilization

HPLC High-pressure liquid chromatography

hrh1 Histamine receptor subtype 1




IHC Immunohistochemistry

IP3 Inositol triphosphate

11

MAO Monoamine oxidase

MAPK Mitogen-activated protein kinase

mENK Methionine-enkephalin

MID2cm Mauthner neuron homologue, middle dorsal 2 contralateral medial longitudinal fasciculus

interneuron in rhombomere 5

MID3cm Mauthner neuron homologue, middle dorsal 3 contralateral medial longitudinal fasciculus

interneuron in rhombomere 6

MO Morpholino oligonucleotide

mRNA Messenger RNA

NF-κB Nuclear factor κ-light-chain-enhancer of activated B cells

nMLF Medial longitudinal fasciculus

o/n Overnight

PCPA p-Chlorophenylalanine

PD Parkinson’s disease

pen-2 Presenilin enhancer 2

PFA Paraformaldehyde

PKA Protein kinase A

PKC Protein kinase C

PLC Phospholipase C

PNMT Phenylethanolamine Ν-methyl transferase

psen1 Presenilin1

psen2 Presenilin2

qPCR Quantitative real-time polymerase chain reaction

RM ANOVA Repeated measures ANOVA

RT Room temperature

RT-PCR Real-time polymerase chain reaction

SEM Standard error of mean

slc17a6a Solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 6a,

or vesicular glutamate transporter 2b

slc17a6b solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 6b,

or vesicular glutamate transporter 2a

slc17a7 solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 7,

or vesicular glutamate transporter 1

sOVA Succinylated ovalbumin

std Standard

TALEN Transcription activator-like effector nuclease

TeTx Tetanus toxin

TH Tyrosine hydroxylase

TILLING Targeting induced localized lesions in genomes

TL Tubingen longfin

TMN Tuberomammillary nucleus

TPH Tryptophan hydroxylase

TRH Thyrotropin-releasing hormone

TSA Tyramide signal amplification

UAS Upstream activating sequence

VMAT2 Vesicular monoamine transporter 2

WISH Whole-mount in situ hybridization

WT Wild-type

ZFN Zinc finger nuclease

ZT Zeitberger time

Introduction

12

1 Introduction

The basis of modern neuroscience was laid about a century ago when the Spanish

neuroanatomist Santiago Ramon y Cajal suggested that neurons are the

anatomical and functional units that together form the central nervous system

(CNS). His work focused on the structure and organization of the CNS and

contributed to the acceptance of the neuron doctrine. The neuron doctrine states

that neurons are the fundamental structural and functional units of the CNS, are

distinct units that are comprised of dendrites, somata, and axons, and that

information flows along the neuron from dendrites to axon. Specific behaviors are

produced by integration of neuronal networks and sensory inputs. Failure to

produce appropriate responses to certain environmental stimuli can be

detrimental for the individual. Hence, it is of the outmost importance that the

individual responds in a correct way. Coordination of all the neurons in the CNS is a

complex task and therefore elucidating how these networks of molecular and

neuronal mechanisms interact is a field of constant study and holds many

unrevealed aspects.

The histaminergic system is linked to many neuronal pathways and, by interaction

with receptors in different brain areas, is involved in several complex behaviors,

from hormonal control to cognition. Although the histaminergic system has been

studied for decades, the development of the neurons and the exact functions and

molecular mechanisms of the interactions with other neurotransmitter systems

are still poorly understood. Diseases in which the histaminergic system has been

implicated are Alzheimer’s disease (AD), Parkinson’s disease (PD), schizophrenia,

narcolepsy, and epilepsy. Whether the histaminergic system is involved in the

pathogenesis of these diseases is still unknown. An analysis of linkage in a two-

generation pedigree showed that a mutation in histidine decarboxylase (hdc), the

rate-limiting enzyme in histamine synthesis, is associated with Tourette’s

syndrome (Ercan-Sencicek et al. 2010). This is the first evidence that histamine is

indeed associated with the etiology of a specific disease. These results open an

avenue for speculations and one might argue that histamine plays a crucial role in

modulating and fine-tuning of motor functions. If this is the case, this could

partially explain why the histaminergic transmission and system is altered in

diseases that affect motor functioning, such as in PD. In PD, cognitive impairments

are also evident. Taken together, it is still unclear why the histaminergic system is

altered in diseases with cognitive decline, such as AD and PD, in the sleep disorder

Introduction

13

narcolepsy, or in alertness- and neuronal excitability-associated diseases such as

schizophrenia and epilepsy, respectively. Although the role of histamine in

neuronal plasticity, e.g. during neurodegeneration, has been studied, there are still

many unanswered questions to be solved.

In this thesis, I addressed the factors that affect the development of the

histaminergic system and how this system contributes to mediating of specific

behaviors. I set out to study the neuronal histaminergic system in a vertebrate

species, the zebrafish Danio rerio. The zebrafish has, during the recent decades,

emerged as a highly suitable vertebrate model for biomedical and neuroscientific

research, due to its main features: ex utero development, amenability to genetic

manipulation, and cost-effectiveness. Neuronal and molecular networks

underlying behaviors have been studied successfully in the invertebrates

Caenorhabditis elegans and Drosophila melanogaster, and vertebrate model

organisms, but some unanswered questions still remain.

Review of the literature

14

2 Review of the literature

One of the many challenges in the field of medicine is the identification of specific

connections of neuronal networks that underlie and mediate specific behaviors.

Since the human brain is formed of billions of neurons, it is challenging to pinpoint

the changes in neuronal pathways and the exact mechanisms mediating different

behaviors. Addressing these questions in a vertebrate model organism with lower

neuronal complexity, i.e. the zebrafish, has therefore emerged as an attractive

alternative approach.

2.1 Neuronal networks and plasticity

Neuronal networks, in the biological sense, are those that specific pools of neurons

form with each other to execute certain physiological functions. The ability of the

individual to adapt to a changed environment, as well as the capacity to learn and

remember, are dependent on how well the nervous system is able to form new

and undo old connections, defined as neuronal plasticity. The concept of neuronal

plasticity was initially described in 1890 by James, and the concept was later

refined by several scientists, including Cajal and Hebb (as cited in Berlucchi &

Buchtel 2009). The connections between neurons are formed by specialized

junctions, i.e. synapses. Strengthening or weakening of the synapses and neuronal

circuits underlie learning and memory (Kandel, Schwartz & Jessell 1991).

Alterations in the morphology of the synapses have also been linked to

neurological disorders (Penzes et al. 2011).

The neuronal circuits that underlie behavior in mammals have been extensively

characterized, but the roles of individual neurons in bringing bout precise

behaviors remain to be elucidated (Kandel, Schwartz & Jessell 1991). Behaviors can

vary from simple reflexes to those that require the cooperation of several brain

areas such as the brainstem, telencephalon, cerebellum, basal ganglia, and/or

thalamus (Figure 1). The cortico-striato-thalamic system and cerebellum therefore

control and modulate the functions of the upper motor neurons in mammals. The

release of acetylcholine from the α-motor neurons at myoneuronal junctions

induces muscle contraction and the produced behavior can be recorded. A new

cycle is initiated when somatosensory receptors are activated and the newly

generated information is processed through the spinal cord via modulatory

systems to the motor cortex.


15

Figure 1. Neuronal circuits that underlie behavior (modified from Kandel, Schwartz &

Jessell 1991). The neuronal pathways that underlie behavior are organized in a hierarchical

manner. The cerebral cortex is the master regulator, integrating motor output control and

somatosensory information. Behaviors can be modulated by the thalamus, basal ganglia,

cerebellum, brain stem, and spinal cord. Dotted lines indicate the connections known in

mammals but still unknown in zebrafish.

2.1.1 Aminergic neurotransmitter systems

Histamine belongs to the group of biogenic amines, together with dopamine,

serotonin, adrenaline, and noradrenaline (Figure 2). These neurotransmitter

systems constitute together with γ-aminobutyric acid (GABA) and glutamate, the

classical neurotransmitter systems and networks in the brain. All these

neurotransmitter systems interact to produce complex behaviors such as cognition

and movement, and regulate wake-sleep states (Saper, Scammell & Lu 2005, Haas,

Sergeeva & Selbach 2008, Eriksson et al. 2010). The amines are modulatory

transmitters, since they in general alter the excitability of neurons that are either


16

excitatory or inhibitory, and mainly exert their actions through volume

transmission (Agnati et al. 1995, Cragg et al. 2001).

Figure 2. Chemical structure of the aminergic neurotransmitters histamine, serotonin, and

dopamine.

Histamine, a biogenic amine, was discovered by Dale and Laidlaw in 1910 and is

one of the phylogenetically oldest neurotransmitters. The first indication that

histamine functions as a neurotransmitter was by the French professor Schwartz in

1970 when he identified the properties and distribution of the histamine

producing enzyme HDC in the rat brain (Schwartz, Lampart & Rose 1970).

Histaminergic neurons were later identified in the tuberomammillary nucleus

(TMN) in the posterior part of the hypothalamus in rat brain independently by two

research groups in 1984 (Panula, Yang & Costa 1984, Watanabe et al. 1984). In the

periphery of the body, histamine is the main mediator of allergic responses acting

through histamine receptor subtype 1 (hrh1). Histamine is involved in an array of

different physiological functions and states, ranging from cognition to hormonal

homeostasis (Haas & Panula 2003, Haas, Sergeeva & Selbach 2008). It was only

recently that a strong association of the neurotransmitter system with a specific

human neuronal disease, Tourette’s syndrome, was established (Ercan-Sencicek et

al. 2010) and shown to be hrh1-mediated (Fernandez et al. 2011). In zebrafish, the

role of histamine has lately been studied in dominance and aggression (Filby et al.

2010, Norton et al. 2011, Pavlidis et al. 2011).

2.1.2 Development of the histaminergic neurotransmitter system

The development of the histaminergic system has been studied in several species,

including zebrafish. In the developing rat brain, histamine is transiently expressed

in the serotonergic raphe nucleus (Vanhala, Yamatodani & Panula 1994). In

zebrafish, the first histaminergic neurons appear in the ventral hypothalamus at 3

days postfertilization (dpf) and efferent projections from these neurons innervate

the dorsal telencephalon by 4 dpf (Eriksson et al. 1998). The histaminergic system

consists of a relatively small, specific, and localized neuron population in the

posterior part of the hypothalamus in zebrafish, and no transient system has been


17

detected (Eriksson et al. 1998) as in mammals. Considering that the histaminergic

system consists only of a few neurons in the larval zebrafish, the fiber projections

that arise from these neurons are quite elaborate. A detailed study of the intact

fiber network could reveal the precise three-dimensional (3D) innervation

patterns. Although histamine is involved in and regulates many physiological

functions, the factors that regulate the development of this system are not known.

2.1.3 Synthesis, storage, release, and catabolism of the aminergic

neurotransmitters

Histamine is derived from ι-histidine by HDC (EC 4.1.1.22) (Garbarg et al. 1974) and

is packed into vesicles by vesicular monoamine transporter 2 (VMAT2) (Figure 3)

(Merickel & Edwards 1995). After release of histamine into the extracellular space,

histamine is bound to its receptors or inactivated by methylation into tele-

methylhistamine, a process that is mediated via histamine N-methyltransferase

(HNMT, EC 2.1.1.8) (Brown, Tomchick & Axelrod 1959). Tele-methylhistamine is

further oxidized by monoamine oxidase (MAO, EC 1.4.3.4) to methylimidazole

acetaldehyde (Hough & Domino 1979). Two forms of MAO are known, of which

MAO-A metabolizes serotonin (Nicotra et al. 2004) and MAO-B metabolizes

histamine (Schwartz et al. 1991). As in other species, MAO in zebrafish is also

found in the histaminergic neurons (Sallinen et al. 2009), suggesting that MAO may

be responsible for the inactivation of histamine in this species. In nonneuronal

tissue in mammals, histamine is oxidized by diamine oxidase (DAO, EC 1.4.3.3)

(Schayer 1953). In contrast to mammals, DAO expression has been observed in

zebrafish neuronal tissue, specifically in the ventral habenula, which corresponds

to the mammalian lateral habenula (Amo et al. 2010). Histamine synthesis can be

inhibited by α-fluoromethylhistidine (α-FMH), which is an irreversible inhibitor of

HDC (Garbarg et al. 1980). So far, no reuptake system for histamine has been

identified.


18

Figure 3. Synthesis, storage, release, and catabolism of histamine. ι-Histidine is taken up

into neurons, where it is converted to histamine by HDC. Histamine is packed into vesicles

by VMAT2 in the presynaptic terminals, and after release histamine binds to hrh1, hrh2,

and hrh3 receptors expressed in the brain. In neuronal tissue, histamine is cleared from the

extracellular space by HNMT and MAO. Histamine is also degraded by DAO (pathway

indicated in gray).

Serotonin is derived from ι-tryptophan by two modifications mediated by

tryptophan hydroxylase (TPH, EC 1.14.16.4) (Lovenberg, Jequier & Sjoerdsma

1967) and aromatic ι-amino acid decarboxylase (AADC, EC 4.1.1.28) (Lovenberg,

Weissbach & Udenfriend 1962). Two forms of TPH exist, of which TPH2 is

expressed in the brain and is responsible for the production of neuronal serotonin

(Walther et al. 2003). Serotonin is packed into vesicles by VMAT2, transported to


19

the nerve terminals, secreted, and bound to serotonin receptors. Serotonin can be

taken up into the cells by serotonin reuptake transporters (Torres, Gainetdinov &

Caron 2003). Inactivation of serotonin is mediated by MAO (Sjoerdsma et al. 1955)

by formation of 5-hydroxyindoleacetic acid (5-HIAA). The synthesis and catabolism

of serotonin can be manipulated by different chemicals, such as p-

chlorophenylalanine (PCPA), a selective irreversible inhibitor of TPH (Koe &

Weissman 1966, Jequier, Lovenberg & Sjoerdsma 1967), ι-deprenyl, which is a

MAO inhibitor (Fowler, Oreland & Callingham 1981), and fluvoxamine, a selective

serotonin reuptake inhibitor (Claassen et al. 1977).

Catecholamines (dopamine, noradrenaline, and adrenaline) are all derived from

the same amino acid, ι-tyrosine, by the following four enzymatic reactions:

tyrosine hydroxylase (TH, EC 1.14.16.2) forming ι-dihydroxyphenylalanine (ι-DOPA)

(Nagatsu, Levitt & Udenfriend 1964), AADC forming dopamine (Lovenberg,

Weissbach & Udenfriend 1962), dopamine β-hydroxylase (DβH, EC 1.14.17.1)

forming noradrenaline (Levin, Levenberg & Kaufman 1960) and

phenylethanolamine Ν-methyl transferase (PNMT, EC 2.1.1.28) forming adrenaline

(Axelrod 1962). The main enzymes in the degradation of amines are MAO and

catechol-O-methyl transferase (COMT, EC 2.1.1.6).

An interesting feature of rodent histaminergic neurons is that they express the

dopamine-producing enzyme, ι-DOPA (Yanovsky et al. 2011). Furthermore

histaminergic neurons take up ι-DOPA, the precursor of dopamine, and are

dopamine-immunoreactive (Yanovsky et al. 2011), suggesting that these neurons

can also produce dopamine.

2.1.4 Histaminergic receptor-signaling system

Aminergic neurotransmission is mediated through G-protein-coupled receptors.

