Upregulation and changes in expression pattern of Arg ... · 2.2 Vasotocin (AVT) Gene Structure The...

Aus dem Institut für Tierzucht, Mariensee

der Bundesforschungsanstalt für Landwirtschaft (FAL)

Upregulation and changes in expression pattern of

Arg-vasotocin and galanin after water deprivation in male

chickens

INAUGURAL - DISSERTATION

Zur Erlangung des Grades einer Doktorin

der Veterinärmedizin

-Doctor medicinae veterinariae-

(Dr. med. vet.)

Vorgelegt von

Secil Cabuk

(Ankara)

Hannover 2007

Wissenschaftliche Betreuung: Prof. Dr. Dr. Nahid Parvizi

Dir & Prof. Dr. Roland Grossmann

1. Gutachter: Prof. Dr. Dr. Nahid Parvizi

2. Gutachter: Jun. Prof. Dr. Silke Rautenschlein

Tag der mündlichen Prüfung: 22.05.2007

Mit freundlicher Unterstützung der Bundesforschungsanstalt für Landwirtschaft

(FAL) und Gesellschaft der Freunde der Bundesforschungsanstalt für

Landwirtschaft

For my family

Contents

CONTENTS

1 INTRODUCTION…………………………………………………………...…….11 2 REVIEW OF LITERATURE………………………………………………..….12

2.1 Hypothalamo-neurohypophysial System (HNS)..........................................12

2.1.1 Historical Perspective of HNS…….…………………………………….…......12

2.1.2 Posterior Pituitary Hormones……………………………………………...…...12

2.2 Vasotocin (AVT) Gene Structure………………………………………….......14

2.3 Pre- and Postnatal Development of the Avian AVT System……………..…16

2.4 Functions of AVT………………………………………………………….…..…17

2.5 The Role of AVT in Body Fluid Homeostasis………………………….…......19

2.6 Chemistry of Galanin…………………………………………………………....21

2.7 The Ontogenic Development of Galanin System in the Avian Brain…........22

2.8 Distribution of Galanin-immunoreactive Structures in Diencephalon…...…23

2.9 Coexistence of Galanin ……………………………………………………......24

2.9.1 Characterization of Galanin Coexistence………..………………………...…24

2.9.2 Coexistence of Galanin with Hormones and Neurotransmitters in the

Hypothalamus …………………………………………………...…………......25

3 AIM AND OBJECTIVES……………………………….…...……………….….27

4 MATERIALS AND METHODS……………………..…………....……….……28

4.1 Animals........................................................................................................28

4.2 Experimental design and tissue preparation....……….................…….……28

4.3 Immunohistochemistry……………………………………………………….....30

4.3.1 Basic Principles of the Immunohistochemistry………………………..…. .…30

4.3.2 Procedure of the Immunohistochemistry and Laser Scanning

Confocal Microscopy………………………………………………….….…......31

Contents

4.4 Radioimmunoassay of AVT…………………………………………………..…..35

4.5 Processing and analysis of data………………………………….……...…..….36

5 RESULTS………………………………………………………………………..…37

5.1 Effects of dehydration on blood parameters…………………………...….…...37

5.2 AVT and GAL Co-localization in magnocellular neurons of the

hypothalamic nuclei supraopticus (SON)…………………………..…………..38

5.3 Lack of AVT and GAL Co-localization in PVN Neurons……….....….……….49

6 DISCUSSION…………………………………………………………………..…54

7 SUMMARY…………………………………………………………………….….59

8 ZUSAMMENFASSUNG………………………………………………...……….61 9 REFERENCES…………………………………………………………..….……63 10 APPENDIX…………………………………………………………………….….90

10.1 List of Abbreviations………………………………………………..…………...90

10.2 Tables……………………………………………………………………...……...93

10.3 Chemical Reagents……………………………………………………..…….….94

10.4 Instruments…………………………………………………………………..…....95

10.5 Composition of Solutions and Buffer……………………………………..….....96

11 ACKNOWLEDGMENT…………………………………………….……….…....98

Introduction

1 INTRODUCTION

Arginine-vasotocin (AVT) is a submammalian neuropeptide that like the

mammalian counterpart vasopressin was originally identified as the antidiuretic

hormone. Arginine-vasotocin is pruduced in the magnocellular neurons of the

hypothalamic nuclei supraopticus (SON) and paraventricularis (PVN) and released at

the axonal terminalis in the neurohypophysis, finally reaches into the bloodstream to

act on peripheral targets and regulate plasma volume and plasma osmolality, etc.

These projections form the classical hypothalamo-neurohyophysial neurosecretory

system makes the major contribution to the peptide content in the circulation. Galanin

(GAL), a 29 amino acid peptide, is distributed throughout the central nervous system

and is particularly concentrated within the basal forebrain, hypothalamus, septal

region and amygdala in the rat and monkey (MERCHENTHALER et al. 1993) and

found to be a potent modulator of neuroendocrine regulation.

Dehydration of both cellular and extracellular compartments is inducible in

terrestrial animals by water deprivation and also has been shown to be an effective

stimulus for AVT from the posterior pituitary gland of avian species. One of the main

actions of AVT is the stimulation of renal water reabsorbtion at the distal convolute

tubules and the collecting ducts in the kidney (STALLONE and BRAUN 1986a,b).

There are several reports demonstrating that either water deprivation

(MÜHLBAUER et al. 1992; CHATURVEDI et al. 2001) or the administration of

hypertonic saline (KOIKE et al., 1986; STALLONE and BRAUN 1986a,b) results in

an increase of plasma osmolality and plasma AVT levels in chicken.

Little is known about the distribution of hypothalamic AVT in birds using

modern histological methods. Arginine-vasotocin and galanin immunoreactive (-ir)

neurons were determined utilizing a novel technique of ultrastructural

immunocytochemistry. Distribution and co-localization of arginine-vasotocin and

galanin are studied during osmotic challenge in the male adult chicken. Thus, present

study was undertaken to investigate the effect of dehydration on the GAL and AVT

co-localizations and galanin expression in the chicken hypothalamo-

neurohypophysial system.

11

Review of Literature

12

2 REVIEW OF LITERATURE 2.1 Hypothalamo-Neurohypophysial (HNS) 2.1.1 Historical Perspective of HNS

It was only in 1849 that direct evidence of a role of the hypothalamus in the

endocrinology was provided by Claude Bernard when he demonstrated that injury to

the floor of the 3th ventricle, the piqure diabetique caused polyuria and glucosuria.

Subsequently, numerous studies in the late 19th century and early 20th century

confirmed that the hypothalamic-posterior pituitary system was the site of production

of a major osmoregulatory substance (GRAY and SIMON 1954).

The concept of neurosecretion was suggested in the 1940s by SCHARRER and

SCHARRER (1954) and BARGMANN and SCHARRER (1951), who demonstrated

that peptides, synthetizied by neurons in the supraoptic and paraventricular nuclei of

the hypothalamus passed through nerve fibers, was stored in nerve endings in the

posterior pituitary and subsequently was released into the general circulation. This

was the beginning of the research concerning oxytocin and vasopressin the two

nonapeptide hormones of the neurohypophysis. Progress in the understanding of

hypothalamic and pituitary endocrinology is achieved utilizing numerous techniques

which have allowed the identification of hormones, their isolation, characterization

and localization (LANDRY et al. 2003; LENG et al. 1992).

2.1.2 Posterior Pituitary Hormones

The major production sites of neurohyhpophysial hormones are the nucleus

paraventricularis (PVN) and nucleus supraopticus (SON) (Fig. 1). The paraventricular

nuclei lie adjacent to the third ventricle and ventromedial to the fornix. The supraoptic

nuclei are magnocellular nuclei of hypothalamus; they have no significant

parvicellular component and they overlie the anterior optic tract into anterolateral and

posteromedial components, united by a thin stream of cells.

Neurosecretory neurons appear to be present in all vertebrates and invertebrates. It

is established that supraoptic and paraventricular neurons conduct neural pulses like


13

other neurons, and that these neural pulses trigger increased Ca ion permeability in

the axon endings, with the Ca ion triggering hormone secretion by exocytosis

(BISSET and CHOWDREY 1988; BROWNSTEIN et al. 1980; NORMANN 1983).

Posterior lobe hormones are synthesized in the endoplasmic reticulum of the cell

bodies of the neurons. The newly synthesized peptide chains of precursor hormone

(see 2.2) are transferred to the Golgi apparatus where they are packed into secretory

granules, and the granules are transported to the axon terminals at a flow rate of 8

mm/hr which is faster than normal axoplasmic flow and are released into the capillary

spaces into the posterior pituitary (POULAIN et al. 2001).