Histamine acts through the four G-protein-coupled histaminergic receptor

subtypes 1, 2, 3, and 4 (hrh1, hrh2, hrh3, and hrh4) (Haas & Panula 2003, Haas,

Sergeeva & Selbach 2008). Of the known receptors, hrh1, hrh2, and hrh3 are

expressed in the vertebrate brain. hrh4 has also been detected in the mammalian

brain, but the highest level of hrh4 messenger RNA (mRNA) is found in the spinal

cord (Strakhova et al. 2009). hrh1 is mainly implicated in regulation of wakefulness

in rodents (Monti, Pellejero & Jantos 1986, Yanai et al. 1998) and hrh2 is mainly

involved in cognitive functions in rodents (Dai et al. 2007). hrh3 is expressed in

mammalian TMN histaminergic neurons and activation of hrh3 reduces the firing

rate of the neurons and thereby reduces the release of histamine (Arrang, Garbarg


20

& Schwartz 1983, Arrang, Morisset & Gbahou 2007). Furthermore, hrh3 can act as

a presynaptic heteroreceptor (Schwartz et al. 1990) and regulates the release of

several other neurotransmitters (Blandina et al. 1996, Yamamoto et al. 1997,

Schlicker, Werthwein & Zentner 1999, Jang et al. 2001, Pillot et al. 2002b).

Histamine-mediated neuronal excitation is achieved mainly via activation of hrh1

and hrh2 receptors in mammals, whereas hrh3 activation inhibits histamine

release in rats (Arrang, Garbarg & Schwartz 1983). Of the histaminergic receptors,

the crystalline structure of the human HRH1 together with a first-generation hrh1

antagonist, doxepin, has been determined (Shimamura et al. 2011), providing

detailed information on the molecular properties necessary for specific binding of

ligands to hrh1. The histaminergic receptors signal through different intracellular

signal transduction pathways (Haas, Sergeeva & Selbach 2008). In Table 1 the

features of the different histamine receptors are compared. The properties of the

zebrafish histamine receptor subtypes are not well characterized and require

further study.

Table 1. Properties and role of histamine receptors in mammals (modified from Haas,

Sergeeva & Selbach 2008). For abbreviations in the table, please see list on page 8–9.

Feature hrh1 hrh2 hrh3

G protein Gq/11 Gαs Gi/o

Signal transduction PLC, IP3, DAG, Ca2+

,

PKC, AMPK, NF-κB

AC, cAMP, PKA, CREB AC, cAMP, MAPK,

Akt/GSK3

Cellular function Postsynaptic

excitability

and plasticity

Postsynaptic excitability

and plasticity

Presynaptic transmitter

release

and plasticity

Systemic function Behavioral

state and

reinforcement

(arousal),

working memory,

feeding rhythms,

energy metabolism,

endocrine control

Learning

and memory

Blood-brain barrier

control, behavioral

state and

reinforcement

Pathophysiology Disorders of sleep,

eating, memory,

mood, addiction,

pain and neuro-

inflammation

Schizophrenia, pain and

neuro-inflammation

Disorders of sleep,

mood, memory, eating,

addiction, pain and

neuro-inflammation


21

2.2 Neurotransmitter systems under normal conditions and disease

2.2.1 Alertness and wakefulness

Alertness and wakefulness are specific states and important for the survival of the

individual. Almost a century ago, the Viennese neurologist von Economo noted

that lesions in the junction between the telencephalon and mesencephalon were

associated with profound sleepiness in his patients (von Economo 1930). Later, it

was found that this area harbors the aminergic and peptidergic systems that

constitute the ascending arousal system (AAS) and activate the cerebral cortex and

thalamus (Saper, Scammell & Lu 2005). Histamine in the TMN is part of the AAS

and histaminergic neurons fire during the waking period in mammals (Mochizuki et

al. 1992, Takahashi, Lin & Sakai 2006). The rest of the components of the AAS

include the dopaminergic ventral tegmental area, serotonergic raphe nucleus,

noradrenergic locus coeruleus (Saper, Scammell & Lu 2005), and the

hypocretin/orexin (hcrt) of the lateral hypothalamus (de Lecea et al. 1998). Hcrt

signals through two G-protein-coupled receptor subtypes 1 and 2 (hcrt1 and hcrt2)

in rodents and fish (Trivedi et al. 1998, Panula 2009). Of these two receptors, hcrt2

is expressed at high densities in mammalian histaminergic neurons (Yamanaka et

al. 2002). In rodents histamine and hcrt may be responsible for different aspects of

arousal/wakefulness: histamine mainly responsible for cognitive and motivated

behaviors and hcrt for motor activity (Anaclet et al. 2009). Reduction in the hcrt

level causes narcolepsy in humans (Peyron et al. 2000). The symptoms of

narcolepsy have only been reproduced in hcrt-/- mice, not in hdc-/- mice (Anaclet

et al. 2009). Serotonin, dopamine, galanin, GABA, thyrotropin-releasing hormone

(TRH) and methionine-enkephalin (mENK) are also known to affect wakefulness by

either promotion or inhibition of the AAS (Cirelli 2009). The sleep-promoting

center in the brain is the ventrolateral preoptic nucleus, which consists of

GABAergic and galaninergic neurons (Gaus et al. 2002). These neurons innervate

the nuclei of the AAS (Sherin et al. 1998) and are active during sleep (Szymusiak et

al. 1998). Together with the AAS clusters, these sleep-promoting neurons regulate

alertness and wakefulness in mammals.

Although the above-mentioned neurotransmitter nuclei are located in close

proximity, not many of the neurotransmitters are colocalized. To better

understand the role histaminergic neurons play in different behaviors or

physiological states, the cotransmitters of histaminergic neurons have been

identified in different species to varying degrees. Histamine is not colocalized with

serotonin, dopamine, or hcrt in adult zebrafish and the systems have different


22

innervation areas (Kaslin & Panula 2001, Kaslin et al. 2004). Serotonergic fibers and

dopaminergic fibers mainly innervate the ventral telencephalon, whereas

histaminergic fibers innervate the dorsal telencephalon (Kaslin & Panula 2001,

Kaslin et al. 2004). The coexistence of histamine with galanin, GABA, TRH, and

mENK has been studied in rodents (Airaksinen et al. 1992, Kukko-Lukjanov &

Panula 2003), but not in zebrafish. In rats, histamine coexist with galanin, TRH, and

GABA in histaminergic neurons (Airaksinen et al. 1992). In guinea pig, histamine is

coexpressed with GABA and mENK in histaminergic neurons, whereas in mice

histamine is colocalized with galanin and GABA in histaminergic neurons

(Airaksinen et al. 1992). In human TMN, the coexistence of histamine with galanin

and GABA was studied, and it was found that histamine is colocalized with GABA,

but not with galanin (Trottier et al. 2002).

2.2.2 Histamine and neurodegenerative diseases

Aminergic neurotransmitters have been indicated in several neurological diseases,

including narcolepsy, AD, PD (Eriksson et al. 2010), and most recently in Tourette’s

syndrome (Ercan-Sencicek et al. 2010). Narcolepsy is a neurodegenerative disease

characterized by fragmented sleep and unpredictable sleeping bouts throughout

the day, and to date there is no cure for this disease (De la Herran-Arita, Guerra-

Crespo & Drucker-Colin 2011). The neurological background is a deficient hcrt

system in humans (Peyron et al. 2000). The etiology is unknown, although research

indicates an autoimmune origin (Scammell 2003, Hallmayer et al. 2009, De la

Herran-Arita, Guerra-Crespo & Drucker-Colin 2011). It is thought that the

mechanism by which hcrt induces wakefulness is by exciting histaminergic neurons

via hcrt2 receptors (Bayer et al. 2001). Cataplexy, i.e. loss of muscle tone, is often

associated with narcolepsy and is usually triggered by emotional excitation (Anic-

Labat et al. 1999). Narcoleptic patients have traditionally been treated with

stimulants, such as amphetamine (Zaharna, Dimitriu & Guilleminault 2010). Due to

the addictive potential of amphetamine, a new class of drugs has been designed.

Modafinil and armodafinil are the best available drugs to treat narcolepsy,

although the mechanism of action is not known (Zaharna, Dimitriu & Guilleminault

2010). The European Medicines Agency has approved selective serotonin reuptake

inhibitors and tricyclic antidepressants for treatment of cataplexy, while the

American Food and Drug Administration has not (Zaharna, Dimitriu &

Guilleminault 2010).


23

Narcolepsy has been modeled in zebrafish by disruption of the hcrt system, either

by hcrt2 receptor null mutation, hcrt2-/-, (Yokogawa et al. 2007) or hcrt

overexpression (Prober et al. 2006). Both models produce a similar insomnia

phenotype. In humans affected with sleep disorders, histamine and hcrt

concentrations are both reduced (Nishino et al. 2009) and patients with

neurodegenerative disorders display insomnia (Dauvilliers 2007). A comparative

representation of both neurotransmitter systems and their projections in human

and zebrafish brain is available in Figure 4.

Figure 4. Comparative presentation of hcrt and histamine in the central nervous system of

humans (left) and zebrafish (right). The histaminergic and hcrt neurotransmitter systems

are conserved between the two species. The fibers from each neurotransmitter system

innervate the neurons of the other in both humans and zebrafish. The projection pattern of

the neurons is similar in these species, since the fibers innervate the majority of the brain

regions. Please note that the brains are not depicted in correct scale in regard to each

other.

AD is the most common neurodegenerative disease worldwide, with cognitive

decline and dementia as the main manifestations. The neurological hallmarks

include reduced brain size, neurofibrillary tangles, and amyloid plaques (Bishop, Lu

& Yankner 2010). The majority of AD cases are sporadic (Blennow, de Leon &

Zetterberg 2006) and affects persons older than 65 years. The early-onset familial

form of AD has an onset around 30 years (Blennow, de Leon & Zetterberg 2006).

The familial forms of AD are due to mutations in the following genes: amyloid

precursor protein (app), presenilin1 (psen1) and presenilin2 (psen2). Both PSEN1

and PSEN2 are part of the γ-secretase complex (Kimberly et al. 2003), which is

expressed within the membranes of the neurons, including the cell membrane,

endoplasmic reticulum, Golgi apparatus, and mitochondria (Kovacs et al. 1996, Xu

et al. 2002, Hansson et al. 2004) (Figure 5). The cognitive decline associated with


24

AD is mainly due to reduced acetylcholine levels in the brain, which results from

reduced numbers of neurons and neuronal dysfunction (Whitehouse et al. 1982).

Figure 5. The γ-secretase complex is composed of four proteins, PSEN1 or PSEN2,

presenilin enhancer 2 (PEN-2), nicastrin and anterior pharynx-defective 1 (APH-1), and

resides in the membranous organelles of the cell and the cell membrane. These proteins

are not depicted in the correct relation to each other in the figure.

The γ-secretase complex is thought to have many functions, including cleavage of

APP (Borchelt et al. 1996), regulation of the calcium (Ca2+) homeostasis (Guo et al.

1996, Stutzmann et al. 2004), cleavage of the Notch receptor (Levitan & Greenwald

1995), and involvement in Wnt/β-catenin signaling (Serban et al. 2005). Notch

signaling is important in embryonic neural development and in the adult brain

(Ables et al. 2011). In zebrafish, inhibition of γ-secretase with DAPT (N-[(3,5-

Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethylester) affects

Notch signaling (Geling et al. 2002). PSEN1 functions also independently of the γ-

secretase complex. The regulation of Wnt/β-catenin signaling is an example of this

(De Strooper & Annaert 2010). In this case, PSEN1 is important for degradation of

β-catenin (Kang et al. 2002) and lack of PSEN1 is associated with an increase in β-

catenin signaling (Xia et al. 2001). Wnt/β-catenin signaling, as is Notch signaling, is

an important signaling cascade during embryonic development, but also plays a

role in the adult CNS (Inestrosa & Arenas 2010) and in the progression of AD

(Boonen, van Tijn & Zivkovic 2009).

Interestingly, amyloid formation is regulated by hcrt and the sleep-wake cycle,

indicating that there may be a correlation between the AAS and pathogenesis of

AD (Kang et al. 2009). Kang et al. (2009) showed that lack of hcrt would protect

against AD, but a study on the coexistence of narcolepsy with AD found that also

narcoleptics are affected by AD (Scammell et al. 2011). In postmortem brains of AD

patients, the histamine content is reduced (Panula et al. 1998, Shan et al. 2012).

The underlying mechanism for this alteration in histamine content is not known.


25

One possible mechanism may be associated with hypoxia, since human HDC

expression is regulated by hypoxia-inducible factor 1 (Jeong et al. 2009) and

hypoxia in general is associated with the progression of neurodegenerative

disorders, such as AD (Zlokovic 2011). So far, it is not known if neuronal HDC can

be induced by hypoxia. Furthermore, histaminergic neurons exert neuroprotective

effects in organotypic slice preparations during excitotoxic insults (Kukko-Lukjanov

et al. 2006). Other possible mechanisms include alteration in neurogenesis and/or

proliferation during development (Wen et al. 2002, Chevallier et al. 2005,

Rodriguez, Jones & Verkhratsky 2009) or activity-dependent neurotransmitter

specification (Spitzer 2012), that later on in adulthood may affect the balance of

neurotransmitter systems in normal and diseased brains. Histamine receptor

expression has been examined in the prefrontal cortex of AD patients. In human

brain samples, hrh1 binding was decreased (Higuchi et al. 2000), hrh2 mRNA was

unaltered (Perry et al. 1998, Shan et al. 2012), and hrh3 mRNA was increased (Shan

et al. 2012). The increase in hrh3 mRNA was only observed in female AD patients

(Shan et al. 2012). Taken together, understanding of the mechanisms that underlie

altered histaminergic neurotransmission will provide further insight into AD

progress. There is no cure for AD currently, and treatments can only ease the

symptoms of the disease. Acetylcholinesterase inhibitors and N-methyl-D-aspartic

acid antagonists (Wollen 2010) are widely used treatments. Stimulatory therapies,

which include physical exercise, socialization, cognitive training, and music, are

emerging as new therapeutics (Wollen 2010). Since the life expectancy of our

population is increasing, new therapies, but especially better understanding of the

etiology of the disease, are needed to combat the progression of AD. In zebrafish,

the role of psen1 has been studied by transiently inhibiting gene expression.

Transient knockdown of psen1 in zebrafish (Nornes et al. 2003, 2008, 2009)

phenocopies the features reported for the psen1 knockout mice (Shen et al. 1997),

i.e. disrupted CNS formation and Notch signaling.

2.3 Zebrafish as a vertebrate model organism

The zebrafish belongs to the bony fishes (Teleosts) of the ray-finned fishes

(Actinopterygii), which comprise the majority of all living fish. The actinopterygian

fishes and mammals shared an ancestor about 500 million years ago (Metscher &

Ahlberg 1999, Grillner & Jessell 2009). The zebrafish is a vertebrate model

organism that was initially used in developmental biology studies during the 1970s.

During the last decade, the popularity of this vertebrate model has grown in the

field of neuroscience (Panula et al. 2006, 2010, Sager, Bai & Burton 2010). The


26

main advantages, such as its small size (approximately 4 cm in length) and

transparent ex utero development in combination with feasibilities of genetic

manipulation, have rendered it a good model for studying neuronal circuits

underlying behavior. Importantly, the neuroanatomy of zebrafish correlates well

with that of other vertebrates studied, which is one of the reasons it can be

utilized as a model for studying the mechanisms underlying human disease. In

addition, tracing and lesion studies have also been undertaken to elucidate the

neuroanatomy and the role of different brain areas in fish and how these can be

correlated with other vertebrates. The similarity in organization of various

neurotransmitter systems among several vertebrate species has further motivated

the use of zebrafish as a model in neuroscience. Due to the small size of the

zebrafish, 3D imaging of whole neurotransmitter systems is possible. These

methods, combined with targeted stimulation or ablation of single neurons, render

the zebrafish a powerful tool for studying the role of specific neurons (Burgess &

Granato 2007b). For example, Ca2+ imaging can be used to correlate the activity of

a neuron with specific stimuli (O'Malley, Kao & Fetcho 1996, Orger et al. 2008).

Other possible tools that are currently available include optogenetics, which allows

noninvasive activation of neurons within millisecond ranges (Szobota et al. 2007,

Douglass et al. 2008), and high-throughput pharmacological experiments

combined with a behavioral assay (Rihel et al. 2010). The availability of a large

number of transgenic and mutant fish lines combined with the above-mentioned

appealing approaches for studying behavior makes this an excellent model

organism for use in neuroscience.

2.3.1 Comparative neuroanatomy of fish and mammals

The telencephalon develops in actinopterygian fishes via eversion, whereas in

other vertebrates it develops via evagination. The difference in development was

postulated to be the reason for the nonlaminar telencephalon in actinopterygian

fishes (Ito & Yamamoto 2009). In actinopterygian fishes, the telencephalon can be

grossly divided into the dorsal telencephalon, i.e. the pallium, and the ventral

telencephalon, i.e. the subpallium (Nieuwenhuys 1963, 2010, Nieuwenhuys, ten

Donkelar & Nicholson 1998) (Figure 6). Based on tracing and gene expression

studies the basal ganglia, including the striatum, was suggested to be located in

the ventral telencephalon (Rink & Wullimann 2001, Mueller, Wullimann & Guo

2008, Ganz et al. 2011). Toxicity studies, neuronal connectivity, and tracing studies

have suggested that the teleost correlate to the mammalian substantia nigra,

which is part of the basal ganglia, is located in the diencephalon of larval zebrafish


27

(Kaslin & Panula 2001, Rink & Wullimann 2004, Sallinen et al. 2009). The dorsal

part of the dorsal telencephalon (Dd, Figure 6) in goldfish may correspond to the

mammalian cerebral cortex (Vargas, Lopez & Portavella 2009). Recent results

indicate that the Dd region does not exist in zebrafish and that the central part of

the dorsal telencephalon (Dc) corresponds to the mammalian cortex (Mueller et al.

2011). Based on lesion studies of the medial pallium, the area corresponding to the

mammalian amygdala may be located in the medial region of the dorsal

telencephalon (Dm, Figure 6) in goldfish (Portavella et al. 2002, Portavella & Vargas

2005). Similar lesion studies of the lateral pallium suggest that the phylogenetically

conserved area that corresponds to the mammalian hippocampus in goldfish is in

the lateral region of the dorsal telencephalon (Dl, Figure 6) (Portavella, Torres &

Salas 2004, Portavella & Vargas 2005). The dorsal telencephalon is highly

innervated by histaminergic fibers (Kaslin & Panula 2001). In contrast,

dopaminergic, serotonergic, and hcrt fibers innervate the ventral telencephalon

(Kaslin & Panula 2001, Kaslin et al. 2004).

Figure 6. Comparative analysis of the telencephalic areas in fish (left) and mammals (right).

Dd = dorsal area of dorsal telencephalon, Dm = medial area of dorsal telencephalon, Dl =

lateral area of dorsal telencephalon, Dc = central area of dorsal telencephalon, Dp =

posterior area of dorsal telencephalon. In zebrafish, the area that is suggested to

correspond to the mammalian cortex is Dc, whereas studies in goldfish proposed Dd.

The thalamic complex in mammals is a major structure of the diencephalon and

consists of three regions: prethalamus, zona limitans intrathalamica, and thalamus.

The development of the thalamus is evolutionarily conserved, as suggested by a

comparative analysis of mouse, chicken, and zebrafish. The study demonstrated

that homologous genes are responsible for development of the thalamic nuclei in

these three different species (Scholpp & Lumsden 2010).


28

The hypothalamus is located below the thalamus and is connected to the pituitary

(Nieuwenhuys, ten Donkelar & Nicholson 1998). By release of neuropeptides and

neurotransmitters, the hypothalamus maintains the homeostasis of the organism

and is involved in endocrine and metabolic control of body functions and

behaviors. For example, arousal is tightly regulated by the nuclei in the

hypothalamus (Scammell 2003). The mammalian and zebrafish hypothalamus are

not anatomically very alike, but the equivalents of the neuronal nuclei that are

found in the mammalian hypothalamus are also found in zebrafish (Machluf,

Gutnick & Levkowitz 2011). The ventrocaudal hypothalamus is the site of the

histaminergic neurons, and some of the serotonergic, dopaminergic, and hcrt

neuron populations are also located in this region in zebrafish (Kaslin & Panula

2001, Kaslin et al. 2004). To evaluate the role of the cerebellum in modulation of

motor behaviors, the cerebellar anatomy has been described and mutations that

affect the development of the cerebellum have been produced (Bae et al. 2009).

2.3.2 Zebrafish behavior and motor functions

At as early as 17 hours postfertilization (hpf), zebrafish larvae show spontaneous

movement (Brustein et al. 2003). The activity of the fish increases during the

course of a few days and at 4–5 dpf, a mature beat-and-glide swimming pattern

can already be observed. At this age, the larvae begin to hunt for food, since the

nutrients of the yolk sac have been almost completely consumed. Fundamental

behaviors, such as prey capture (Budick & O'Malley 2000) and sleep (Prober et al.

2006, Zhdanova 2006), can be assessed in zebrafish larvae.

The neuronal circuits in the spinal cord and brainstem that underlie behavior have

been studied in zebrafish (discussed below). In contrast, the involvement of the

telencephalon in execution of behaviors has not been assessed (Figure 1). To date,

there are no identified neuronal circuits that connect the telencephalon with the

brainstem (Figure 1). The optic tectum of zebrafish corresponds to the superior

colliculus of mammals and in both cases the main task is to integrate visual

information with motor output (Isa 2002, Sato et al. 2007, Del Bene et al. 2010).

The brainstem nuclei regulate the lower motor neurons and mediate modulatory

inputs from the telencephalon and cerebellum. The spinal cord harbors the motor

and sensory connections and it is this area that coordinates several reflexes.

To link specific neurons with neuronal networks and to identify the circuits

underlying behavior, specific behaviors can be dissected into smaller units. This

allows the identification of which movement patterns zebrafish larvae combine to


29

produce specific behaviors. Dependent on the behavior studied, movement

patterns can be identified by different methods. Identification of the movement

patterns zebrafish combine to produce behavior, such as prey capture or startle

response, requires high-frequency imaging (500–1000 frames per second). Others,

such as sleep, place preference, and general activity, can be assessed with low-

frequency imaging (5 frames per second or less). Different stimuli elicit distinct

responses in zebrafish larvae. Acoustic/vibrational stimuli induce a rapid escape

response (Burgess & Granato 2007b), whereas dark flashes induces a slower

response (Burgess & Granato 2007a). For navigational purposes, adjustment

moves such as turns and scoots (Burgess, Schoch & Granato 2010) and slow-

swimming with the pectoral fins can be detected (Budick & O'Malley 2000).

The reticulospinal neurons in the brainstem of larval zebrafish project to the spinal

cord to regulate execution of many behaviors. The Mauthner neuron (Figure 7) is

the best known and is thought to be the sole driver of the escape response, which

is also called the startle response. The startle response of freely moving larval

zebrafish can be divided into short-latency and long-latency startle responses

(Burgess & Granato 2007b). Both types of startle responses last for several

milliseconds and are completed in less than 1 second after stimulation. The short-

latency startle response is mediated by the Mauthner neuron, whereas the long-

latency startle response is not (Burgess & Granato 2007b). Other neurons of the

brainstem, such as MID2cm (Mauthner neuron homologue, middle dorsal 2

contralateral medial longitudinal fasciculus interneuron in rhombomere 5) and

MID3cm (Mauthner neuron homologue, middle dorsal 3 contralateral medial

longitudinal fasciculus interneuron in rhombomere 6) cooperate with the

Mauthner neuron in giving the escape/startle response a direction (Figure 7)

(O'Malley, Kao & Fetcho 1996). Stimulation of the head causes both the Mauthner

neuron and MID2cm or MID3cm neuron to respond, whereas when the tail is

stimulated, only the Mauthner neuron is activated (O'Malley, Kao & Fetcho 1996).

Among the medial longitudinal fasciculus (nMLF) neurons active during forward

swimming have been identified (Orger et al. 2008). In the same study, neurons that

execute right or left movements were also identified. A schematic drawing of the

anatomical position of the neurons described is presented in Figure 7 and their

connections via commisural interneurons to the lower motor neurons in the spinal

cord are also illustrated.


30

Figure 7. Lateral view of a zebrafish brain. The main brain areas are indicated and the

nMLF, Mauthner neuron, MID2cm, MID3cm, and the relationship to the histaminergic

neurons and their projections are illustrated. Furthermore, the interaction of the lower

motor neurons with the interneurons is visualized.

In both larval and adult zebrafish, the response to changes in the environment can

be assessed. This can either be studied by moving white and black stripes

unidirectionally under the freely moving fish, which causes an optomotor

response, or by fixing the animals and monitoring their eye movements, which in

turn causes an optokinetic response. These two assays have successfully been used

to screen for visual system defects in mutants (Brockerhoff et al. 1995, Muto et al.

2005). Changes in illumination mediate a Mauthner neuron-independent

behavioral response that has been classified as an О-bend, which may be strictly

navigational (Burgess & Granato 2007a, Burgess, Schoch & Granato 2010). In this

study, we assessed a similar response, i.e. an increase in locomotor activity when

zebrafish larvae are exposed to sudden changes in illumination. We call this

response a dark-induced flash response.

2.3.3 Behavioral pharmacology

Pharmacological experiments are easily performed in zebrafish larvae, since they

can be immersed in drugs. High-throughput screening of thousands of molecules

combined with a behavioral readout has been successfully developed to study

sleep-wake regulation in zebrafish larvae (Rihel et al. 2010). Depending on the

properties of the drug, an appropriate solvent must be used to ease the uptake of

the drug into the larvae. Furthermore, the blood-brain barrier in zebrafish begins

to develop at 3 dpf (Xie et al. 2010), but is not fully functional until 9 dpf (Jeong et

al. 2008). The timeline for the development and maturation of the blood-brain

barrier therefore offers a window when drugs are easily taken up into the brain.


31

2.3.4 Modification of gene expression with morpholino oligonucleotides

To transiently inhibit gene expression, morpholino oligonucleotides (MOs) have

been developed. The MOs can be designed to inhibit gene expression via two

different approaches: 1) by inhibiting the binding of the ribosomal subunits to the

mRNA initiation site by targeting the MO upstream of the initiation site or 2) by

interfering with the splicing of the premature mRNA by targeting the MO to the

exon-intron or vice versa boundary. The MOs are injected into zebrafish embryos

at the 1–4-cell stage. Since the effect of the MO is only transient and usually ceases

within the first week of life, the effect of transient downregulation can only be

assessed in larval zebrafish. Care must be taken that the expression of the gene of

interest is downregulated throughout the observation time. This can be done by

verifying loss of the protein by immunohistochemistry (IHC) or Western blotting. If

a splice site MO is used, then the expression of mature mRNA can be assessed by

real-time polymerase chain reaction (RT-PCR). MOs quite often also induce off-

target effects, such as edema and increased cell death (Robu et al. 2007, Eisen &

Smith 2008). Different approaches to control the specificity of the MO have

therefore been proposed. For example, several MOs could be used to induce the

same phenotype and activation of the p53 pathway could be assessed by

quantitative real-time polymerase chain reaction (qPCR) or by coinjecting MO the

that targets the p53 pathway, and rescue of the phenotype is recommended by

injection of target gene mRNA. There are some problems with these approaches,

since some target genes may indeed be involved in the upregulation of cell death

pathways (Sallinen et al. 2010).

2.3.5 Modification of gene expression by inducing mutations into the genome

Chemical mutagenesis was applied to zebrafish almost two decades ago (Driever et

al. 1996) and this approach required forward genetics to identify the mutation-

carrying gene. To speed up the process, the TILLING (Targeting induced localized

lesions in genomes) tool was developed. TILLING is a reverse genetic approach by

which knockout animals can be obtained. TILLING was initially set up for

Arabidopsis thaliana (McCallum et al. 2000, Colbert et al. 2001) and later modified

to allow target-selected mutagenesis in zebrafish (Wienholds et al. 2002, 2003).

Random gain-of-function or loss-of-function mutations are induced into genomes

by treating male zebrafish with a highly potent mutagen, N-ethyl-N-nitrosourea

(ENU, chemical formula C3H7N3O2), which targets the spermatogonial stem cells

(Driever et al. 1996). After a recovery period the ENU-treated males are mated


32

with healthy wild-type (WT) female zebrafish to produce an F1 offspring

generation. Genomic DNA is prepared from fin clips of this live library or from

sperm kept in a frozen library, and the individuals are screened for mutations in a

specific gene. The plant endonuclease CEL-I can be used for detection of the

mutations, since it detects all of the currently known sequence variants, including

deletions, insertions, and missense alterations (Oleykowski et al. 1998). Since ENU

is a highly potent mutagen that induces random mutations throughout the

genome, it is advisable to outcross identified carriers for several generations to

reduce the number of background mutations.

Designed targeted mutagenesis is possible via the zinc finger nuclease (ZFN) and

TALEN (Transcription activator-like effector nuclease) methods (Caroll, Zhang

2011). These two methods have higher specificity for the genomic target sequence

and thereby produce fewer off-target effects (Doyon et al. 2008, Meng et al. 2008)

than random mutagenesis induced with ENU. ZFN and TALEN function in a similar

manner. They are heterodimers that consist of a catalytic unit (FokI endonuclease)

that introduces double-stranded breaks at specific genomic sites, and a targeting

unit that is a DNA-binding unit consisting of several DNA-binding modules. The

double-stranded breaks are then repaired by the DNA repair machinery, which

introduces small insertions or deletions that can cause frameshift knockout

mutations (Bibikova et al. 2003). The difference between ZFN and TALEN is that

each ZFN DNA-binding module recognizes three nucleotides, whereas the TALEN

DNA-binding module recognizes only one, resulting in higher specificity (Caroll &

Zhang 2011). The ZFN method has been available for a decade (Smith et al. 2000),

but due to the high uncertainty with the ZFN design, it has not been used

extensively in zebrafish research. Due to the favorable aspects and increased

knowledge of the methods, the ZFN and TALEN methods will most likely also

become more commonly used in zebrafish research.

2.3.6 Spatially and temporally targeted gene expression

Methods for targeted gene expression in desired tissues have also been described.