Fig. 1: Hypothalamo-hypophysial System (a= Chiasma opticum; b= Nucleus

supraopticus; c= Preoptic area; d= Nucleus paraventricularis; e= Mediobasal

hypothalamic area; f= Tractus hypothalamo-neurohyphysis; g= The end of axons in

the perivascular area. Modified after RIJNBERK et al. 1988).


14

The neurohypophyseal hormones (Arginine vasopressin (AVP); arginine

vasotocin (AVT), lysine vasopressin (LVP); phenylpressin; oxytocin (OT); mesotocin,

valitocin, isotocin, aspartocin) are nonapeptides. AVT is the basic nonapeptide in all

vertebrates except mammals. Most adult mammals have AVP, all pigs have in

general LVP; macropod marsupials have LVP and phenylpressin, whereas phangerid

marsupials have AVP (THORNTON et al. 1986).

2.2 Vasotocin (AVT) Gene Structure

The bifunctional osmoregulatory and reproductive role of AVT is in contrast to

that of analogous mammalian arginine vasopressin, the latter being associated

primarily with osmoregulation (VERNEY 1947). The osmotic stimuli led to AVT

secretion from the axon terminals of magnocellular hypothalamic neurons in the

neurohypophysis and, thus to an increase of plasma AVT concentration (KOIKE et

al. 1979). The loci of hormone production, however, are the nuclei supraopticus and

paraventricularis of the hypothalamus (BROWNSTEIN et al. 1980; KORF et al.

1988). Results from ROUILLE et al. (1989) indicate that in contrast to mammalian

prohormone, which gives rise to three peptides after cleavage during axonal transport

(arginine vasopressin, neurophysin II also called nicotin-stimulated neurophysin and

a glycopeptide), in birds neurophysin II remains linked to the glycopeptide (IVELL et

al. 1983). Sequencing of the chicken AVT cDNA and its gene (HAMANN et al. 1992)

support this avian difference. Despite this difference in cleavage procedure the

genomic structure of the neurohypophysial peptide hormones is highly conserved

among vertebrates (NOJIRI et al. 1987). Thus the tripartite gene is divided into three

exons (A,B and C) and two introns (Fig. 2). Exons A and B in arginine-vasotocin and

arginine-vasopressin genes show a high degree of homology (LAND et al. 1982;

IVELL and RICHTER 1984; RUPPERT et al. 1984). Since the glycopeptide coding

region at the distal 3` end of exon C is lacking in the oxytocin gene (RUPPERT et

al. 1984), arginine vasopressin gene-specific probes are preferably targeted to that

sequence (LEHMANN et al. 1990). Assuming similar genetic conditions in birds, a

cloned 270 bp fragment (HAMANN et al. 1992) was chosen from the 3`end of the

chicken DNA sequence to serve as an AVT-specific gene probe.


15

Fig. 2: Schematic structure of AVT-gen and AVT-precursor and nonapeptide

structures in birds and mammalian (modified from ACHER et al. 1970; IVELL 1987)

SP: Signalpeptid; VT:Vasotocin; GP:Glycoprotein.


16

2.3 Pre- and Postnatal Development of Avian AVT System

An ontogenic series of chick embryo brains was coronally sectioned and

investigated expression of the Arginine-Vasotocin/ Mesotocin-gene (MILEWSKI et al.

1989). In that case, a 39-mer oligonucleotide recognizing the Arg-

vasotocin/Mesotocin encoding sequence of the respective mRNA`s was constructed

employing optimised codon usage and was used for in situ hybridisation (MILEWSKI

et al. 1989). Arg-vasotocin/ Mesotocin-mRNA expressing neurons were first detected

at embryonation (E) day 6 adjacent to the third ventricle (MILEWSKI et al. 1989). At

the same age, AVT can be detected in the brain extracts by radioimmunoassay

(MÜLHBAUER et al. 1993), while immunocytochemical methods gave some positive

results from E8 (TENNYSON et al. 1986). By embryonation day 9 the periventricular

nucleus had expanded in size but the hybridisation signal was weaker and also

these results suggest a migration of cells in all directions away from the third ventricle

into the diencephalon, some pericarya were even observed at the lateral pial

surface far above the optic chiasm (MILEWSKI et al. 1989). By embryonation day 12,

brain differentiation had advanced to distinct hypothalamo-neurohypophyseal nuclei

as well as to accessory groups expressing Arg-Vasotocin/ Mesotocin-mRNA. At this

age, two cell types were distinguishable in the paraventricular nucleus (PVN): the

specific mRNA was expressed in PVN as a weak hydridization signal (MILEWSKI et

al. 1989). Detectable amounts of AVT in the circulating blood appear from at least E

14 onward (MÜLHBAUER et al. 1993a). E 15 embryos respond to osmotic challenge

by increasing AVT concentration in the blood (KLEMPT et al. 1992). DONEEN and

SMITH (1982 a, b) reported that in pigs hypophysectomy by partial decapitation at

E12 leads to a decrease in the volume of the allantoic fluid and at E 16 leads to an

increase in sodium and chloride concentration. The injection of AVP has the opposite

effect on chloride content and also stimulates Ca-ATPase in the metanephros.

(DONEEN and SMITH 1982 a,b)

Remarkable changes in the electrophysiological characteristics of identified

magnocellular hypothalamic neurons are observed during the perinatal period

(GROSSMANN and ELLENDORF 1986 a, b). The extracellular single-unit activities


17

of identified magnocellular neurons in the PVN of the chicken embryo after 18 days

incubation and one hundred and four magnocellular neurons were identified by

antidromic stimulation from the neural lobe (GROSSMANN and ELLENDORF

1986a).

In the posthatched development of the avian AVT system, the main changes

seem to be connected with sexual maturation, since AVT not only participates in the

regulation of water balance as vasopressin does, but also plays a role in contraction

of oviduct smooth muscles, similar to the effect of mammalian oxytocin (NIEZGODA

et al. 1973; SAITO et al. 1987).

Environmental factors play an important role during the ontogeny of the

endocrine systems, especially those participating in adaptation. The influence of low

humidity during incubation (LHI) on arginine vasotocin secretion in male and female

White- Leghorn chicken embryos and chicks also was examined and the results

showed that LHI is more effective in males than females (KISLUIK and

GROSSMANN 1994). Suboptimal conditions during incubation, e g., a low relative

humidity, modulate the activity of AVT system during late embryonal and early

posthatched development (KISLUIK and GROSSMANN 1994).

2.4 Functions of AVT

In birds, AVT is involved in the regulation of water and electrolyte balance

(SIMON-OPPERMANN et al. 1988; RAMIERI and PANZICA 1989), in the regulation

of body temperature (HASSINEN et al. 1994), in the regulation of blood pressure

(SZCZEPANSKA-SADOWSKA et al. 1985; SHIMADA et al. 1986; KOIKE et al.

1988), in some cognative adaptative activities (DAVIS et al. 1982; DAVIS and PICO

1984) and in the activation of some aspects of reproductive behaviour

(copulatory behaviour: KILHSTRÖM and DANNIGE 1972; singing behaviour:

VOORHUIS et al. 1991; sexual motivation: BERNROIDER and LEUTGEB 1994).

Numerous reports indicate that AVT is also involved in oviposition in laying hens and

has an oxytocic activity (RZASA 1970; SAITO and KOIKE 1992) and the injection of

AVT can induce premature oviposition in laying hens (RZASA and EWY 1970).

In addition, plasma levels of AVT increase at the time of oviposition (ARAD and


18

SKADHAUGE 1984; NOUWEN et al. 1984; TANAKA et al. 1984; SHIMADA et al.

1986; SAITO et al. 1987).

Among all investigated avian species, AVT immunoreactive (-ir ) cell bodies in

extrahypothalamic areas were observed only in the brain of songbirds, canary and

zebra finch. In both species clustered AVT-ir perikarya were located in the Bed

Nucleus of Stria Terminalis (BnST: JURKEVICH et al. 1999; KISS et al. 1987;

VOORHUIS et al. 1991; VOORHUIS and DE KLOET 1992) on the border between

the lateral septal (SL) nucleus and the dorsal diencephalon. This cell group and AVT-

ir parvocellular neurons in the dorsal part of the diencephalic paraventricular region

exhibit a clear sexual dimorphism in the canary brain. No signs of immunoreactivity

were observed within corresponding regions of the female brain. Also osmotic stress

induced by water deprivation for 48h had no influence on the number of

immunoreactive or AVT mRNA containing parvocellular cell bodies in the Bed

Nucleus of Stria Terminalis (JURKEVICH et al. 1997).