The medaka (Oryzias latipes) Tol2 transposon system in combination with the

Gal4/UAS (upstream activating sequence) system has been used to express genes

downstream of the UAS in specific neurons (Halpern et al. 2008, Asakawa &

Kawakami 2009, Suster et al. 2009). In this manner, the neural circuits underlying

the touch response in zebrafish were unraveled. Inhibition of neurotransmitter

release from Gal4-positive hindbrain interneurons and Rohon-Beard sensory


33

neurons by expression of tetanus toxin (TeTx) under the UAS resulted in lack of a

touch response (Asakawa et al. 2008, Asakawa & Kawakami 2009). This method for

spatially targeted gene expression can also be further modified to express genes in

a temporal manner (Halpern et al. 2008). This method requires, as does TILLING,

large facilities for housing and screening the different Gal4 fish lines that later on

are crossed with UAS fish.

2.3.7 Appropriateness of modeling human disease in a simple vertebrate

The molecular pathways underlying many disorders in humans are still unknown

and new inexpensive models are needed to contribute in the quest to reveal these

mechanisms. Invertebrates such as Caenorhabditis elegans and Drosophila

melanogaster have already provided insight into some of the mechanisms

underlying human disease processes. The zebrafish was initially used as a model

organism in developmental studies but has during the past decade become more

widely used to model various human diseases, ranging from disorders of the

nervous system, e.g. attention deficit hyperactivity disorder (Lange et al. 2012), to

leukemia (Ridges et al. 2012). The zebrafish is, due to its small size, less expensive

to keep in large quantities than are rodents. In addition, zebrafish may by the

public be considered as a more ethical choice than mammals as disease models.

The major drawback of zebrafish in modeling human disease is that it has

undergone the genome duplication event, and therefore some of the genes in the

zebrafish are duplicated, whereas in other cases one gene in zebrafish carries the

properties of both human homologues. An example of this is the enzyme

responsible for converting ι-tyrosine to ι-DOPA, TH, which in zebrafish is duplicated

(Chen, Priyadarshini & Panula 2009). So far, the function of zebrafish TH2 is

unknown and zebrafish TH1 converts ι-tyrosine to ι-DOPA, just as in humans.

Another example is MAO in zebrafish, which carries features of both human MAO-

A and MAO-B (Anichtchik et al. 2006). Naturally, the novel insights obtained in

zebrafish must be studied in mammalian model organisms and humans before

conclusions regarding the findings are applicable to humans and human disease.

Taken together, the above-mentioned results indicate that the zebrafish is an

appropriate tool for modeling human disease to answer mechanistic questions.

Aims of the study

34

3 Aims of the study

The main goal of this thesis project was to study the functions of neuronal

histamine; the interactions of the system with other neurotransmitter systems, its

role in neuronal plasticity, and the neuronal circuits underlying behavior. The

zebrafish was chosen as a model organism and various methods for quantifying

larval zebrafish behavior were established.

The specific aims of the thesis were the following:

I Identify and characterize the histamine receptors in zebrafish and

the behavioral effects of their ligands,

II Identify the cotransmitters of the histaminergic neurons,

III Study the role of histamine in alertness and wakefulness,

IV Elucidate the factors that regulate the development of histamine

neurons by characterizing the system in psen1 knockout fish, and

V Assess the role of MAO in behaviors driven by amines.

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4 Methods

Please find more detailed information regarding the materials and methods in the

original publications.

4.1 Animals

Several zebrafish strains were used in this thesis. For the studies in I–III and V, a

WT strain that originated from Abo Akademi University and which was used in the

following studies (Kaslin & Panula 2001, Peitsaro et al. 2003, Kaslin et al. 2004,

Sallinen et al. 2009, 2010) was used. For the results in unpublished form (IV), a

homozygote TILLING mutant line carrying a premature stop codon in the psen1

gene (hu2547) was used. This line was a kind gift from Dr. Zivkovic (Hubrecht

Institute of Developmental Biology and Stem Cell Research, Royal Netherlands

Academy of Arts and Sciences, Amsterdam, and University Medical Center,

Utrecht, The Netherlands). The mutation was induced in the Tubingen longfin (TL)

strain and we therefore used the TL strain as the control WT line in studies with

the mutant. Fish handling and housing (+26–28 °C, 14:10 light-dark cycle) were

done as previously described (Westerfield 2007). The permits for the experiments

were obtained from the Animal Experiment Board of the Regional State

Administrative Agency for Southern Finland, in agreement with the ethical

guidelines of the European Convention.

4.2 Manipulation of gene expression by morpholino oligonucleotides (III)

We designed an MO that targeted the initiation site of the hdc mRNA (atg), 5’-

CATCCCGTCACTCAGAGAAGATTAG-3’. A standard (std) control MO was used as the

control (both MOs from Gene Tools). At 5–6 hpf, the success rate of the injection

was observed in a fluorescence microscope, since the MOs were tagged with

fluorescein. Any uninjected embryos within the MO-injected groups were excluded

from the experiment. The injected embryos were grown until 5 dpf or older, when

the experiments were conducted. Uninjected WT animals were used as additional

controls.

4.3 Pharmacological treatments

For the pharmacological studies, zebrafish larvae were dechorionated with forceps

at 24 hpf and raised in 6-well plates with 3 ml of embryo medium (E3: 5 mM NaCl,

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0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, all reagents from Sigma-Aldrich) to

which the drug was added. The group size varied between n=10–35. If the drug

treatment was started at 5 dpf or later, the fish were raised in petri dishes until the

appropriate age. The E3 medium with the drug was usually changed once daily.

4.3.1 Treatment with histamine receptor ligands or α-fluoromethylhistidine (III)

Zebrafish larvae were treated with 1, 10, or 100 μM pyrilamine (Sigma-Aldrich),

cimetidine (Sigma-Aldrich), thioperamide or immepip. The thioperamide and

immepip were kind gifts from Professors Rob Leurs and Henk Timmerman (Vrije

Universiteit, Amsterdam, The Netherlands). The control groups were kept in E3

medium. The treatments were initiated at 2–3 hpf, 1 dpf, or 2 h before the

behavior was assessed. The locomotor activity was observed at 5 dpf. The α-FMH

was a kind gift from Dr. Kollonitch (Merck) and the treatments were 1 mM for 1–5

or 1–7 dpf before behavior (please see 4.4) or hcrt whole-mount in situ

hybridization (WISH, please see 4.9).

4.3.2 Deprenyl, p-chlorophenylalanine, and fluvoxamine treatments (V)

Fish exposed to solutions containing deprenyl (Sigma-Aldrich) at 1, 10, or 100 μM

were further divided into three groups. The fish were exposed to the drug for

different times, either 0–7 dpf, 0–5 dpf or only 2 h preceding testing. For deprenyl

and PCPA (Sigma-Aldrich) treatment, the fish were divided into six groups and

exposed to 150 μM or 1.5 mM PCPA for 1–5 dpf and/or 100 μM deprenyl 0–5 dpf.

The fluvoxamine (Sigma-Aldrich) treatments were also done in combination with

deprenyl (100 μM 0–5 dpf) or alone by exposing zebrafish to 100 μM fluvoxamine

2 h before the animals were killed at 5 dpf. The control groups received no drugs.

4.3.3 γ-Secretase inhibitor DAPT treatments (IV)

The DAPT in solution (Tocris Bioscience) was diluted in dimethyl sulfoxide (DMSO)

to reach a final concentration of 2.5 mM. At 1 dpf, 30 fish per treatment group

were dechorionated, moved to 6-well plates, and kept in 3 ml of E3 medium

containing either 1% DMSO or DAPT. After the treatment, the fish were killed and

fixed with 4% 1-ethyl-3,3(dimethyl-aminopropyl) carbodiimide (EDAC; CMS

Chemicals) diluted in 0.1 M phosphate buffer, pH 7.0.

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4.4 Behavioral tests

To assess the effect of histamine on behavior, several behavioral assays were

developed during the course of this thesis. The behavioral experiments were done

during the light period of the day, unless otherwise mentioned. The zeitberger

time (ZT) onset was at 9.00 in the morning (ZT0) and lights went off at 23.00 in the

evening (ZT14). The behavioral data were either collected by i) observing the fish

and manually scoring the specific behaviors or ii) a video camera and further

analyzed with EthoVision 3.1 software (Noldus Information Technology). Advanced

calibration of the arenas was applied in each case when the EthoVision software

was used. All behavioral experiments were done at room temperature (RT) and

repeated three times with different fish.

4.4.1 Locomotor activity of zebrafish (I, III–V)

The larvae were placed in individual hemisphere-shaped chambers (diameter 4 cm,

volume 12 ml) or in the wells of a 48-well plate (volume 1 ml) and analyzed for 10

min at a sample rate of 5–25 frames per second. The total distance moved (cm),

turn angle (degrees), and movement (percentage of time fish spent moving) were

calculated from the coordinates acquired, using the EthoVision software. System

noise was removed, using an input filter of 0.2 cm for minimum distance moved.

The tracks were excluded if the sample size (i.e. the number of frames in which the

animal was detected) did not exceed 90% of the maximum sample size or if

reflections were detected. The locomotor activity (total distance moved, cm) of

adult zebrafish was analyzed in the same manner as for the larvae, with the only

difference being that the arenas used were larger (diameter 22 cm, volume 2 l).

4.4.2 Place preference (IV)

Place preference was assessed in the same arenas as the locomotor activity. The

arenas were divided into several zones (in EthoVision) and the time the fish spent

in each zone during the tracking period was determined.

4.4.3 Place preference in a water column (V)

A vertical place preference test was done in a 2-ml cylinder filled with E3 medium.

Two groups were used: a control group without drug treatment and a ι-deprenyl

group with treatment of 100 μM ι-deprenyl for 0–5 days. Ten 5-day-old larvae

were placed in a cylinder and allowed to accommodate for 5 min. The cylinder was

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divided into upper and lower parts. The number of fish in the lower part was

counted every 5 min for 25 min.

4.4.4 Locomotor activity of larval zebrafish over a 24-h period (III)

The activity and frequency of movement were observed during a 24-h period. The

zebrafish larvae (5 dpf) were placed individually in the wells of white 96-well plates

5 hours before tracking, which was started at ZT14. A video camera connected to a

computer with EthoVision software was used for tracking the fish illuminated by

infrared lights. Two parameters were analyzed: frequency of movement and total

distance moved per hour.

4.4.5 Dark-induced flash responses in larval zebrafish (III, IV)

In this behavioral test, we studied whether light per se affects the activity of fish.

We set up an assay to approach this, which was similar to assays in previous

studies in which related behaviors were assessed (Burgess & Granato 2007a,

Burgess & Granato 2007b, Emran, Rihel & Dowling 2008). Light-induced decrease

in locomotor activity and dark-induced flash response of morphant fish were

assessed by observing 5 dpf fish during 2 min periods of lights on and off for 10

min. The fish were transferred to 48-well plates on the day of observation,

illuminated by infrared lights, and their movement was recorded with the same

system as described above.

4.4.6 Optomotor response (III)

An optomotor response assay was developed for the initial zebrafish mutation

screen to identify visual system defects in mutants. We used a simplified version of

the optomotor assay by manually moving a paper with black and white stripes (1-

cm-wide stripes) for 30 seconds under a plate with 6–7 dpf zebrafish larvae and

observing whether or not the larvae adjusted their direction of swimming in the

direction of movement of the stripes.

4.5 Lipophilic dye DiI tracing (III)

Micropipettes were filled with 20% carbocyanine dye DiI (Molecular

Probes/Invitrogen Corporation) solution (diluted in 1:1 ethanol:DMSO). After

anesthetization of the fish, the needle was placed perpendicularly to the body axis

and 2 nl of the tracer was injected, by PV830 Pneumatic PicoPump (World

Precision Instruments Inc.) at a pressure of 30 psi for 5 ms, through the skin to the

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superficial part of the right dorsal telencephalon of 9 dpf larvae. The injection was

done under a stereomicroscope and targeting was verified immediately with a

Leica MZFBL III fluorescence microscope (Leica Microsystems GmbH). After the

injection, the larvae were kept alive 1 h, killed and fixed with ice-cold 4% EDAC.

4.6 Real-time polymerase chain reaction

4.6.1 Cloning of zebrafish hrh1, hrh2, and hrh3 receptors in adult zebrafish (I)

Sanger Institute zebrafish blast pages (http://www.ensembl.org/Danio_rerio) were

used to identify sequences that are similar to the human hrh1, hrh2, and hrh3

receptors. The coding sequences predicted for the receptors were amplified, using

PCR. RNA isolated from adult zebrafish brains was used as a template for the

reactions. The RNA was extracted with RNAwiz™ (Ambion Inc.) and

complementary DNA (cDNA) was reverse-transcribed, by the First-Strand Synthesis

Kit (Amersham Biosciences) or by the SuperScript™ First-Strand Synthesis System

(Invitrogen Corporation) according to the manufacturers’ instructions. The

following primers were used: hrh1 (DQ647806) forward 5′-

TGTCTCTCCTCACCGTCATC-3′ and reverse 5′-CAATCCCTTCATTTACCCGCTCT-3′; hrh2

(DQ647808) forward 5′-ATGCAATTTATATTCAGCGAT-3′ and reverse 5′-

ACTACCTTTTACTCCCATTAGC-3′; hrh3 (DQ647807) forward 5′-

ATGGAGAGAGAAAACGCG-3′ and reverse 5′-AATACTTCTGGTCAATGTTC-3′. The

amplified PCR products were sequenced from both directions (PRISM 310; Applied

Biosystems Inc.).

4.6.2 Expression of hrh1, hrh2, and hrh3 mRNA (I)

RNA was extracted from fish at different developmental stages (3, 6, 11, 24, 48,

and 72 hpf, 5, 7, and 10 dpf) from freshly isolated brains, eyes, gills, intestine,

heart, liver, muscle, and spleen from 10 adult fish and reverse-transcribed as

described above. cDNA originating from 0.25 μg total RNA was used for the PCR

reactions, which were carried out with the same primer pairs as above for hrh1,

hrh2, and hrh3.

4.6.3 Cloning full-length hdc cDNA and synthesis of capped hdc mRNA (III)

The 5' half of zebrafish hdc cDNA was amplified with primers, forward 5’-

ATGCAGCCGCAGGAGTACAT-3’ and reverse 5’-GGCTGCCGGGTCACGACT-3’, with

the partial hdc clone (Yokogawa et al. 2007) as PCR template. The 3' half was

amplified with forward 5’-GTCCTGAGCTGCGCTATTTC-3’ and reverse 5’-

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GTTATTTAGTAGGCAGCGGTGTG-3’ primers by RT-PCR from total RNA extracted

from adult zebrafish hypothalamus. The full-length hdc cDNA was acquired by

splice overlap extension (SOE) PCR, with forward 5’-ATGCAGCCGCAGGAGTACAT-3’

and reverse 5’-GTTATTTAGTAGGCAGCGGTGTG-3’ primers. All the above-

mentioned PCR reactions were done with Phusion high-fidelity DNA polymerase

(Finnzymes), which makes blunt end PCR products. The 3’ end of the full-length

fragment acquired was modified to increase the stability of the fragment with an

adenosine-tailing procedure that adds 3´ adenosine overhangs. This modification

was accomplished with GoTaq master mix (Promega), followed by

thymine/adenine-cloning. The modified full-length hdc cDNA was cloned into a

pGEM-Teasy vector and sequenced. It was inserted into a pMC vector at the EcoRI

and SpeI restriction enzyme sites (Fink et al. 2006). Capped RNA (mRNA) was

synthesized with T7 mMESSAGE mMACHINE (Ambion) after linearization of the

cloned plasmid with a NarI and purified with Qiagen RNeasy Mini Kit (Qiagen),

after DNase I digestion for removing template DNA. The purified capped hdc RNA

(mRNA) was injected into the yolk of embryos at the 1–4 cell stage.

4.6.4 Quantitative real-time polymerase chain reaction (III, IV)

RNA was isolated from 30 pooled 5 dpf larvae using a Qiagen RNeasy® Mini Kit,

following the protocol supplied by the manufacturer (Qiagen). RNA quality and

amount were analyzed spectrophotometrically and cDNA was prepared by

SuperScript III® (Invitrogen), primers used for qPCR were the following: hcrt A

(NM001077392) forward 5’-TCTACGAGATGCTGTGCCGAG-3’ and reverse 5’-

CGTTTGCCAAGAGTGAGAATC-3’ (Kaslin et al. 2004), galanin (NM_015973) forward

5’-GACCAACTGATACTCAGGATGCA-3’ and reverse 5´-

ATCCCGAGTGTTTCTGTCAGAA-3’, and β-actin (AF057040) forward 5’-

CGAGCAGGAGATGGGAACC-3’ and reverse 5’-CAACGGAAACGCTCATTGC-3’ (Keegan

et al. 2002).

4.6.5 Touchdown RT-PCR for identification of mutation in psen1-/- (IV)

Adult WT and psen1-/- (hu2547) zebrafish were anesthetized in clove oil and

genomic DNA was isolated from finclips with buffer (50 mM KCl, 2.5 mM MgCl, 10

mM Tris-HCl, 0.45% NP40, 0.45% Tween 20, 0.01% gelatine, and 1.6 µg/ml

poteinase K). For identification of the mutation, standard touchdown RT-PCR was

performed with the following primer pairs: forward 5’-GAAACCCATTTGGGAAGTG-

3’ and reverse 5’-AAATACAAGCCTACACACAACC-3’, and forward 5’-

AGGAAACAGCTATGACCAT GAACGCAGAAGAATGAACG-3’ and reverse 5’-

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TGTAAAACGACGGCCAGT CAAGTTTGACATTTGCATGG-3’. The mutation was located

to exon 5, where it induced a premature stop codon.

4.7 Sequence comparisons and analysis of histamine receptors (I)

Multisequence alignments were performed, using ClustalW on Web pages

provided by the European Molecular Biology Laboratory-European Bioinformatics

Institute, Hixton, Cambridgeshire, UK, and the sequence analyses were carried out

with the GENSCAN Web server at Massachusetts Institute of Technology,

Cambridge, MA, USA. Sequence comparisons were carried out with ALIGN

provided by European Molecular Biology Laboratory-European Bioinformatics

Institute.

4.8 [125I]iodoaminopotentidine binding to hrh2 (I)

[125I]iodoaminopotentidine was synthesized as described (Leurs et al. 1994) and

was a kind gift from Professors Leurs and Timmerman (Vrije Universiteit).

4.9 Whole-mount in situ hybridization (III, IV)

Zebrafish MAO (AY185211) (Anichtchik et al. 2006), hcrt (NM_001077392) (Kaslin

et al. 2004), hrh1 and hrh3 (DQ647806 and DQ647807) (Peitsaro et al. 2007) were

cloned into pGEM-Teasy vector as described earlier. The partial coding region of

hdc (NM001102593) (Yokogawa et al. 2007) was cloned into pGEM-Teasy vector,

using forward 5’-GCAGCCGCAGGAGTACATGC-3’ and reverse 5’-

GGCTGCCGGGTCACGACT-3’ primers. gad1 (NM194419.1), gad2 (NM001017708.2),

slc17a6a (NM001009982.1), slc17a6b (NM001128821.1), and slc17a7

(NM001098755.1) (the last five abbreviated in the list on page 8–9) were cloned

and were kind gifts from Dr. Higashijima (Higashijima, Mandel & Fetcho 2004)

(State University of New York, Albany, NY, USA). The Notch1a (NP_571516.1) clone

was a kind gift from Dr. Brand (Center for Regenerative Therapies Dresden,

Biotechnology Center and University of Technology Dresden, Germany). Antisense

and sense digoxigenin-labeled RNA probes were synthesized using the digoxigenin

RNA labeling kit (Roche Diagnostics), following the instructions of the

manufacturer. In situ hybridization was performed as previously described (Thisse

& Thisse 2008) with some modifications as described (Chen, Priyadarshini & Panula

2009, Kudo et al. 2009).

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4.10 Immunohistochemistry (II–V)

For optimal staining for histamine IHC, it was necessary to use the bifunctional

fixative EDAC, due to the properties of the anti-histamine antibody (Auvinen &

Panula 1988, Panula et al. 1989, 1990). For histamine IHC, the samples were fixed

in 4% EDAC and 0.1% paraformaldehyde (PFA; Sigma-Aldrich) in 0.1 M phosphate

buffer, pH 7.0, overnight (o/n) at +4 °C. For other antibody staining, PFA fixation (2

or 4%) in 0.1 M phosphate buffer, pH 7.4, o/n at +4 °C was used. Brains from larval

zebrafish were dissected after fixation and processed for IHC immediately. Adult

brains that were intended for sectioning were incubated in 20% sucrose in 0.1 M

phosphate buffer, pH 7.4, o/n at +4 °C, then embedded and sectioned at 20 µm

with a Leica cryostat. The sections were stored at -20 °C until further processing

for IHC. No sucrose treatment was performed on the brains that were processed as

whole-mounts. The preabsorption controls for some of the primary antisera were

reported earlier (Panula et al. 1990, Kaslin & Panula 2001, Kaslin et al. 2004).

4.10.1 IHC with antibodies from different species on whole-mount larval brains

(II–V)

The following primary antibodies were used and incubated o/n at +4 °C: rabbit

anti-histamine 19C (Panula et al. 1990) 1:10 000, mouse anti-TH 1:1000

(LOT22941, DiaSorin Inc.), rabbit anti-serotonin 1:1000 (S5545, Sigma-Aldrich),

rabbit anti-active caspase 3 1:500 (clone C92-605, 559565, BD Biosciences), rabbit

anti-hcrt A 1:1000 (AB3704, Millipore/Chemicon), rabbit anti-β-catenin (clone

2206, LOT056K4763, Sigma-Aldrich), and rabbit anti-galanin antibody 1:1000–

1:5000 (AB1985 and AB5909, Millipore/Chemicon). The secondary antibodies were

highly cross-purified Alexa-fluorophore-conjugated goat antirabbit or antimouse

antibodies (with 488, 561, or 647 fluorophores; Molecular Probes) incubated o/n

at +4 °C.

4.10.2 IHC on whole-mount adult brains (II, IV)

IHC on whole-mount adult brains was performed as for larval brains; the only

difference was that the incubation times were prolonged (primary antibody

incubations 2–4 days). The following primary antibodies were used: rabbit anti-

histamine 19C (Panula et al. 1990) 1:10 000, mouse anti-TH 1:1000 (Diasorin Inc.),

and rat anti-serotonin 1:250 (MAB352, Millipore/Chemicon). The secondary

antibodies were highly cross-purified Alexa-fluorophore-conjugated goat

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antirabbit, antimouse and/or antirat antibodies (with 488 or 561 fluorophores;

Molecular Probes).

4.10.3 IHC with two antibodies from the same species on cryosections (II, III)

A tyramide signal amplification kit (TSA kit; PerkinElmer Life and Analytical

Sciences) was used to enable double-staining of samples with primary antibodies

from the same species. The preabsorption controls are listed in Table 2. IHC with

TSA was performed according to the manufacturer's instructions. After the TSA-

IHC, the samples were stained to obtain the desired double-staining and mounted

in 1:1 glycerol in phosphate-buffered saline.

Table 2. Preabsorption controls for the antibodies used in the cotransmitter study (more

detailed information can be found in II).

Preabsorption controls

Antigen Preabsorption

conjugate/peptide

Manufacturer,

catalog number

Dilution used

(of conjugate/peptide)

GABA GABA-sOVA Karhunen et al., 1993 20 µg/ml

Galanin Human galanin peptide BACHEM, H-8230 10 µM

Galanin Porcine galanin peptide BACHEM, H-1365 50µM

Histamine Histamine-sOVA Panula et al., 1984 2 µg/ml

mENK mENK peptide Sigma-Aldrich, E-5757/ M-

6638

100 µM

TRH TRH peptide Pennisula Laboratories, H-

4915

10 µM

The following primary antisera and antibodies were used: rabbit anti-histamine

19C 1:50 000 (Panula et al. 1990), rabbit anti-GABA 1H 1:1000 (Karhunen et al.

1993), rabbit anti-TRH #4319 1:1000 (Airaksinen et al. 1992), rabbit anti-mENK #36

1:1000 (Airaksinen et al. 1992), rabbit anti-hcrt A 1:1000 (AB3704,

Millipore/Chemicon), and rabbit anti-galanin antibodies 1:1000–1:5000 (AB1985

and AB5909, Millipore/Chemicon). The secondary antibody was a biotinylated goat

antirabbit antibody 1:750 (Vectastain ABC kit, Vector Laboratories Inc.). Avidin-

conjugated Texas Red (Vector Laboratories) 1:500 and streptavidin-conjugated

fluorescein 1:50 (Molecular Probes) and highly cross-purified Alexa-fluorophore-

conjugated goat antirabbit antibodies (with 488 or 561 fluorophores, Molecular

Probes) were used to visualize the primary staining.

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4.10.4 Enzyme histochemistry and activity assay (V)

An MAO oxidase enzyme histochemistry and activity assay was carried out, as

described (Anichtchik et al. 2006).

4.11 High-pressure liquid chromatography

4.11.1 Catecholamine and serotonin HPLC (V)

Catecholamines and their metabolites were measured from snap-frozen samples

that had been stored at -80 °C. A total of 15–25 larvae were pooled and

homogenized in 150 μl of cold solution containing 0.4 M perchloric acid, 0.1%

Na2S2O5, and 0.1% ethylenediaminetetraacetic acid. After centrifugation, an

aliquot of the supernatant was injected into the HPLC system. The HPLC system

that was used for dopamine, noradrenaline, adrenaline, 3,4-dihydroxyphenylacetic

acid, 5-HIAA and 3-methoxytyramine measurements comprised a Waters pump

model 515 (Waters Corporation), Waters Autosampler 717plus, and an ESA

Colochem 5100A electrochemical detector (ESA). The HPLC system used for

serotonin measurements included a Waters pump model 510, Waters

Autosampler 717plus and Concorde electrochemical detector (Waters

Corporation).

4.11.2 Histamine HPLC (III)

Histamine was assayed with HPLC, as described earlier (Eriksson et al. 1998).

Briefly, samples were collected by pooling 10 or 30 larvae from which the eyes and

tails were removed. These samples were frozen on dry ice and stored at -80 °C

until further processing for HPLC. The samples were then homogenized by

sonication in 2% perchloric acid, centrifuged for 30 min at 15 000 g at +4 °C, and

filtered through a 0.45 µm polyvinylidene diflouride filter (Pall Life Sciences) before

loading into the HPLC system. The protein concentration was measured by the

BCA-kit (Thermo Fisher Scientific Inc.) to normalize the results from HPLC

measurement.

4.12 Microscopy

Samples of MAO WISH were examined, using an Olympus microscope (Olympus

Corporation) connected through a charge-coupled device camera to the AnalySIS

software (Soft Imaging Software GbmH). WISH samples of hdc, hrh1, hrh3, hcrt,

gad1, gad2, slc17a6a, slc17a6b, and slc17a7 were analyzed with an inverted Leica

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DM IRB microscope. The images were acquired with a Leica DFC490 camera and

Leica Application Suite, MultiFocus option (Leica Microsystems).

The IHC samples were all analyzed with a Leica TCS SP2 AOBS confocal microscope.

The excitation wavelength of the argon laser for samples visualized with

streptavidin-fluorescein and the Alexa 488 fluorophore was 488 nm and the

emission was collected at 500–550 nm. The excitation wavelength of the diode

laser used for samples stained with the Alexa 568 fluorophore was 561 nm and the

emission was collected at 600–700 nm (narrower emission spectra when triple-

staining was used to prevent overlap). A helium/neon laser was used to excite the

Alexa 647 fluorophore at 633 nm and the emission was collected at 640–750 nm.

In all cases, sequential scanning between the frames and frame averaging were

applied to minimize cross-talk between the channels. For the 3D analysis, the

optimal step size calculated by the Leica software was applied.

4.13 Image analysis

The brains were scanned with optimized step size and averaging to allow 3D

rendering of the data in Imaris software (Bitplane AG). In general, maximum

projections of the stacks acquired were produced with Leica software for

comparative analysis. The images were further processed using ImageJ or Fiji. All

processing was applied to whole images. The 3D-rendered images were exported

as snapshots and as video files. Quantification of the histamine neuron number

was done by counting the neurons from all the stacks of images that had been

acquired and scanned with optimal settings.

For the colocalization study (II), a single sagittal section was studied from each

adult zebrafish brain. The section with the most histamine-immunoreactive

neurons was chosen. All the histamine-immunoreactive neurons were counted, as

well as the number of cells containing both histamine and the other neuronal

markers of interest. We were interested in which neurotransmitters are

colocalized with histamine in histaminergic neurons, and not the surrounding

fibers. Thus, we counted the neurons and utilized no software for assessing

colocalization, because it would have detected each pixel and not distinguish

between the fibers and the somata of the neurons. Single focal planes of the

overlay images were analyzed for colocalization with Leica software or open-

source image analysis software Fiji (http://pacific.mpi-

cbg.de/wiki/index.php/Main_Page). The maximum projections of the stacks

acquired were produced with Leica software for comparative analysis.

Methods

46

The anatomical structures were identified and/or numbered, using the atlas of the

adult zebrafish brain (Wullimann, Rupp & Reichert 1996), and the atlas based on

the location of the aminergic neurons in adult zebrafish (Kaslin & Panula 2001). All

pictures were compiled into panels in CorelDraw (Corel Corporation).

4.14 Statistical methods

For statistical analysis between two groups, the Student’s t-test or Mann-Whitney

U-test were used (GraphPad Prism Software or Microsoft Office Excel 2007). For

statistical analysis between several groups, one-way analysis of variance (ANOVA)

was applied with Tukey’s Multiple Comparison post hoc test in GraphPad. To

assess the interaction between time and treatment two-way repeated measures

(RM) ANOVA was applied to the material in GraphPad Prism. The graphs were

plotted as mean values and the variation was indicated with standard error of the

mean (SEM), unless otherwise described.

Results

47

5 Results

Please consult the original publications in the appendix for more detailed

information.

5.1 Identification and characterization of the histamine receptors in

zebrafish and the behavioral effects of their ligands (I, III)

The innervation pattern of the histaminergic neurotransmitter system was studied

previously in zebrafish (Eriksson et al. 1998, Kaslin & Panula 2001). In contrast, the

distribution of the histamine receptors has so far been unknown in zebrafish and

the specific site of action for histaminergic innervation is unknown in any species.

Thus, a detailed map of the distribution of the receptors could reveal the site of

action of the histaminergic neurons. The mRNA of the three histamine receptors

hrh1, hrh2, and hrh3, were already expressed from 3 hpf onwards in the

developing zebrafish embryo, as detected with RT-PCR. Histamine receptor mRNA

in situ hybridization revealed expression patterns in different parts of the zebrafish

brain (Figure 8). hrh1 was mainly expressed in the dorsal telencephalon, with low

levels of expression in the ventral telencephalon and diencephalon of larval fish

brain. hrh2 binding was detected with [125I]iodoaminopotentidine in adult fish

brain sections in the optic tectum, brainstem, and caudal hypothalamus, which

also harbors the histaminergic hdc-positiv, neurons. The receptor with the most

restricted neuronal expression pattern was hrh3, being located only in the dorsal

telencephalon and ventrocaudal hypothalamus of larval zebrafish. Furthermore,

we identified the histamine receptor-dense area as glutamatergic, because

markers for the glutamatergic neurons were mainly found in the dorsal

telencephalon and the GABAergic markers in the ventral telencephalon.