Osmotic and hemodynamic stress are the two primary regulators of

vasopressin or arg-vasotocin release from the posterior pituitary. The pathways

providing information about plasma osmolality and blood pressure or blood volume

are distinct and utilize different receptors (BURAZIN et al.2001) and

neurotransmitters. There are two different vasopressin receptors (V1- and V2-

receptors: FAHRENHOLZ 1984). V2-receptors mediate an antidiuretic effect and V1-

receptors mediate an vasocontractional effect on blood vessels. Osmotic regulation

of vasopressin or arg-vasotocin release is depending upon afferents from the lamina

terminalis region (CIOSEK 2000). Furthermore, hemodynamic information is

transmitted to the vasopressin neurons via multisynaptic pathways from the

brainstem with A1 catecholamine neurons of the ventrolateral medulla providing to

the final link for the information on decreases in blood pressure and volume. Several

neurotransmitters and neuropeptides are expressed in the A1 neurons including

norepinephrine, glutamate, neuropeptide Y and substance (SLADEK 2004).


19

2.5 The Role of AVT in Body Fluid Homeostasis

The body-intrinsic, extracellular fluid represents the compartment responsible

for the controlled exchange between the intracellular milieu and the external

environment in birds as all other classes of vertebrates. With regard to ionic

composition, sodium (Na) and chloride (Cl) contribute to the osmotic pressure of the

extracellular fluid. Maintenance of the sodium concentration (tonicity) and volume of

the extracellular fluid under varying external conditions are necessary prerequisites

for cellular and integrative functions, and a homeostatic circuit controls these

extracellular fluid parameters. Minute alterations in either extracellular fluid tonicity or

volume are perceived by systemic osmo- and volume receptive elements as well as

central nervous osmo- and sodium sensors which are well characterized in mammals

and birds. All these signals are finally conveyed to the hypothalamus of the

diencephalon, where complex integration takes place in mammals (ANDERSSON

1978; BIE 1980) and birds (SIMON 1982; GERSTBERGER et al.1994). Besides a

centrally mediated alteration in the drive to drink water, peripheral osmoregulatory

target organs such as the kidneys (AMER and BROWN 1995), the cardiovascular

system or supraorbital salt secreting glands in the case of marine birds are

subsequently activated or inhibited via the autonomic nervous system and (neuro-)

hormones to re-establish extracellular fluid tonicity or volume in avian species.

Altered release of the arginine-vasotocin (AVT) represents together with

arginine vasopressin as antidiuretic principle one of the major routes to readjust

extracellular fluid tonicity or volume (DANTZLER 1980, 1989).

Released from the axons of the magnocellular neurons terminating within the

neurohypophysis, AVT circulates as free hormone in the blood at concentrations of

5-30 pg/ml in various birds (GRAY and SIMON 1983; STALLONE and BRAUN

1986a,b; GRAY and ERASMUS 1988) with a short biological half-life of 1-3 minutes

(it has two component dissapearance curve with a fast t ½ which is less than 1 min

and a second component of 2-3 min). Being the major osmoregulatory hormone in

birds besides angiotensin II and the atrial natriuretic factor (WILSON 1984; SCHUTZ

and GERSTBERGER 1992; SIMON et al. 1992), AVT proved to be mainly under

osmotic rather than volume control in avian species. Thus, increasing plasma


20

osmolality due to altered sodium concentration during water deprivation, led to a

linearly correlated rise in plasma AVT with a sensitivity of 0.3-0.4 pg/ml per mOsm/kg

in the duck (GRAY and SIMON 1983) and even 1.3 pg/ml per mOsm/kg in the

dehydrated chicken (STALLONE and BRAUN 1986a,b). Hypo- or hypervolemia

resulted in a modest alteration of the slope relating extracellular fluid tonicity and

plasma AVT. Using molecular techniques, MÜHLBAUER et al. (1992) reported

increase expression of the AVT gene in the chicken hypothalamus already 19h after

onset of water withdrawal, with plasma osmolality still being near-basal values.

Surprisingly, in chicken challenged by hypertonic saline drinking for 5 days, a

dissociation between plasma osmolality and AVT plasma concentration was visible:

extremely high plasma osmolalities were accompanied by only moderate plasma

AVT concentrations (MÜHLBAUER et al. 1992). In addition, SAITO and

GROSSMANN (1998) reported that plasma osmolality did not change during the first

4h of water deprivation, but had increased significantly at 6h and 8h. Plasma sodium

levels, did not change but plasma potassium concentrations gradually decreased

during dehydration. Plasma AVT levels and hypothalamic AVT mRNA levels

increased significantly after 8h.

Systematically administrated AVT reduced renal water elimination in the pekin

duck, Kelp gull and the domestic fowl (BRAUN 1991; GERSTBERGER et al. 1985;

STALLONE and BRAUN 1985; GRAY and ERASMUS 1988). Tracer dilution

techniques indicating both glomerular and tubular actions of neurohormone revealed

that AVT (BRAUN and DANTZLER 1974; GERSTBERGER et al. 1985; GRAY and

ERASMUS 1988) applied to conscious White Leghorn under conditions of steady-

state diuresis driven by systemic infusion of hypotonic NaCl. AVT infusion markedly

reduced urine flow rate and slightly enhanced natriuresis, quite different from

experiments in the duck and gull, where antidiuresis was accompanied by

pronounced antinatriuresis (BRUMMERMANN and BRAUN 1995).

Interestingly in White Leghorn chicken with diabetes insipitus, AVT application

induced a strongly stimulated natriuresis, at least partially due to augmented

glomerular filtration rate, at only slightly changed urine flow rate (BANKIR et al. 1989;

BOYD and MOORE 1990; BRUMMERMANN and BRAUN 1995). On the other

hand, in the freshwater-acclimated duck, the antidiuretic activity of


21

exogenously applied AVT in physiological concentrations showed a clear

dependence on the sodium status of the extracellular fluid.

Furthermore, the dehydration-induced changes in plasma osmolality and in

electrolyte concentrations shown in the study of SKADHAUGE (1981) were closely

associated with increased plasma AVT and prolactin levels. The significant

correlation between plasma AVT and prolactin and between both hormones and Na

and Cl and osmolality, indicate an osmoregulatory trigger for AVT and prolactin

secretion. Water deprivation has been shown to increase plasma prolactin

concentration in the fowl (HARVEY et al. 1979).

2.6 Chemistry of Galanin

Galanin, a 29 amino acid peptide, was first isolated from porcine small

intestine and named galanin because of its amino-terminal glycine and amidated

carboxyl-terminal alanine residues (TATEMOTO et al. 1983). The amino acid

sequence of GAL is known for 6 group of vertebrates: porcine (TATEMOTO et al.

1983), bovine (RÖKAEUS and CARLQUIST 1988), rodent (VRONTAKIS et al. 1987;

KAPLAN et al. 1988), ovine (SILLARD et al. 1991), chicken (NORBERG et al. 1991)

and human (EVANS and SHINE 1991; SCHMIDT et al. 1991). The sequence of cow,

rat and human GAL were deduced from the cDNA sequences. Whereas the pig,

sheep and chicken GAL were originally purified from small intestine and from brain

and sequenced directly. The cDNAs encoding porcine, rat, bovine and human GAL

have been cloned (RÖKEAUS and BROWNSTEIN 1986; VRONTAKIS et al. 1987;

KAPLAN et al. 1988; RÖKAEUS and CARLQUIST 1988; EVANS and SHINE

1991). The deduced amino acid sequence indicates that GAL is derived, through

proteolytic processing and, in most species carboxyl-terminal amidation, from a larger

precursor protein known as prepro-GAL. Southern and Northern blot analyses in

several tissues reveal that GAL is encoded by a single-copy gene and that its mRNA

migrates as a single band of ~900 nucleotides (including a poly-A tail). Prepro-GAL

mRNA encodes a 123-124 amino acid precursor protein and the amino terminus of

prepro-GAL includes a hydrophobic sequence of approximately 20 amino acids,

characteristic of a single peptide for transport of the prohormone into the

endoplasmic reticulum (KAPLAN et al. 1988).


22

Northern blot analysis revealed that avian galanin mRNA was expressed, as a

shorter transcript, in the quail brain, ovary, and intestine, unlike the liver and oviduct.

A larger mRNA of avian galanin may be further expressed only in the ovary

(KOHCHI and TSUTSUI 2000).

The GAL sequence consists of amino acids 33-62 in the precursor and is

flanked by two pairs of basic acids (Lys-Arg) which are cleavage sites. Whereas the

sequence of the amino-terminus of GAL is highly conserved, the carboxy-terminal

region exhibits substantial species variability which might be responsible for the

species specificity of GAL effects on several endocrine system (GILBEY et al. 1989;

MIRALLES et al. 1990). The high conserved aminoterminus might be responsible for

the receptor interaction. The fragment 1-16 of GAL has full agonistic properties in

inhibiting muscarinic agonist-mediated stimulation of phosphatidyl inositol turnover in

slices of rat ventral hippocampus (FISONE et al. 1989).

Presence and the localization of galanin and its binding sites in the quail brain

were examined (AZUMAYA and TSUTSUI 1996; LISMAA and SHINE 1999). The

number of galanin-binding sites when compared on the basis of unit weight was

maximal in the interbrain including the preoptic and hypothalamic regions and

minimal in the cerebellum (AZUMAYA and TSUTSUI 1996; GUNDLACH et al.

2001). The binding of [125I] avian galanin was also evident in oviduct in Japanese

quail and the galanin binding was specifically inhibited as a function of the

concentrations of both avian and rat galanins (TSUTSUI et al. 1997).

2.7 The Ontogenic Development of Galanin System in Avian Brain

The ontogenic development and differention of the galanin-immunoreactive

(GAL-ir) neuronal system in the avian brain was studied by immunocytochemistry

and the first traces of GAL-ir can be detected already on day 2 of embryonic life

within the neural tube. And the system appears to be fully developed around day 16

(JOZSA and MESS 1994). GAL-ir neuronal system develops parallel in the

hypothalamic and in extrahypothalamic sites and the first-defined groups of GAL-ir

perikarya are visible within the diencephalon and medulla oblangata as early as day


23

6. In the embryonic hypothalamus, the periventricular and the tuberal area are

especially rich in GAL-ir neurons. Since similarities have been observed in

developmental patterns of the GAL-ir system and the hypothalamic hormonal

systems, it has been assumed that the galaninergic system has a functional

relationship with these neural structures during development (JOZSA and MESS

1994).

2.8 Distribution of Galanin-immunoreactive Structures in the Diencephalon

The anatomical distribution of GAL containing neurons has been studied in the

central nervous system of the rat (MERCHENTALER et al. 1993) and major

populations of immunostained perikarya were detected in several brain areas. In the

rat, a prominent cluster of cells is present in the ventrolateral portion extends from the

rostral margin of the optic chiasm to the supraoptic nucleus and another group of

cells is present in the preoptic suprachiasmatic nucleus. A third group of

immunoreactive cells is seen in the periventricular preoptic nucleus, the fourth group

occupies the lateral aspects of the medial preoptic area extending into the

medial forebrain bundle and the anterior hypothalamic nucleus (MERCHENTHALER

et al. 1993). In the same species, large numbers of GAL-ir perikarya occupy the

dorsal aspects of the anterior hypothalamus, starting at the level of the anterior

commissure and continuing to the level of SON and the both magno- and

parvicellular subdivisions of the PVN (SWANSON and KUYPERS 1980) contain

immunoreactive cells. Galanin in these perikarya is co-localized with a large number

of other neuropeptides and neurotransmitters.

Some of the immunoreactive cells in the parvocelllular subdivision project to

the median eminence (MERCHENTHALER 1991), to the brainstem and to the spinal

cord. Some of the galaninergic neurons in the arcuate nucleus innervate other

galaninergic cells, thereby providing the anatomical foundations for an ultrashort

feedback mechanism, by which GAL can regulate its own secretion into the portal

circulation (LOPEZ et al. 1992).


24

Some of the immunoreactive cells in the magnocellular subdivisions project

to the posterior lobe of the pituitary and to the brainstem (SWANSON and

KUYPERS 1980; MEISTER et al. 1990b; VILLAR et al.1990). The supraoptic

nucleus, including the retrochiasmatic portion, contains large intensely-stained cells.

Cluster of closely packed GAL-ir perikarya are seen in the accessory magnocellular

nuclei and the largest group of immunoreactive perikarya is located in the

dorsomedial nucleus (MERCHENTHALER et al. 1993). The majority of these GAL-ir

cells projects to the external zone of the ME (NIIMI et al. 1990; MERCHENTHALER

1991) and the fibers in the external zone contact the capillaries of the portal system.

Thereafter, GAL is released into the portal circulation through which it reaches the

anterior pituitary and alters the secretion of several anterior pituitary hormones. The

immunoreactive fibers in the internal zone project to the posterior pituitary where GAL

is released into the general circulation. In quail brain galanin-like immunoreactivity

was found in several restricted regions throughout the brain, the most intense

immunoreaction was present in the diencephalic region (AZUMAYA and TSUTSUI

1996; JOZSA and MESS 1993), the ventral hypothalamus, the median eminence,

the central gray of the brain stem, and the dorsomedial caudal medulla (JOZSA and

MESS 1993).

2.9 Coexistence of Galanin 2.9.1 Characterization of Coexistence

The studies on coexistence has identified one or more neuropeptides within

the same neuron usually containing one of the (classic) neurotransmitters, GAL

within cholinergic neurons in the Diagonal band of Broca (MELANDER et al. 1986).

For example, the coexistence of AVP and corticotropin-releasing hormone in the

parvicellular subdivisions of the PVN can be seen in adrenalectomized rats (TRAMU

et al. 1983; KISS et al. 1984; SAWCHENKO et al. 1984).


25

In general, neuropeptides may interact with classical neurotransmitters and

probably with other neuropeptides at a variety of sites and in many ways.

Neuropeptides could affect the synthesis, storage, release, receptor binding and/ or

degradation of the coexisting substance in the presynaptic cell. In the postsynaptic

cell, among other mechanisms, the coexisting partner could alter binding to the

receptor or the second messenger cascade triggered by the other substance; for

example Calcitonin gene-related peptide can inhibit the substance P endopeptidase

(LE GREVES et al. 1985) and thus prevent the breakdown of substance P,

prolonging its half-life.

2.9.2 Coexistence of Galanin with Hormones and Neurotransmitters in the

Hypothalamus

In rats, the parvicellular PVN contain a small number of GAL-ir neurons

corticotropin-releasing hormone or neurotesin and an even smaller group of GAL-ir

perikarya contains both of these peptides (CECCATELLI et al. 1989). Within the

magnocellular PVN, GAL coexist with AVP (RÖKAEUS et al. 1988; GAYMANN and

MARTIN 1989; GAI et al. 1990; MEISTER et al. 1990b; VILLAR et al. 1990), oxytocin

(GAYMANN and MARTIN 1989; MEISTER et al. 1990b; LANDRY et al. 1991),

dopamine, dynorphin, cholecystokinin, enkephalin, corticotropin-releasin hormone,

peptide histidine isoleucine, and thyrotropin releasing hormone (MEISTER et al.

1990b). The degree of colocalization is high with AVP, dynorphin, and

cholecystokinin; moderate with enkephalin; and low with oxytocin, cortocotropin-

releasing hormone, peptide histidine isoleucine and thyrotropin releasing hormone

(MEISTER et al. 1990b).

In general the detection of GAL and most of other neuropeptides, with the

exception of AVP and oxytocin requires the use of salt loading or hypophysectomy

(BJÖRKSTRAND et al. 1993). The large degree of coexistence in the magnocellular

and, to a lesser extent, in the parvicellular, neurosecretory system indicates that

these neurons are able to release a cocktail of neuropeptides and neurotransmitters

into the general or portal circulation. The precise functional signification of the

coexistence of the above mentioned substances with GAL remains to be clarified.


26

The majority of GAL-ir nerve terminals is derived from neurons in the arcuate

nucleus and only a third of these terminals originates in the PVN and medial preoptic

area (MERCHENTHALER 1991).

Co-existence of neurotransmitters and neuropeptides may be sexually

dimorphic; the degree of colocalization of luteinizing hormone-releasing hormone and

GAL is four-to five-fold higher in the female than in the male rat

(MERCHENTHALER et al. 1991). Only a small population of luteinizing hormone-

releasing cells in the diagonal band of broca and medial preoptic area of the male

rats is lightly immunopositive cells for GAL (COEN et al. 1990; MERCHENTHALER

et al. 1990). Although large number of studies has investigated the patterns of

coexistence, with few exceptions the physiological meaning of this phenomenon

remains unclear.