Figure 8. Histamine receptor expression in the zebrafish brain. Lateral view of the brain.

The hrh3 mRNA had the most restricted expression pattern, whereas hrh1 mRNA was

expressed both in the telencephalon and diencephalon. The data is a merged presentation

of the findings in I and III.

Results

48

To assess the functionality of the receptors, the zebrafish larvae were treated with

histamine receptor ligands. Treatment with different concentrations of the hrh1

antagonist pyrilamine, hrh2 antagonist cimetidine, and hrh3 inverse agonist

thioperamide and hrh3 agonist immepip resulted in altered behavior, which

suggested that the receptors were functional. Each of the histamine receptor

ligands tested significantly reduced the locomotor activity of zebrafish larvae

during a short 10 min tracking period, whereas the turn angle of the fish was not

altered. Pyrilamine at the highest dose, 100 µM, caused the strongest effect

compared with that of control animals (p ≤ 0.001). Surprisingly cimetidine

decreased the movement at all doses in comparison to that of control animals (1

µM, p ≤ 0.05, 10 µM, p ≤ 0.05 and 100 µM, p ≤ 0.001). Both thioperamide and

immepip strongly affected on movement with the highest dose (100 µM, p ≤ 0.001

and p ≤ 0.05, respectively), compared with the control animals.

5.2 Cotransmitters of the histaminergic neurons (II, III)

A quantitative comparison of the histaminergic system in the brains of adult male

and female zebrafish was done to rule out sex-related differences in the animals.

No significant difference between the sexes was found (p ≤ 0.57) and therefore

further studies were done on groups of fish in which both sexes were represented.

To assess the similarity of zebrafish histaminergic neurons with other species, the

cotransmitters of these cells were studied. We found that the pattern of the

various neurotransmitters in zebrafish was similar to that documented in rats

(Airaksinen et al. 1992). Galanin was the most commonly observed neuropeptide

that coexists with histamine, since about 30% of the histaminergic neurons

observed were positive for both markers. GABA and histamine were colocalized in

14% of the neurons and TRH coexisted with histamine in about 7% of the neurons.

mENK and hcrt were never observed to coexist with histamine.

In the whole-mount adult brain, the contacts between the histaminergic system

and the serotonergic and dopaminergic systems were assessed. Histamine neurons

surround the caudal zone of the periventricular hypothalamus (Hc), which harbors

both serotonergic and dopaminergic neurons (Kaslin & Panula 2001). Histamine-

immunoreactive fibers are scarce in the region that harbors the serotonergic and

dopaminergic neurons. We observed that the histaminergic fibers innervated only

the ventrolateral part of the Hc.

Results

49

5.3 Histaminergic system in alertness/wakefulness (III)

Histamine neurons are active during the waking period and silenced during sleep

(Lin et al. 2011). Translation inhibition of hdc, which is essential for the conversion

of the amino acid ι-histidine to histamine, with MOs resulted in significantly lower

histamine levels in zebrafish larvae at 5 dpf when morphants were compared with

control animals (75% reduction in hdc MO group, p ≤ 0.05). Furthermore, the

histamine immunoreactivity was almost completely abolished in hdc MO-injected

animals, compared with both WT controls and std MO-injected fish. At 24 hpf and

6 dpf, the hdc MO-injected fish showed no abnormal gross phenotypes. At 5 dpf,

cell death was not increased, nor was any defects observed in other related

neurotransmitter systems, such as the dopaminergic system. Inhibition of hdc

impaired locomotor activity during the light period (Figure 9). Two-way RM ANOVA

revealed a significant interaction between the WT and hdc MO groups (p ≤ 0.001,

Figure 9), whereas the std MO did not induce a significant effect. These results

indicate that the effect was specific to the hdc MO.

Figure 9. Transient inhibition of hdc induced a decline in locomotor activity during the light

phase of a 24-h period in 5–6 dpf old larvae. Findings compiled from III.

Since the activity of the larvae was altered during the light period, we

hypothesized that the decreased activity would be due to attenuated alertness

rather than a motor dysfunction. We assessed the optomotor response of the hdc

MO-injected fish and found that it was not significantly altered compared with that

of WT siblings. To assess whether the decreased activity attenuated alertness, we

subjected the zebrafish larvae to periods of lights on and off. The dark-induced

flash response (consisting of an O-bend (Burgess & Granato 2007a)) is elicited in

WT fish as a response to the onset of the dark period and lasts for less than a

second. Inhibition of hdc interfered with the response of larval fish to

environmental cues and the dark-induced flash response was therefore impaired in

hdc MO-injected fish when compared with noninjected WT control fish (Figure 10).

Results

50

This difference was confirmed by a significant time and treatment interaction (p ≤

0.05, Figure 10) when WT and hdc MO fish were compared. In line with these

observations, the same impairment of the response to environmental cues could

be detected when WT larvae were treated with the hrh1 antagonist pyrilamine (p ≤

0.001, Figure 10). The dark-induced flash response was recovered in hdc MO-

injected larvae that were coinjected with hdc mRNA (p>0.05).

Figure 10. Histamine drives the dark-induced flash response in larval zebrafish in a hrh1-

dependent manner. The data are compiled from III.

The strongest mRNA expression of hrh1 was detected in the dorsal telencephalon

of larval zebrafish and this area is highly innervated by histaminergic afferent

fibers. We hypothesized that the dorsal telencephalon would be the main area

that controls alertness-associated behavior. To further study the neuronal circuits

that underlie this behavior, we traced the connections of the dorsal telencephalon

by injecting a lipophilic dye, DiI, into this area. We found that the fibers from the

dorsal telencephalon innervated the histaminergic ventrocaudal hypothalamus.

The distribution of TH-immunoreactive neurons was used as an anatomical map to

locate the targets of the DiI-labeled fibers. No fibers were positive for both DiI and

histamine. This fact supports the idea that the target area of the dorsal

telencephalon would indeed be the ventrocaudal hypothalamus, since it was not

the histaminergic fibers per se that were labeled with DiI.

5.4 γ-Secretase regulates the development of histamine neurons (IV)

The histaminergic neurons play an important role in alertness and many

neurological disorders (Haas, Sergeeva & Selbach 2008). However, little is known

about which factors mediate the development of this system. We found that,

when γ-secretase functioning is disturbed due to a loss of function mutation in

psen1, the development of the histaminergic neurotransmitter system was altered

(Figure 11). Expression analysis of hdc mRNA at 7 dpf revealed significantly fewer

Results

51

hdc-positive neurons in psen1-/- than in WT controls (p ≤ 0.001). Similarly, at 7 and

8 dpf the histamine-immunoreactive neuron number in the psen1-/- fish was lower

than in WT fish (p ≤ 0.001 and p ≤ 0.01, respectively).

Figure 11. Loss of function mutation of psen1, which is part of the γ-secretase complex,

impaired the development of the histaminergic neurotransmitter system at the larval

stage. DAPT treatment induced the same phenotype. The γ-secretase complex consists of

PSEN1, nicastrin, APH-1, and PEN-2. The psen1 mutant zebrafish carries a premature stop

codon in the third extracellular loop. The part of psen1 that is missing in the mutant is

visualized in the image as a dotted line. Unpublished results, IV.

The same impairment of development of the histaminergic neurons was found

when larval zebrafish were treated with an inhibitor of γ-secretase, DAPT (Figure

11). DAPT treatment for 1–5 dpf negatively affected development of the

histaminergic neurons and when compared with WT and DMSO treated siblings

there were significantly fewer histamine-positive neurons in the DAPT-treated

animals (p ≤ 0.05). A shorter treatment time with DAPT from 6–8 dpf had the same

effect on development of the histaminergic system (p ≤ 0.05).

To verify that psen1-/- specifically affected the development of the histaminergic

system, we also assessed the development of the galaninergic (mRNA and protein

level) and dopaminergic (by TH-IHC) systems. There was no difference in the

development of these systems when WT and psen1-/- animals were compared at 7

dpf. Furthermore, we could not detect an increase in cell death in the psen1-/-

compared with the WT siblings.

Results

52

5.5 Neuronal plasticity and the histamine system (III, IV)

We found that in psen1-/- zebrafish the histaminergic neurotransmitter system

was temporally and spatially altered in the brain compared with WT fish (Figure

12). At the larval stages, fewer histamine-positive neurons in the psen1-/- fish

were observed than in the WT (p ≤ 0.001). At increasing age, the difference

between the WT and the mutants was reduced. In 2-month-old animals, the

histamine neuron number in the psen1-/- fish was not significantly different from

that in the WT (p > 0.05). This state was not stable and changed with time, since

the histamine neuron number was significantly (about 30%) increased in psen1-/-

fish compared with the WT controls (p ≤ 0.001) when the animals were 1 year old

(Figure 12).

Figure 12. The development of the histaminergic neurotransmitter system is temporally

altered in psen1-/- zebrafish. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Unpublished results, IV.

At the age when the psen1-/- fish had significantly lower numbers of histaminergic

neurons, the behavior of the animals was also changed. At 6 dpf the psen1-/-

larvae failed to exhibit the dark-induced flash response, a histamine-mediated

behavior. Furthermore, at 7 dpf the psen1-/- larvae swam significantly less than

the WT fish (p ≤ 0.001). The locomotor activity of adult psen1-/- fish was unaltered

compared with the WT controls. However, the mutants exhibited an increased

thigmotaxis (p ≤ 0.05), since they avoided the inner zone of the arena. The

behavioral phenotypes were associated with the changes in the histaminergic

system.

After hdc knockdown, we found that the hcrt system did not develop as in WT

siblings. Histamine regulated the development of the hcrt neurons, since hdc MO-

injections reduced hcrt mRNA, as measured both by in situ hybridization (Figure

13, p ≤ 0.05, WT and hdc MO) and qPCR (p ≤ 0.05). The hcrt neuron number was

Results

53

rescued when the embryos were coinjected with hdc MO and hdc mRNA. Injection

of 800 pg of hdc mRNA alone induced an increase in the hcrt neuron number

compared with WT controls (p ≤ 0.05).

Figure 13. Rescue of hcrt neurons in zebrafish after injection of hdc mRNA. *p ≤ 0.05, **p ≤

0.01. The data are compiled from III.

Treatment of WT larvae with the inhibitor of HDC, α-FMH, induced the same

reduction in hcrt neuron number (Figure 14 b, p ≤ 0.05) that was observed after

inhibition of hdc with MO. In situ hybridization of hcrt and hrh1 in separate

samples revealed that these two transcripts are found in the same region (Figure

14 a). This finding led us to question whether hcrt neurons could be regulated in a

hrh1-dependent manner. Indeed, the regulation of the developing hcrt

neurotransmitter system was hrh1-dependent. This was evident, because the hrh1

antagonist pyrilamine reduced the hcrt neuron number (Figure 14 c, p ≤ 0.001).

Results

54

Figure 14. Development of the hcrt neurons in zebrafish. a) Histamine receptor mRNA

expression in comparison to hcrt neurons. b) Treatment of larvae with α-FMH caused

impairment in the developing hcrt system. c) Histamine regulated hcrt neuron

development in a hrh1-dependent manner, since the hrh1 antagonist pyrilamine interfered

with the development of the hcrt system, *p ≤ 0.05, ***p ≤ 0.001. The data are compiled

from III.

5.6 Monoamine oxidase affects locomotor activity via serotonin (V)

In zebrafish, MAO is found in dopaminergic, serotonergic, and histaminergic

neurons (Sallinen et al. 2009). The role of MAO was assessed in amine-driven

behaviors, such as locomotor activity. Chronic (0–7 dpf) treatment of zebrafish

larvae with the MAO inhibitor ι-deprenyl reduced the locomotor activity of larval

zebrafish in a dose-dependent manner at 7 dpf. When the larvae were allowed to

recover for 2 days, the behavior also returned to the level of the controls. Acute (2

h) treatment just prior to assessment of behavior induced a significant decline in

movement. We also assessed the vertical place preference in a water column of ι-

deprenyl-treated larvae and found that these swam closer to the water surface

than the controls (p ≤ 0.0001). Interestingly, we found that inhibition of MAO

increased the serotonin content 100-fold, but did not affect the metabolism of

other amines (dopamine and noradrenaline) that were measured with HPLC. The

increase in serotonin was associated with a decline in locomotor activity and an

increase in ectopic serotonin-positive neurons (Figure 15).

Results

55

Figure 15. MAO drives serotonin-mediated behaviors in larval zebrafish. ι-Deprenyl inhibits

MAO and thereby the conversion of serotonin to its metabolite, 5-HIAA. This causes an

increase in serotonin concentration and decrease in locomotor activity. PCPA is an inhibitor

of TPH and by exposure of larvae to both chemicals (ι-deprenyl and TPH), PCPA inhibits the

elevation of serotonin. The data are compiled from V.

To verify whether serotonin was responsible for the phenotypes observed, we

treated larvae with drugs that interfere with serotonin synthesis (TPH inhibitor

PCPA) and uptake (selective serotonin reuptake inhibitor fluvoxamine). The

behavioral deficit was rescued by treatment of the ι-deprenyl-exposed larvae with

PCPA. HPLC analysis revealed that PCPA treatment rescued the effect that ι-

deprenyl had on serotonin increase and therefore allowed serotonin to remain at

control levels. The ectopic serotonin-positive neurons were abolished when the ι-

deprenyl-treated animals were treated with the selective serotonin reuptake

inhibitor fluvoxamine.

Discussion

56

6 Discussion

6.1 The methods – drawbacks and strengths

To date, there are several available zebrafish strains; however the interstrain

differences have not been studied extensively. In zebrafish, the differences in the

genetic backgrounds of the various WT strains do not affect spontaneous

locomotor activity (Bretaud et al. 2011). The zebrafish strain that was used for four

of the publications in this thesis has previously been used in several studies

(Eriksson et al. 1998, Kaslin & Panula 2001, Kaslin et al. 2004, Anichtchik et al.

2006, Sallinen et al. 2009, 2010, Perala et al. 2010) and the behaviors observed for

the adult and larval fish are reproducible (above-mentioned publications

compared with results in I–IV).

Genetic manipulation of gene expression can either be transient through MOs or

then stable by induction of mutations into genomes, followed by forward or

reverse genetic screening of the animals for mutations. At a glance, the MO

approach seems to be the easiest. The main strength with MOs is that they

constitute a quick and inexpensive approach to study the function of specific

genes. The main problem is that many of the MOs have short-lasting effects that

are no longer effective when the fish are 1-week-old and start to exhibit

quantifiable behaviors. Furthermore, many MOs induce unspecific off-target

effects and therefore it has been stressed that several MOs should be used to

reproduce the phenotypes of interest (Robu et al. 2007). We did this, but found

that the splice site blocking MOs did not recapitulate the phenotype observed with

initiation site blocking MOs, although the phenotype was phenocopied by other

means of intervention of histaminergic signaling (III). Therefore many different

approaches to recapitulate the same phenotype provide a good approach. The

behavioral analysis was done mainly in 1-week-old larval zebrafish. At this age, the

fish already hunt for food, since they have consumed the nutrients from the yolk

sac, and show many quantifiable behaviors. The drugs can be taken up at this late

stage (I, III), because the blood-brain barrier of the zebrafish is not fully functional

at 7 dpf and the initiation site blocking MO we used was still effective at this age

(III). The behavioral effects observed were therefore due to the MO and/or

pharmacological interventions used. In the behavioral experiments, the number of

individuals used per group in each experiment was quite high and was needed to

reduce the standard deviation and variation seen within the groups. This is one of

Discussion

57

the major drawbacks with the behavioral assay approach, but can quite easily be

circumvented since fish lay many eggs per clutch.