Aims and Objectives

3 AIMS AND OBJECTIVES

As the literature review indicates, the studies reported in this dissertation were

undertaken to further elucidate hypothalamic distribution of arginine vasotocin and

galanin and their co-localization after water deprivation. Besides the control of water

balance, AVT is strongly involved in the mechanisms leading to egg laying in female

birds. Thus, to avoid any influence with egg laying only male chickens were used. To

this end we evaluated the upregulation in the expression pattern and signal

intensities of AVT and galanin in hypothalamic SON and PVN neurons. Following

questions were addressed using the male chicken as the experimental model.

- Are GAL and AVT co-localizations in hypothalamus affected in response to an

osmotic challenge? And

- Is galanin in hypothalamus affected by dehydration in the male chicken brain?

27

Materials and Methods

28

4 MATERIALS AND METHODS 4.1 Animals

For all experiments male chickens of Lohman-Selected Leghorn (LSL; Gallus

gallus domesticus) were obtained from a commercial breeder (Lohman Tierzucht

GmbH, Cuxhaven) at the age of approximately 18 weeks. This breed is fully grown at

age of 28-36 weeks. Adult male chickens were housed in individual cages under

conditions of 12 hours of light and 12 hours of darkness. They received standard

layer diet with food and tap water provided for ad libitum consumption. Animal

maintenance and the experiments were conducted in accordance with the relevant

laws and regulations that govern the treatment of experimental animals (AZ: 5096-

4250213). All studies were performed at the Institut für Tierzucht Mariensee der

Bundesforschungsanstalt für Landwirtschaft.

4.2 Experimental design and tissue preparation

A total of 16 adult male chicken were used at the age of 22-35 weeks. One

group of cockerels (n=8) was deprived from water for 48h beginning at 9 a.m. At the

end of the dehydration period one blood sample (1ml) from each animal (8

normohydrated and 8 dehydrated) was withdrawn from the wing vein (vena ulnaris)

into a heparinized tube. Plasma was separated by centrifugation (3.000 x g, 15 min,

4°C) for measurement of AVT, plasma osmolality and plasma sodium and potassium

concentration. Plasma osmolality was determined by vapor pressure osmometry

(Wescor 5500, Wescor Inc., Logan, USA), plasma levels of sodium and potassium

were measured in blood using blood gas analysing system (Rapid lab 865, Bayer

Healthcare, Fernwald, Germany). Immediately after the withdrawal of the blood

sample, chickens were euthanasied under deep anesthesia with Narkoren

(Merial, 85399 Hallbergmoos, 16% Pentobarbital-Natrium in PBS solution; 40-80 mg/

kg body weight iv) and perfused. Perfusion was performed via the carotid arteries

with 150 ml 0.1 M phosphate buffered saline (PBS, pH 7.4) containing 0.2% heparin

followed by 360ml Zamboni`s fixative (4% paraformaldehyd and 150 ml saturated

picric acid in 0.1 M PBS, pH 7.4). The brains were removed, postfixed for 6-8 hours

in the same fixative and placed in 25% sucrose solution in 0.1 M PBS for


29

cryoprotection. They were stored at –80°C until cut into transverse serial sections of

40µm using a cryostat (Reichert-Jung, 2800; Frigocut-E). Fifty sections from each

brain, corresponding to the levels of A 8.2- A 7.6 of the chicken brain atlas (Fig.3)

(KUENZEL and MASSON 1988) were collected in 0.02 M PBS and processed for

immunohistochemistry.

Fig. 3: A section from brain chicken atlas (modified from KUENZEL and MASSON

1988). A: Archistriatum, AA: Archistriatum Anterior, BnST: Bed Nucleus of Stria

Terminalis, E: Ectostriatum, FA: Tractus Fronto-Archistriticus, FDP: Fasciculus

Prosencephali Lateralis, INP: Nucleus Intrapedindiculuslaris, LMD: Lamina Medullaris

Dorsalis, LSO: Organum Septi Laterale,, LH: Lamina Hyperstriatica, LT: Lamina

Terminalis, MPO (d,m,v): Nucleus magnocellularis preopticus, pars dorsalis,

medilais, ventralis, OVLT: Organum Vasculosum Lamina Terminalis, POM: Nucleus

Preopticus Medialis, POP: Nucleus Preopticus Periventricularis, PP: Paleostrium

Primitivum, PV: Nucleus Posterioventralis Talami, QF: Tractus Quntofrontalis, SP:

Nucleus Septalis Lateralis, SO(e,v): Nucleus supraoptic, pars externus, ventralis,

TSM: Tractus septomesencephalicus, VIII: Ventriculus Tertius, VL: Ventriculus

Lateralis.


30

4.3 Immunohistochemistry

4.3.1 Basic principles of the immunohistochemistry

Immunohistochemistry utilizes antibodies to localize specific peptides in tissue

sections. In this method, the tissue section is incubated with a labelled antibody, the

section is then washed and the site of antigen-antibody reaction is identified by

visualizing the label. Aside from its power as a tool in research,

immunohistochemistry has found an increasing attention in diagnostic histopathology

(OBERMANN et al. 1996).

In general, there are two methods of using immunohistochemistry: a) direct

method of detecting a protein in a tissue section utilizing a labelled antibody and b)

the indirect method in which the primary antibody is left unlabelled and then the label

is attached to a different reagent or second antibody which is then used to detect the

primary antibody. Figure 4 illustrates the basic procedure of direct and indirect

methods. In the direct method the first antibody is linked to the trace, in the indirect

method the second labelled antibody is used as tracer.

Fig. 4: The direct and indirect methods of visualising the reaction site of an antibody

on a tissue section. 1, primary antibody; 2, labelled second antibody.


31

In the present study the indirect method with use of a fluorescent label was

utilized for laser scanning confocal microscopy. A fluorescent label should have a

high quantum yield, good separation between the wavelengths of excitation and

emission, a wavelength of maximal absorption close to a strong line from mercury arc

lamp and an emission wavelength suitable for photographic film and the human eye

(HELL and STELZER 1992). The advantage of using a fluorescent label is its speed.

Once the slide has been incubated with labelled antibody, the coverslip can be

mounted and at the end the results can be visualized.

4.3.2 Procedure of the Immunohistochemistry and Laser Scanning

Confocal Microscopy

Immunohistochemical procedures were performed essentially as described by

JURKEVICH et al. (1999). Briefly, sections were washed six times in 0.02 M PBS at

room temperature. To reduce the nonspecific binding sites sections were incubated

for 30 min in 0.02 M PBS containing 10% normal goat serum (Dako, Hamburg) and

0.2 % Triton X-100. Thereafter the sections were incubated at 4°C with rabbit

polyclonal antibody against Arg-vasotocin diluted 1:30000 and guinea pig antibody

against galanin (Peninsula laboratories, California) diluted 1:600 in 0.02 M PBS with

1% normal goat serum, 0.2% Triton X-100 and 0.1% sodium azide for 22 hours at

4°C. The sections were then rinsed in 0,02 M PBS and incubated with goat anti-

rabbit IgG coupled with ALEXA 555 (Vector laboratories, Burlingame, TI-1000)

diluted 1:400 and goat anti-guinea pig IgG coupled with fluorescein (FITC, Vector, FI-

7000) diluted 1:400 in 0,02 M PBS with 0.2% Triton X-100, 1% normal goat serum

and 0.1% sodium azide in the dark at room temperature for 90 min. After washing,

the sections were rinsed again in 0.02 M PBS with 0.2% Triton X-100 and mounted

on gelatine coated slides with vectashield mounting medium (Vector, H-1000) and

then sealed with coverslips using colorless nail polish. The slides were stored at 4°C

and protected from the light until confocal imaging microscopy was performed as

multitracking of FITC (galanin) and ALEXA 555 (AVT). Argon laser (488 nm) exited

for FITC and emission was seleted with bandpass of 505 to 520 nm. Helium/ Neon

green laser (543nm) exited for Alexa 555 and emission was selected


32

with long pass of 585 nm. Optical slices (of 3 to 4 consecutive 40 µm sections from

each brain) were focused in Z direction to 2 µm using a 20x objective with 512 pixels

and to 1 µm in Z direction using a 40x objective. Number of cells expressing one or

both of peptides were counted on 3D projection of 1 µm optical slices in total stacks

of 30 µm thick central sections of 325 square µm.

The obtained digital data were transferred to the photoshop 6.0 software

(Adobe San Jose, CA) for the preparation of the images for the presentation.

The AVT antibody was kindly provided by Dr. D. Gray (Max Planck Institute for

Physiological and Clinical Research, Bad Nauheim, Germany). This antiserum and

galanin antiserum used in this experiment have been successfully used in other

chicken brain immunohistochemical studies conducted at the same time or previously

in the Department of Functional Genomics and Bioregulation (BARTH et al. 1997;

JURKEVICH et al. 1997; KLEIN et al. 2006). All necessary control incubations such

as preincubation of galanin antiserum (produced against full-length galanin) with

porcine galanin, change of the order of the incubation with primary antibodies

(KLEIN et al. 2006) were conducted. The primary antibodies were omitted in some of

the incubations to control the background. The galanin antibody used in this

experiment has been shown to have no crossreactivity to vasoactive intestinal

peptide, secretin and human insulin.

The confocal laser scanning microscopy is routinely performed in the

Department of Functional Genomics and Bioregulation. The instrument used in this

study is LSM 510 (Carl Zeiss, Göttingen) and scanning is performed by servo-

controlled galvanometers and the scanning time is 2 sec/ field of 512X512 pixels and

a microprocessor controls the scanning process. After digitization, the signal is led to

a frame memory the read-out of which is in video frequency. Consequently the image

on the monitor is stationary provided (HELL 1996). A continuously variable

magnification can be obtained by a zoom unit, the laser scan microscope was used

with a conventional as well as with laser light source (HELL and STELZER 1992).

Both incident illumination (for reflectance and fluorescence) and transmitted

illumination for absorbance, phase- and differential interference contrast) was

obtained with laser source. To provide better focusing and searching possibilities in


33

the preparation and to allow investigation of double-stained probes, conventional epi-

illumination device has been added to the laser scanner. An example (HELL 1997) of

the separation of blue (460 nm) from green (530 nm) light is given in figure 5.

Fig. 5: Excitation and emission spectra of fluorescein isothiocyanate (FITC).

Wavelength of excitation; solid line and emission; dotted line (modified from

SCHRADER and HELL 1996).

Similar chromatic beam splitters (CBS) as a dichroic mirrors exist for

separation of other regions of the light spectrum, from the UV (300nm) to the red

(700nm). Figure 6 demonstrates an example of an absorption and an emission

spectrum. At a given wavelength, the most intense fluorescence can be obtained

when the specimen is irradiated with a wavelength close to the peak of the excitation

of the wave (SCHRADER and HELL 1996).


34

Fig. 6: Separation of excitation and fluorescence light in incident illumination. The

CBS is placed at a 45° angle to the light path. This CBS reflects the bleu (wanted)

excitation light onto the preparation and transmits the green fluorescence light

towards the eyepieces. The blue (unwanted) excitation light is partly reflected

backwards into the direction of the light source and only a small part is transmitted

towards the eyepieces (modified from HELL 1997).


35

4.4 Radioimmunoassay of AVT

Arginine vasotocin radioimmunoassay was performed according to XU (1991)

with some modifications. In short, aliquots of the chicken plasma samples (200µl)

were extracted with cold acetone (-20°C) and centrifuged at 4.000 x g for 10 minutes

at 4°C. The supernatant was decanted and mixed with 800 µl petroleum benzene

and shaken 30 seconds and then left at room temperature for 30 minutes. The ether

phase was discarded and the aqueous layer extracted once again with petroleum

benzene. After discarding the ether phase, the aqueous phase was dried under

vacuum in a SpeedVac (Savant, New York). The dried extract was stored at –20°C

until assayed. The RIA was performed in duplicate using synthetic AVT (Sigma,

Steinheim) as a standard. Just before conduction of the assay, the dried extracts

were redissolved in 200µl of 0.1 M Tris-HCl, pH 7.4 and 200 µl of the antiserum

were added. Standard curves were obtained by adding 200µl of dilutions of standard

AVT and 200µl of the antiserum at working dilution giving a final dilution of 1:200.000.

The AVT antiserum was kindly supplied by Dr. Gray, Max-Planck-Institute for

Physiological and Clinical Research, Bad Nauheim (GRAY and SIMON 1983). The

crossreactivity with mesotocin and angiotensin II were below 1% (MÖHRING et al.

1980; GRAY and SIMON 1983).

The unlabeled peptide and antiserum were incubated for 48 hours at 4°C

before the addition of labelled tracer [¹²I] (Specific activity 109 mCi/ml, Amersham,

Buckinghamshire, UK). Fifty µl (3.000-4.000 cmp) of labelled tracer was added and

the incubation continued at 4°C for a further 24 hours. Control tubes containing

either tracer alone (200µl of 0.1 M Tris-HCl, pH 7.4 and 200µl AVT antiserum) or

buffer alone (400µl of 0.1 M Tris-HCl, pH 7.4) were used in every assay. The

separation of bound and free tracer was done by the rapid addition of 800µl absolute

ethanol followed by mixing and centrifugation at 3.500 rpm for 20 minutes at 4°C.

The supernatant were removed by aspiration and radioactivity in the pellets was read

by using the gamma counter spectrometry (1277 Gamma Master, LKB Wallac). The

results were analysed with RiaCalc Program (Pharmacia, LKB Wallac). The detection

limit of the assay was 1.5 pg/ml. All samples were measured in the same assay. The

intraassay variation coefficient was 7.2±0.2%.


36

4.5 Processing and analysis of data

The success of dehydration of the adult male chickens was based on the

analysis of plasma osmolality and plasma concentrations of AVT, Na and K. Thus,

the student `s t-test was employed to determine the differences in plasma AVT,

osmolality, Na and K between the control and the dehydrated group. Pearson

correlation test was used to verify correlation between plasma AVT and osmolality

levels in dehydrated and normohydrated animals. Statistical analysis was performed

using a software package (Sigma Stat, Version 2.0). No attempt was undertaken to

statistically analyse the neuroanatomical data. These data were obtained as

described in 4.3.2 and are described as observed in all animals.

Results

5 RESULTS

5.1 Effects of dehydration on blood parameters

Dehydration for 48 h resulted, as expected, in a significant increase in plasma

levels of AVT (p=<0.001); sodium (p=<0.001) and plasma osmolality (p=<0.001),

whereas, potassium levels were not significantly different from the plasma levels

measured in the control group. The results are summarized in the table 1. The

concentrations in individual animals in each group are presented in the appendix

table 1. Neither in normohydrated (r=0.41; p>0.05) nor in dehydrated (r=0.009;

p>0.05) animals was a significant correlation detected between plasma AVT and

plasma osmolality levels.

Table 1. Effect of 48 hours water deprivation on plasma osmolality and plasma

concentration of arg-vasotocin (AVT), sodium (Na) and potassium (K) in adult male

chickens.

Treatment

Osmolality (Mmol/l) mean±SEM

Sodium (Na) (Mmol/l) mean±SEM

Potassium (K) (Mmol/l) mean±SEM

(AVT) (Mmol/l) mean±SEM

Dehydrated

312.25±2.65* (n=8)

166.56±1.84* (n=8)

3.65±0.20 (n=8)

44.19±5.23* (n=6)

Control

295.00±3.12 (n=8)

155.05±1.29 (n=8)

3.57±0.15 (n=8)

15.39±2.91 (n=6)

*Values indicate mean ±S.E.M ; n=8 (for AVT n=6) ; *p=<0.001 (Student `s t-

test) dehydrated vs. control group.

37

Results

5.2 AVT and GAL Co-localization in magnocellular neurons of the

hypothalamic nuclei supraopticus (SON)

The distribution of Arg-vasotocin immunoreactive (AVT-ir) and galanin

immunoreactive (GAL-ir) neurons in the hypothalamus of White Leghorn cockerels

were determined by double labelling immunocytochemistry. AVT and GAL were

distributed ventral regions of the diencephalon in clusters of neurons that correspond

structurally to the brain chicken atlas (KUENZEL and MASSON 1988) at the level of

A 8.8 (Fig. 7)

Fig. 7: Differential Interference Contrast (DIC) and coronal levels of the chicken brain

atlas (KUENZEL and MASSON 1988). Red frames mark the investigated (Nucleus

supraopticus, pars ventralis) microscopic field in the corresponding section. Scale

bar= 1 cm; TSM: Tractus Septomesencephalicus, SOv: Nucleus Supraoptic pars

ventralis, POP : Nucleus Preopticus Periventricularis, TSO : Tractus

Occipitomesencephalicus.