IHC with two primary antibodies from the same species was used in the

colocalization study (II). The drawback of this approach is that we had to use

antibodies from the same species, which could have resulted in some false-positive

detection. This approach worked, because we used the appropriate controls

required. We also ensured that there was no overlap between the light paths,

neither excitation nor emission, of the confocal microscope. While scanning the

images, care was taken to ensure that the best possible resolution was used and

also that the averaging of each channel was performed to reduce background

interference and thereby excess detection. In a few cases, deconvolution was

applied to the images, but we observed that this did not affect the final outcome

of the image, suggesting that the IHC staining procedure and confocal microscope

settings were optimal.

6.2 Neuronal circuits underlying behavior (III, V)

In this thesis, we observed that histamine and serotonin affected behavior. We

found that hdc knockdown abolished the dark-induced flash response and that

pharmacological inhibition of hrh1 resulted in the same behavioral phenotype.

These results indicated that histamine via hrh1 is an important mediator of rapid

behavioral responses that are elicited by sudden changes in the environment. In

agreement with our results, studies using hdc knockout mice have shown that in

situations when increased alertness is required, hdc knockouts fail to respond

(Ohtsu et al. 2001, Parmentier et al. 2002, Dere et al. 2004). Knockout of hrh1 in

mice results in reduced locomotor activity (Inoue et al. 1996). Contrasting results

have been reported in rats, since hrh1 antagonism with pyrilamine increases the

startle response and attenuates prepulse inhibition (Roegge et al. 2007). This

discrepancy between the genetic and pharmacological models may be due to

differences in uptake of or sensitivity properties of the hrh1 ligand. The dorsal

telencephalon showed the highest hrh1 and hrh3 mRNA expression in zebrafish.

The expression patterns of inhibitory GABAergic and excitatory glutamatergic

neuron markers revealed that the dorsal telencephalon was mainly glutamatergic.

IHC analysis showed that the dorsal telencephalon in zebrafish larvae also harbors

GABAergic neurons (Perala et al. 2010). Taken together, these data suggest that

hrh1 and hrh3 are mainly expressed in glutamatergic postsynaptic

nonhistaminergic neurons in the dorsal telencephalon in zebrafish. Previous

Discussion

58

studies with mammals showed that histamine receptors are expressed in a laminar

manner in the cortex of rodents (Pillot et al. 2002a, Haas, Sergeeva & Selbach

2008) and humans (Jin & Panula 2005, Jin, Anichtchik & Panula 2009). The dorsal

telencephalon of the zebrafish brain is highly innervated by histaminergic fibers

(Kaslin & Panula 2001). Our tract-tracing study of the projections from the dorsal

telencephalon with the lipophilic dye DiI revealed that the fibers terminated in the

histaminergic ventrocaudal hypothalamus and other areas in zebrafish. None of

the fibers were doubly positive for both DiI and histamine. Antero- and retrograde

tracing identified the limbic telencephalon as the main input area in rat

histaminergic tuberomammillary neurons (Ericson, Blomqvist & Kohler 1991). Only

a few fibers were reported to enter the core of the TMN (Ericson, Blomqvist &

Kohler 1991) in the same manner as we observed in zebrafish. Based on these

data, we propose that in zebrafish a hypothalamic-telencephalic loop exists that

mediates the alertness-associated behavior we observed as a dark-induced flash

response.

In larval fish, lack of histamine affected alertness-associated behavior, whereas

inhibition of MAO with ι-deprenyl increased mainly serotonin and reduced

locomotion. In mammals, the main substrate for MAO is dopamine, which is one of

the amines that mediate locomotion. PD is a result of degeneration of the

dopaminergic neurons in the substantia nigra. The main symptom of PD is the

motor deficit that emerges when a large proportion of the niagral dopaminergic

neurons have died. Modeling PD with neurotoxins in zebrafish causes a locomotor

deficit that can be partially rescued by ι-deprenyl treatment (Sallinen et al. 2009).

Deprenyl-mediated inhibition of MAO in rats elevated dopamine levels (Molinengo

& Ghi 1997), whereas in zebrafish we found that the main substrate for MAO was

serotonin. In humans, altered serotonin levels are associated with depression,

anxiety, and the serotonin syndrome. The serotonin syndrome is caused by

increased levels of serotonin in the brain, leading to consciousness impairment,

including coma, agitation, rigidity, and tachycardia (Radomski et al. 2000, Ables &

Nagubilli 2010). Administration of the serotonin receptor agonist into the raphe

nucleus in rats reduced spontaneous locomotor activity (Hillegaart 1990) and

injections into the periventricular nucleus of the hypothalamus increased inactivity

(Osei-Owusu et al. 2005). The effect is thought to be mediated via reduced activity

of the serotonergic projections in areas controlling motor function in the

telencephalon (Hillegaart 1990). In our experiments, inhibition of MAO with ι-

deprenyl increased the serotonin level in the brains of zebrafish larvae and was

associated with increased heart rate, reduced locomotion, and preference for

Discussion

59

remaining close to the water surface. The reduction in movement could be dose-

dependently reversed when the larvae were treated with a TPH inhibitor.

Furthermore, the increase in serotonin level measured by HPLC was associated

with expression of ectopic serotonin-immunoreactive neurons in the ventral

hypothalamus. Treatment with a selective serotonin reuptake inhibitor,

fluvoxamine, restored the neuron phenotype by abolished ectopic serotonin

immunoreactivity. Taken together, uptake of serotonin in ectopic neurons of the

ventral hypothalamus induced a behavioral alteration that could be restored by

normalized serotonin levels.

Based on tract tracing and IHC data, we found afferent and efferent projections

that provided connections between the dorsal telencephalon and the ventrocaudal

hypothalamus. This telencephalo-hypothalamic loop may be crucial for execution

of motivated behaviors. Recent studies in zebrafish have identified specific brain

areas that are involved in mediating certain behaviors. For example, filtering of

visual information in the optic tectum by GABAergic interneurons is important in

prey capture behavior (Del Bene et al. 2010). Interestingly, hrh1 (Yanai et al. 1998)

and hdc knockout (Dere et al. 2004) in mice cause increases in serotonin turnover.

These rodent data interlink the role of reduced histamine via hrh1 signaling with

an increase in the serotonin metabolite 5-HIAA. The increase in 5-HIAA

concentration was only observed in the brainstem of hrh1 knockout mice, whereas

the turnover rate of 5-HIAA/serotonin was increased in the telencephalon,

hippocampus, mesencephalon, and brainstem (Yanai et al. 1998). In hdc knockout

mice, serotonin turnover was increased in the frontal cortex, a brain area linked

with attention and alertness (Dere et al. 2004). Collectively, the results from our

study provide novel data regarding two neuronal circuits by which specific

aminergic neurotransmitter systems affect neuronal plasticity and specific

behaviors in the zebrafish.

6.3 The histaminergic system in zebrafish compared with that in

mammals (I–III)

Histaminergic neurons are important in mediating various behavioral responses.

Therefore, it was important to study which neurotransmitters colocalize with

histamine and how similar the neurons of zebrafish are in comparison with

mammalian histaminergic neurons (data summarized in Table 3). First, we

quantified the histaminergic neuron number in both female and male adult

zebrafish brain and observed no differences between the sexes. This allowed us to

use both males and females for the cotransmitter analysis without taking into

Discussion

60

account the gender difference. We found that the cotransmitters of histamine in

histaminergic neurons are galanin, GABA, and TRH. A previous study of

histaminergic neurons revealed that there are discrepancies between different

rodent species (Airaksinen et al. 1992). In rats, the cotransmitters of histaminergic

neurons are galanin, GABA, and TRH (Airaksinen et al. 1992). In mice the

neurotransmitters that coexist with histamine are galanin and GABA, whereas in

guinea pig histamine colocalizes with GABA and mENK (Airaksinen et al. 1992).

Only one study has addressed whether galanin and GABA are cotransmitters of

tuberomammillary histaminergic neurons in humans and it showed that GABA

coexists with histamine (Trottier et al. 2002). In this study on postmortem samples

from both males and females, no galanin was found in histaminergic neurons

(Trottier et al. 2002). Fibers of each of the neurotransmitter systems studied

innervated the histaminergic neurons and may affect the activity of the histamine

neurons. Previous studies in rodents have shown that galanin has an inhibitory

effect on histamine release (Arrang et al. 1991) and that sleep deprivation

increases galanin levels (Toppila et al. 1995). Furthermore, TRH excites both

histaminergic (Parmentier et al. 2009) and hcrt neurons (Gonzalez et al. 2009).

Interestingly, the stimulating effect of TRH is abolished in hdc knockout animals

(Parmentier et al. 2009), which suggests that the wake-promoting effect of TRH is

histamine-mediated. The role of mENK in the histaminergic system has not been

studied. Galanin and GABA may form the main components of the sleep-

promoting system (McGinty & Szymusiak 2003). Neurons of these two systems are

located in the ventrolateral preoptic nucleus. These innervate and exert an

inhibitory effect on the activity of histaminergic neurons (Sherin et al. 1998). It has

long been known that via this projection the GABAergic neurons regulate the

sleep-wake cycle (McGinty & Szymusiak 2003). We also assessed the anatomical

relationship between histamine, serotonin, and dopamine in whole-mount adult

zebrafish brains. In this case, we found that the serotonin and dopamine neurons

are found in the same area, which is devoid of histaminergic neurons.

Furthermore, we could detect no neurons that were positive for both histamine

and TH or histamine and serotonin. This is in accordance with what was reported

from studies on sections of the adult zebrafish brain (Kaslin & Panula 2001) and rat

(Vanhala, Yamatodani & Panula 1994).

Discussion

61

Table 3. Comparison of the cotransmitters in histaminergic neurons in several species.

References to original publications are found in the text for human, rat, guinea pig, and

mouse. Zebrafish data presented as reported in II.

Cotransmitters of histaminergic neurons

Human Rat Guinea pig Mouse Zebrafish

hcrt ? - ? ? -

Galanin + + - + +

GABA - + + + +

mENK ? - + - -

TRH ? + - - +

TH ? - ? ? -

5-HT ? - ? ? -

The histamine receptors were expressed in the zebrafish embryo from 3 hpf

onwards and the histamine receptor subtypes were found throughout the brain.

As in mammals (Haas, Sergeeva & Selbach 2008), zebrafish hrh1 and hrh3 were

mainly expressed in the dorsal telencephalon. Faint expression of hrh1 was

detected in the lateral and ventrocaudal hypothalamus and hrh3 was expressed in

histaminergic ventrocaudal hypothalamus as well. The histamine receptors in

zebrafish larvae seem to be functional, since ligands targeting the receptors had

behavioral consequences in zebrafish. The behavioral consequences observed in

zebrafish were in accordance with those reported for mice (summary in Table 4).

In hrh1 knockout mice, spontaneous locomotor and exploratory behaviors are

reduced during the active period (Inoue et al. 1996) and in stressful situations

(Yanai et al. 1998). The motor coordination of hrh1 knockout animals was reported

to be normal (Inoue et al. 1996, Yanai et al. 1998), suggesting that hrh1 may

mediate the locomotor activities associated with emotion. In line with these

observations, we observed a reduction in spontaneous locomotor activity after

treatment of zebrafish larvae with pyrilamine, suggesting that the functionality of

hrh1 was conserved between the species. hrh2 knockout mice develop normally

and are fertile (Kobayashi et al. 2000). The CNS phenotype of hrh2 knockout mice

results in reduced spontaneous movement, increased restlessness in familiar

environments, since the knockouts exhibit increased rearing behavior, and lower

susceptibility for electrically induced convulsions compared with that in WT mice

(Fukushima et al. 2003). Similarly, in our study on zebrafish larvae, the hrh2

antagonist cimetidine reduced spontaneous locomotor activity and movement at

Discussion

62

low doses. Mice devoid of hrh3 also develop normally, are fertile, and display

overall reduced locomotor activity and normal circadian rhythm (Toyota et al.

2002).

Table 4. Comparison of the rodent and zebrafish histaminergic systems. References to

original publications regarding the mouse are found in the text. Zebrafish data presented

as reported in I and III.

Inhibition either via genetic (mouse) or pharmacological (zebrafish) intervention

Mouse Zebrafish

hdc Fails to respond in situations that require

increased alertness

Fails to respond in situations that

require increased alertness

hrh1 Reduced spontaneous locomotion Reduced spontaneous movement,

impaired response to environmental

cues

hrh2 Reduced spontaneous movement, increased

restlessness in familiar environments

Reduced spontaneous movement

hrh3 Reduced spontaneous movement Reduced spontaneous movement

The activity of hrh3 affects the release of amines, neuropeptides, and other

substances such as GABA and acetylcholine (Haas, Sergeeva & Selbach 2008),

which is why the levels of amines and their metabolites were measured in hrh3

knockout animals (Toyota et al. 2002). Only the histamine level was reduced in the

cortex of hrh3 knockout mice (Toyota et al. 2002). Thioperamide, an unselective

antagonist of hrh3, has been used in pharmacological studies in rats to increase

wakefulness (Monti et al. 1991) and to increase turnover of histamine in the cortex

of rats (Yates et al. 1999). Thioperamide treatment of zebrafish larvae decreased

spontaneous movement, a result in contrast to that reported for rats. Interestingly,

immepip which is a hrh3 agonist, reduced spontaneous locomotor activity. These

results indicate that hrh3 antagonism/agonism in zebrafish must be further

studied, e.g. with receptor binding and electrophysiology, to validate the exact

properties and specificity of the hrh3 receptor. Taken together, the functionality of

the histamine receptors in the zebrafish was generally conserved, since

pharmacological inhibition of two of the receptors produced similar behaviors, as

was previously described in mice.

So far, two studies have assessed the role of histaminergic ligands in larval

zebrafish behavior (I and III) and one study assessed the involvement of

histaminergic ligands in sleep-wake regulation in zebrafish larvae (Rihel et al.

2010). These findings are in line with each other and suggest that the zebrafish can

Discussion

63

be used as a model to study the role of histaminergic neurons under different

conditions.

6.4 The potential role of histamine in neurodegenerative disease (III, IV)

In this thesis study, deletion of psen1 led to altered development of the

histaminergic system that persisted until adulthood. These findings linked

abnormalities in histaminergic system development with AD associated gene

psen1. The main symptom of AD is dementia and cognitive decline, which are due

to progressive degeneration of neuronal tissue (Bishop, Lu & Yanker 2010).