38

Results

Magnocellular neurons of the SON have oval cell bodies, thus, they have the

appearance of the typical hormone-producing cells. These neurons of the

hypothalamo-neurohypophysial system, which synthesize and secrete the peptides,

represent a specialized class of peptidergic neurons (Fig. 8).

Fig. 8: Peptidergic neurons, in SONv, in the normohydrated male chicken.

Scale bar= 50 µm.

39

______

Results

The novelity of the present study is that the confocal analysis of the labelled

neurons was focused on the cell bodies. Clusters of AVT and GAL containing

neurons, extending anteriorily from the Tractus septomesencephalicus (TSM), in

Nucleus supraopticus, pars ventralis (Fig. 9) were analysed. The pattern of the

immunostaining was different for GAL and AVT. In all brain sections of the 8

normohydrated animals, the intensity of the labelling detected in SON was weaker for

GAL than for AVT. Figure 10 and Figure 11 illustrate GAL and AVT immunostained

neurons in SON in brain sections of two normohydrated animals. In fact, no cell

bodies were marked in SON for GAL (Fig. 10E, Fig. 11E). Whereas, there was a

dense staining of cell bodies producing AVT (Fig. 10F, Fig. 11F). Furthermore,

strong labelling of cell endings and nerve fibers were visible concerning AVT.

Interestingly, few neuronal fibers were also stained for GAL. There was no co-

localization of AVT and GAL in SON of all normohydrated animals (Fig. 10H; Fig.

11H).

Fig. 9: The structure of Tractus septomesencephalicus (TSM). A section from adult

male chicken brain from the laser-scaning confocal microscope, Differential

Interference Contrast (DIC). Scale bar= 50 µm.

40

TSM

TSM

Fig. 10: Distribution of AVT in the SONv of the normohydrated adult male chicken. E: No GAL-ir neurons, F:AVT-ir neurons, G:Differential Interference Contrast(DIC), H: No GAL and AVT Co-localizatio. Scale bar= 50 µm.

E F

G H

TSM

41

Fig. 11: Distribution of AVT in the SONv of normohydrated adult male chicken.E:No GAL-ir neurons, F: AVT-ir, neurons, G: Differential Interference Contrast (DIC), H:No AVT and GAL Co-localization Scale bar= 50µm.

E F

G H

TSM

42

A

Fig.12: Single staining immunohistichemistry against GAL in the SONv, in the dehydrated adult male chicken. E: GAL-ir neurons, F:No sign of AVT-staining (red color) G: Differential Interference Contrast(DIC), H:No sign of AVT and GAL Co-localization (yellow color)Scale bar = 50 µm.

E F

G H

__ __

__ __

43

A

Fig. 13: Double staining immunohistochemistry for AVT and GAL in the SONv, in the dehydrated adult male chicken. A: GA L-ir neurons, B: AVT-ir neurons, C:DifferentialInterference Contrast ( DIC), D: Co-localization of AVTand GAL. Scale bar= 50 µm.

D

B

C

____ __

__ __

44

Results

The pattern of the distribution and the intensity of the labelling was thoroughly

different in dehydrated animals. In all eight dehydrated male chicken studied, in

contrast to the normohydrated animals, there were GAL immunostained cells visible

in both single stained (GAL only ; Fig. 12) and double stained (GAL and AVT ; Fig.

13) sections. A clear upregulation of AVT containing cells was also present in SON

sections in all dehydrated animals (Fig. 14 and Fig. 15). The fluorescence intensities

of GAL- and AVT-ir neurons were recorded from the maximum projections within the

AIM-software. In general, both peptides AVT and GAL were homogeneously

distributed in cell bodies (see Fig. 16A and B). Interestingly, there was a co-

localization of GAL- and AVT -ir neurons in dehydrated animals (Fig. 14; Fig. 15).

In intensely labelled neurons, both signals co-localized and merged images appeared

in yellow fluorescence (Fig. 16C).

45

Fig. 14: Distribution of GAL Co-localization with AVT in SONvof the dehydrated adult male chicken. A:GAL-ir neurons, B: AVT-ir neurons, C: DifferentialInterference Contrast (DIC), D: Co-localization of AVT and GAL . Scale bar= 50 µm.

46

AAA B

C D

TSM

Fig.15: Distribution of GAL Co-localization with AVT in SONv of the dehydrated adult male chicken. A: GAL-ir neurons, B: AVT-ir neurons, C: Differential Interference Contrast (DIC), D:Co-localization of AVT and GAL. Scale bar= 50 µm

47

A B

C D

__ __

__ __

TSM

Results

48

Fig. 16: Double staining immunohistochemistry for AVT and GAL in SONv in the

dehydrated adult male chicken; A: AVT-ir neurons; B: GAL-ir neurons; C: Co-

localization of AVT and GAL. Scale bar= 50 µm.

A

C

B

____

____ ____

Results

5.3 Lack of AVT and GAL Co-localization in PVN Neurons

The magnocellular neurons of PVN were also heavily stained for AVT (Fig. 17F;

Fig. 18F) in normohydrated chickens. In contrast to the SON cells, however, a dense

dendritic fiber network was labelled for GAL (Fig. 17E). Dehydration upregulated the

expression of both GAL and AVT, although the intensity was much less than SON

(compare fig. 19 and 20 with fig. 14 and 15). Surprisingly, there was no co-

localization of GAL- and AVT-ir neurons, neither in normohydrated (Fig. 17H; Fig.

18H) nor in dehydrated (Fig. 19H; Fig. 20H) animals.

49

E F

G H

50

Fig. 17: Distribution of AVT in magno- and parvocellular neurons of PVN in the normohydrated male chicken. E: No GAL-ir neurons, F: AVT-ir neurons, G:DifferentialInterference Contrast (DIC), H: No GAL and AVT Co-localization. Scale bar= 50 µm.

E F

G

51

Fig. 18: Distribution of AVT inmagno- and parvo-cellularneurons of PVN in the normohydrated male chicken. E:No GAL-ir neurons, F: AVT-ir neurons, G: Differential InterferenceContrast ( DIC), H: No GAL and AVT Co-localization.Scale bar= 50 µm.

H

A BB

DCFig. 19: No GAL Co-localization with AVT had been found in PVN in dehydrated male chicken. A:No GAL-ir neurons, B: AVT-ir neurons, C: Differential Interference Contrast (DIC), D: No GAL and AVT Co-localization. Scale bar= 50 µm.

52

A B

C DD

Fig. 20: No GAL Co-localization with AVT had been found in PVN in dehydrated male chicken, A: No GAL-ir neurons, B: AVT-ir neurons, C: Differential Interference Contrast ( DIC), D: No GAL and AVT Co-localization.Scale bar= 50µm

53

Discussion

6 DISCUSSION

Water deprivation for 48 hours as an osmotic stimulus caused an increase in the

plasma AVT levels concomitant with enhancement of plasma osmolality. This

indicates that the osmoregulatory system within the hypothalamo-neurohypophysial

axis was stimulated in the animals used in the present study. Similar to mammals,

water deprivation has been shown to be an effective stimulus for AVT synthesis and

AVT discharge into the blood stream in the domestic chicken (ARAD et al. 1985;

NOUWEN et al. 1984). The rate of biosynthesis and axonal transport of AVP (or

AVT) are two major limiting factors for the hypothalamic stores of this neurohormone

(MURPHY and CARTER 1990; ROBERTS et al. 1991). NOUWEN et al. (1984)

observed that plasma AVT levels increased initially after dehydration but when

dehydration was continued beyond 72 to 96 hours the plasma AVT concentration

decreased again. In the present experiment the AVT values were significantly higher

in dehydrated animals than in normohydrated ones, indicating that the animals were

not in condition of progressive dehydration. It is interesting to note that progressive

dehydration leading to extracellular fluid hyperosmolality causes, in addition of a

release of AVP into the blood (BOURQUE et al. 1994; IVANJI et al. 1991 and 1995),

also a release of the hormone into the cerebrospinal fluid (IVANJI et al. 1995).