Histamine receptors are involved in memory formation and hrh3 antagonists have

been implicated in improving cognitive functions (Haas & Panula 2003, Haas,

Sergeeva & Selbach 2008). We found that in psen1-/- larvae the histaminergic

neuron number was lower than in WT siblings. This reduced histaminergic neuron

number was not associated with increased cell death or alterations in other

neurotransmitter systems, such as dopamine or galanin. Notch signaling was

earlier reported to be involved in development of the dopaminergic system in

zebrafish (Mahler, Filippi & Driever 2010). We did not, however, observe any

changes in the dopaminergic neurotransmitter system between the psen1-/- fish

and WT fish. CNS defects and abnormal phenotype are characteristic of psen1

knockout mice, which die at birth (Shen et al. 1997). We observed no major

defects in the gross phenotype of psen1-/- larval fish compared with WT fish. The

role of psen1 has been studied by MO in zebrafish and these studies have shown

that transient inhibition of psen1 and psen2 affects Notch signaling to varying

degrees (Nornes et al. 2009). Notch is a crucial regulator of neural development in

many species (Pierfelice, Alberi & Gaiano 2011) and we found here that the

development of histaminergic neurons was regulated via γ-secretase activity,

which affects Notch signaling. The reduced number of histaminergic neurons in

psen1-/- was associated with a decrease in locomotor activity and lack of robust

response to environmental cues. We furthermore observed that the histamine

neuron number was higher in psen1-/- at 1 year of age than in WT fish. This

neuronal phenotype was also associated with increased thigmotaxis, suggesting

increased alertness or anxiety. Reduced anxiety is observed in adult zebrafish

when the histamine level in the brain is reduced with α-FMH (Peitsaro et al. 2003).

The mechanism underlying the increase in histaminergic neurons may be increased

proliferation. In line with this hypothesis, mutations in psen1 increase proliferation

in mouse models of AD (Veeraraghavalu et al. 2010) and histaminergic signaling

itself potentiates proliferation and neuronal fate in vitro (Molina-Hernandez &

Discussion

64

Velasco 2008). Taken together, we found that the histaminergic system was plastic

throughout life and that this plasticity was mediated by psen1 and Notch1a. Notch

signaling was recently shown to control regeneration of motor neurons in adult

zebrafish (Dias et al. 2012). Alternatively, the plasticity observed in the

histaminergic system in this AD model could be a consequence of

neurotransmitter respecification (Spitzer 2012).

Furthermore, we found that histamine had a bidirectional effect on the

development of hcrt neurons, which is the main neurotransmitter system affected

in narcolepsy. In our studies, hdc knockdown and hrh1 antagonism resulted in

decreased hcrt neuron number in the treated animals compared with WT siblings

at 5 dpf. The deficit was rescued with coinjection of hdc MO and mRNA, whereas

hdc mRNA alone led to an increase in the number of hcrt neurons. In August 2010,

a survey was initiated due to the increased incidents of narcolepsy in Finnish and

Swedish children and the link between Pandemrix, a H1N1 influenza vaccine, and

the most severe form of narcolepsy, i.e. narcolepsy associated with cataplexy, was

established in persons under 20 years of age (Dauvilliers et al. 2010, Kallweit et al.

2011, Kornum, Faraco & Mignot 2011). Narcolepsy is thought to be an

autoimmune disease (Scammell 2003, De la Herran-Arita, Guerra-Crespo &

Drucker-Colin 2011). In the cerebrospinal fluid of narcoleptic patients, the

histamine level is reduced (Nishino et al. 2009) and in hrh1 knockout animals, the

hcrt levels are also reduced (Lin et al. 2002). Previously, it was shown that hcrt

excites histaminergic neurons (Eriksson et al. 2001), whereas the opposite has not.

Histamine and hcrt are responsible for different aspects of alertness, histamine for

motivated alertness and hcrt for locomotion-mediated alertness (Anaclet et al.

2009). Histamine is either neuroprotective (Kukko-Lukjanov et al. 2006) or

neurotoxic (Vizuete et al. 2000). The neurotoxic effect of histamine has been

documented in the dopaminergic neurons in the substantia nigra (Vizuete et al.

2000). Alternatively, histamine could exert a neurotrophic effect, since we found

that lack of histamine interferes with the development of the hcrt system. These

results may have implications for the use of antihistamines during pregnancy.

Since antihistamines are widely used in the treatment of allergy and pass through

the placenta easily from the mother to the fetus, the effect of antihistamine

medication during pregnancy on the developing child should be studied. An

intriguing finding linked AD pathology to hcrt a few years ago, when changes in the

β-amyloid concentration in brain interstitial fluid was correlated with wakefulness

in mice and infusions with hcrt increased the amount (Kang et al. 2009). Here, we

showed that histamine affects the hcrt system that underlies narcolepsy.

Discussion

65

Furthermore, we showed a spatiotemporal alteration in the histaminergic system,

which was due to impaired psen1 function, a gene associated with AD. Therefore,

combining knowledge of the interaction between the histaminergic and hcrt

systems and the effect that hcrt has on the formation of β-amyloids in a circadian

manner, could provide further information on the mechanisms underlying the

progression of neurodegenerative diseases.

Taken together, we identified for the first time a factor that affects the developing

histaminergic neurotransmitter system. These results suggest that histaminegic

neuron development is regulated by γ-secretase, and that histamine itself drives

the development of the hcrt system. These results offer a new avenue for study of

the etiology of AD and narcolepsy, respectively.

Conclusions

66

7 Conclusions

In this thesis the role of the histaminergic neurotransmitter system in neuronal

plasticity was examined (Figure 16).

• We found that the histaminergic system resembles that of other

vertebrates, since the similarity between zebrafish and rodent histamine

receptors is reasonably high. Moreover, histaminergic ligands have

behavioral effects on larval zebrafish that mimic previously documented

behaviors that have been observed in mammals.

• The phenotype of the histaminergic neurons in zebrafish resembles that of

mammals, and the zebrafish histaminergic neurons have the same

cotransmitters as the histaminergic neurons in rats.

• Behavioral experiments revealed that histamine mediated the response of

animals to environmental cues by altered alertness. We proposed that this

rapid response, the dark-induced flash response, was partially controlled

by the dorsal telencephalon. The dorsal telencephalon of zebrafish was

innervated by a dense network of histaminergic fiber projections and

expressed hrh1 and hrh3. This brain area sent afferent projections that

terminated at the histaminergic neurons. Together these formed a

telencephalic-hypothalamic loop. The hrh1 antagonist pyrilamine

abolished the dark-induced flash response.

• We found that histamine deficiency interfered negatively with the

development of the hcrt system in a hrh1-dependent manner. Another

novel finding was the identification of γ-secretase and possibly Notch

signaling as factors that regulated the development of histaminergic

neurons.

• We found that MAO, also expressed in histaminergic neurons, mainly

affects the metabolism of serotonin. We identified increased serotonin

levels and ectopic serotonin immunoreactivity in the ventral hypothalamus

as mediators of decreased locomotor activity in larval zebrafish.

Conclusions

67

In conclusion, the main findings in this thesis are the identification of new

regulatory mechanisms for histamine. Firstly, we found that histamine affects the

development of the hcrt system. Secondly, we found that the developing

histaminergic system itself is regulated in a γ-secretase-dependent manner. These

results are raising new important aspects of study in the etiology of

neurodegenerative disorders, such as narcolepsy and AD.

Figure 16. Summary of the main results presented in this thesis. Histaminergic neurons

containing histamine (HA) and several cotransmitters project from the ventrocaudal

hypothalamus to the dorsal telencephalon. In this area, hrh1 and hrh3 are highly

expressed. The neurons that project from the target area of histaminergic neurons are

mainly glutamatergic and innervate the ventrocaudal hypothalamus. In this region,

serotonergic neurons are also found and ectopic expression of serotonin as a result of

inhibition of MAO decreases locomotor activity. Inhibition of γ-secretase downregulates

the histaminergic neuron number in larval zebrafish, whereas inhibition of histamine

synthesis downregulates the number of hcrt-positive neurons in the lateral hypothalamus

in a hrh1-mediated manner and impairs the behavioral responses to environmental cues.

Future prospects

68

8 Future prospects

Tract tracing revealed a hypothalamic–telencephalic loop in zebrafish similar to

that detected in rodents (Ericson, Blomqvist & Kohler 1991). Recently, a study

reported that the Dc in zebrafish could be the mammalian cortex equivalent

(Mueller et al. 2011). Further studies in comparative neurology and hodology that

can be correlated with behavioral outputs are needed for a better understanding

of neuronal functions in the zebrafish brain. Although the MO approach was

successful in this study, knockout of hdc by TILLING, Gal4/UAS, ZFN, or TALEN

would provide favorable additional models for assessing the role of hdc and the

histaminergic system in development and disease. Furthermore, the role of MAO

in histaminergic neurons could be studied with Gal4/UAS:TeTx transgenic

zebrafish.

We showed that the dark-flash response was mediated by hrh1 and therefore hrh1

knockouts could in future experiments be studied to verify the results of the

pharmacological experiments. Interestingly, increased serotonin turnover was

detected in the brainstem of hdc (Dere et al. 2004) and hrh1 knockout mice (Yanai

et al. 1998). When extrapolated to zebrafish, this could provide a link between the

two neuronal circuits that we identified. Therefore, the serotonin turnover could

be assessed in animals treated with the hrh1 antagonist pyrilamine. Since hrh1

antagonism affected the hcrt neurons, this system should be studied in both hdc

and hrh1 knockouts. The fact that histamine affects the development of the hcrt

neurons allows studies of the role of histamine in development and etiology of

narcolepsy. As pointed out earlier, a mutation in hdc was associated with

Tourette’s syndrome (Ercan-Sencicek et al. 2010) and we found that lack of hdc

abolished the dark-induced flash response in zebrafish larvae. Taken together,

these results, the responsiveness to changes in the environment in zebrafish and

fine-tuning of the motor responses, appear to be mediated at least partially via the

histaminergic neurotransmitter system. Furthermore, these results indicate that

histamine could be needed for fine-tuning of motor functions, since Tourette’s

syndrome patients suffer from involuntary uncontrolled small muscle contractions,

i.e. tics. Therefore, it would be important to study the role of the histaminergic

neurotransmitter system in prepulse inhibition and the startle response in

zebrafish larvae. Since the startle response is driven by Mauthner neurons, the

innervation of histaminergic neuron projections of the Mauthner neurons needs to

be determined before the behavioral studies are conducted.

Future prospects

69

Since psen1 knockout affects the developing histaminergic system, the effect of

this mutation could also disturb the hcrt system. A consensus of increased

incidence of narcolepsy in AD patients is not available, since the two studies

conducted reported contrasting results (Fronczek et al. 2011, Scammell et al.

2011). The effect that psen1 knockout has on cell proliferation in rodents has not

been reported for zebrafish. Therefore, the effect of histaminergic ligands on

behavior and proliferation in psen1-/- and WT zebrafish should be determined.

Finally, we reported that inhibition of γ-secretase with DAPT reduced the

histamine neuron number. In zebrafish, DAPT mainly affects the ability of γ-

secretase to cleave Notch (Geling et al. 2002), and studying the histaminergic

neurotransmitter system in Notch mutants would give a definite answer on

whether or not the impaired development of histaminergic neurons is Notch-

mediated.

Taken together, the results acquired from the pharmacological and transient

knockdown approaches should be verified in stable knockout zebrafish models.

The novel mechanistic findings should further be validated in other model

organisms and humans, to eventually be able to draw conclusions applicable to

humans, based on these specific findings.

Thank you

70

Acknowledgements

This work has been carried out at the Neuroscience Center and Institute of

Biomedicine, Anatomy at the Faculty of Medicine, University of Helsinki. I started

my PhD project in late 2007 and had a break for five months during 2008 to obtain

a teacher’s degree at the Faculty of Pedagogy, Abo Akademi University. My study

was supported by the Academy of Finland, the Finnish Technology Development

Fund, the Sigrid Juselius Foundation, the Helsinki Biomedical Graduate Program,

the University of Helsinki Chacellor’s Funds, the University of Helsinki Funds, the

Magnus Ehrnrooth Foundation, the Swedish Cultural Foundation in Finland, the

European Histamine Research Society, the International Federation of Societies for

Histochemistry and Cytochemistry, and the International Federation of Cell

Biology.

I had the great opportunity and pleasure to be supervised by Professor Pertti

Panula. I am very grateful for your never ending support and encouragement, not

only regarding pursuing a PhD, but also for encouraging me to go through with my

studies to obtain a biology and geography teacher’s degree. Your genuine interest

and passion for science always brought my faith back into my long lasting project.

I would like to thank my thesis committee members Professor Esa Korpi and

Docent Tarja Stenberg for their supportive and constructive comments on the

progress of my thesis. I would also like to thank reviewers Assistant Professors

Sanna Lehtonen and Niclas Kolm for their excellent comments on my thesis, and

opponent Dr. Catherina Becker for accepting the invitation to travel all the way to

Helsinki to discuss my research.

A sincere thank you to all of my coauthors, Dr. Oleg Anichtchik, Dr. Yu-Chia Chen,

Dr. Jan Kaslin, Dr. Denis Khrustalyov, Dr. Hisaaki Kudo, Dr. Nina Peitsaro, Professor

Pertti Panula, Dr. Ilkka Reenilä, Stanislav Rozov, Dr. Ville Sallinen, Pauliina Toivonen

and Gabija Tolekyte. It has been nice to work together with you on our quest to

unravel “the mysteries” within neuroscience!

I would like to thank all the current and past members of the pp-group both at Abo

Akademi University (Abbi, Adrian, Bodil, Dominique, Jan, Jari K, Jin, Kaj, Kimmo,

Levente, Maria Ö, Minna-Maija, Minni, Nina, Oleg, Pirjo, Tina, Tiina-Kaisa,

Veronica) and University of Helsinki (Anna, Anu, Dennis, Gabija, Henri K, Henri P,

Hiroshi, Hisaaki, Ilmari, Jari R, Jenni, Jenny, Juha, Luisa, Maxim, Madhusmita, Maria

Thank you

71

V, Michalis, Michele, Olga, Olli, Oskar, Paula, Pauliina, Piotr, Reeta, Saara, Stanislav,

Susanna, Svetlana, Tiia, Thomas, Tuomas, Veera, Ville, Yarosalv, Yu-Chia, Yumiko)

that I had the pleasure to interact with, for introducing me to the wonderful world

of neuroscience. Thank you for the time spent inside and outside the lab. I really

enjoyed working with and getting to know all of you! I would especially like to

mention Dr. Saara Nuutinen and Dr. Jari Rossi who kindly commented on the first

versions of my thesis. Mikko Liljeström and Dr. Mika Hukkanen at the Biomedicum

Imaging Unit are thanked for their never ending patience regarding questions

about confocal microscopes and image processing. I furthermore would like to

thank all the scientists who visited the pp-group in Helsinki, especially Dr. Mario de

Bono, Dr. Harry Burgess, Professor Robert Cornell, Professor Helmut Haas, Dr.

Marnie Halpern, Dr. Petronella Kettunen, Dr. Donald O’Malley, Professor Hiroshi

Ohtsu, Professor Michalis Pavlidis, Dr. Gonzalo de Polavieja, Professor Manuel

Portavella, and Dr. Maximiliano Suster for the discussions we had regarding

science and zebrafish. Friends in and outside science – you all contributed to this

thesis. I am very grateful for all the fun together, and for having you in my life!

My deepest gratitude goes to my loved ones, to my family including all possible

extensions. Your joyousness, and endless support and faith in me keep on amazing

me. You will always be in my heart.

Maria Sundvik

Helsingfors, 2012

72

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APPENDIX Original publications

Documents

The histaminergic neurotransmitter system : its