A sexual dimorphism in the osmotic control of AVT in the chicken has been

reported by several authors. The previous studies of the Dept. of Functional

Genomics and Bioregulation (JURKEVICH et al. 1997) in accordance with some

other reports (VIGLIETTI-PANZICA et al. 1992, 1994) demonstrated a striking sexual

dimorphism in the AVT expressing neurons in chickens. Such a sexual difference has

been also reported in the Japanese quails (CHATURVEDI et al. 2000). In contrast,

an earlier work by ROBINSON et al. (1990b) showed no sexual dimorphism of basal

plasma AVT levels. It is, however, important to recall that besides the control of

water homeostasis, AVT is modulating the mechanisms controlling the egg

laying. Hence, it is feasible that the female chickens response

stronger to the lack of water than males. Although, both AVT plasma and osmolality

54

Discussion

levels increased in response to the 48 hours water deprivation, there was no

correlation between AVT and osmolality in animals used in this study; neither in

normohydtared nor in dehydrated ones. This is in agreement with data published by

NOUWEN et al. (1984). But, some other researches showed a correlation between

plasma AVT concentration and plasma osmolality during water deprivation (ARAD et

al. 1985; STALLONE and BRAUN 1986) and in normohydrated animals (ARNASON

et al. 1986; CHATURVEDI et al. 2000; ROBINSON et al. 1990a,b). The correlation

has been, however, very low. The discrepancies could be due to the differences in

the breeds, sex, age and housing of the animals used in different experiments.

Similar to mammals and as previously reported by this laboratory (BARTH et al.

1997), dehydration resulted, in a significant increase in plasma sodium concentration

(see results table 1). Whereas, there was no significant changes in concentration of

potassium in the blood plasma. This indicates that the blood sampling and the

handling of the animals did not cause any damaging of the cells. A damage to the

cells would have resulted in flow of intracellular potassium into the blood plasma.

The aim of this study was to determine:

1. Are GAL and AVT co-localizations in hypothalamus affected in

response to an osmotic challenge? And

2. Is GAL in hypothalamus affected by dehydration in the male

chicken brain?

The results of immunohistochemistry combined with confocal laser microscopy

confirm the intensifying of AVT in the SON as well as PVN in response to the 48

hours of water deprivation. This increment indicates 1. that the osmotic challenge has

resulted in activation of AVT expressing in these nuclei and 2. the dehydration has

been moderate and has not induced a depletion of AVT in the hypothalamus. Such a

depletion has been reported in response to a massive dehydration (CIOSEK 2002;

CIOSEK and CISOWSKA 2003). A massive upregulation of GAL was also observed

55

Discussion

in both studied nuclei. Interestingly, a co-localization of GAL and AVT was only

observed in SON cell bodies in dehydrated animals. There was no co-localization of

these two peptides in PVN.

Magnocellular hypothalamo-neurohypohysial system as well as the

parvocellular hypothalamo-anterior pituitary system contain large number of GAL

containing neurons (PALKOVITS et al. 1987). In the rat, cell bodies of GAL neurons

seem to be mainly located in SON and PVN and their axons terminate in the

posterior lobe of the pituitary (ARAD et al. 1990; GUNDLACH and BURAZIN 1998;

PALKOVITS et al. 1987). This seems to be in contrast to chicken, since neither in the

present experiment nor in other studies conducted in the Dept. of Functional

Genomics and Bioregulation (KLEIN et al. 2006) GAL perikarya were observed in

magnocellular PVN neurons. Galanin has been assumed to have an inhibitory action

on the biosynthesis and axonal transport of oxytocin but not vasopressin in

normohydrated rats (CIOSEK 2000). But, interestingly, above authors indicated that

in salt-loaded rats GAL attenuates the secretion of oxytocin and vasopressin. This is

in good agreement with present data showing an upregulation of GAL and AVT and

presence of co-localization of the peptides in dehydrated chickens only.

A recent work of the laboratory (KLEIN et al. 2006) confirm the presence of GAL

dendritic fiber network between the distinct clusters of AVT neurons in PVN of male

and female chickens. They also could not observe any co-localization of GAL and

AVT in PVN. Whereas, in the SON they could determine significantly higher degree

of co-localization in the female than in the male chicken brain. This is in good

agreement with the present findings, which indicate a co-localization, concomitant

with upregulation of both GAL and AVT in SON only when the male chickens were

under an osmotic stimulation.

The present data show for the first time the enhanced expression of GAL and

visualization of co-localization of GAL and AVT in the chicken hypothalamo-

neurohypophysial system due to an osmotic stress. This could explain that AVT is

controlled by paracrine/ autocrine secretion and activity of GAL allowing fine

adjustment of neuronal activity in SON. Interestinly, a subpopulation of magnocellular

56

Discussion

SON neurons expressed AVT only after water deprivation. It is possible that these

cells are the sites of co-localization. It is also interesting to note, that a dense

dendritic fiber network connected the GAL neurons and AVT neurons in both nuclei

investigated. A dendritic release of vasopressin in the hypothalamic magnocellular

nuclei has been shown (LUDWIG 1998; MORRIS et al. 1998; POW and MORRIS

1989; WANG et al. 1995). A dendritic release of GAL is also demonstrated in

magnocellular cells and has been assumed to be the main source of GAL release

(SWANCHENKO and PFEIFFER 1988). Furthermore, the distribution of AVT mRNA

in the hypothalamus of White Leghorn cocks was determined by in situ hybridization

(CHATURVEDI et al. 1993). In control birds that were provided with ad lib, AVT

mRNA was distributed in the paraventricular and lateral region of the hypothalamus.

Water deprivation for 2 and 4 days resulted in an increase in levels of AVT mRNA per

neuron and the number of AVT mRNA-containing cells (CHATURVEDI et al. 1993).

On the other hand, the expression of arginine- vasopressin (AVP) and GAL was

studied by immunocytochemistry and in situ hybridization in the hypothalamus of two

African rodents (LACAS-GERVAIS et al. 2003). In the wild, these animals

experience successive arid and wet seasons that alternately stimulate their

antidiuretic and diuretic systems. In this study, animals were subjected to both

standardized laboratory conditions and to eight days of water-restriction. Under both

sets of conditions, AVP and GAL detected in the SON and PVN. AVP and GAL

responses to water-restriction differed in the two species, as did behavioural

adaptations to the hot-dry seasons. In Taterillus gracilis, AVP and GAL like

immunoreactivity peptides and mRNA levels increased in the SON and PVN. In

Steatomys caurinus, which estivates, in the SON, AVP and GAL mRNA levels

increased, whereas, in the PVN, only AVP mRNA levels increased. In both species,

the changes in the amount of GAL like immunoreactivity peptide appeared to be

closely linked to changes in AVP levels, suggesting that GAL is involved in the

osmoregulatory response to water-restriction (LACAS-GERVAIS et al. 2003).

57

Discussion

In conclusion, the results of the present study show a close functional link

between GAL and AVT neurons. The co-localization and simultaneous upregulation

of these peptides in response to water deprivation may indicate a site specific control

of AVT by GAL. The future studies should clarify, if the difference in the co-

localization of AVT and GAL in SON and PVN and in particular the absence of any

co-localization of these two peptides in PVN is a principle phenomenon or if it is

stimuli specific and changes under other stimulatory conditions such as oviposition.

58

Summary

7 SUMMARY

Secil Cabuk

Upregulation and changes in expression pattern of

Arg-vasotocin and galanin after water deprivation in male chickens

Within the hypothalamo-neurohypophysial-system (HNS) of avian species, the

nonapeptide arginine vasotocin (AVT) is synthesized in neurosecretory neurons and

transported axonally to the neurohypophysis where it is released into the blood. In

chickens AVT has antidiuretic and uterotonic functions. It is therefore equivalent to

both arginine vasopressin (AVP) and oxytocin in mammalian species. Beside this

classical endocrine function also neuromodulatory functions of the nonapeptides

within the CNS are described. Galanin (GAL), a 29 amino acid peptide, is distributed

throughtout the central nervous system and concentrated within the basal forebrain,

septal region and amygdala and found to be a potent modulator of neuroendocrine

regulation.

The studies reported in this dissertation were undertaken to further elucidate

arg-vasotocin and galanin and their co-localization and plasticity after water

deprivation. Besides the control of water balance, AVT is strongly involved in the

mechanisms leading to egg laying in female birds. Thus, to avoid any influence with

egg laying only male chickens were used. To this end the expression pattern and

signal intensities of AVT and GAL in hypothalamic supraoptic (SON) and

paraventricular (PVN) neurons were evaluated.

Following questions were addressed using the chicken as the experimental

model:

- Are galanin and arginine vasotocin co-localizations in hypothalamus affected

in response to an osmotic challenge? And

- Is galanin in hypothalamus affected by dehydration in the male chicken

brain?

59

Summary

Adult (age: 22 to 35 weeks) male Lohmann Selected White Leghorn (LSL)

chicken were used and deprived of drinking water for 48h. The effects of dehydration

were examined by measuring plasma osmolality, plasma levels of potassium, sodium

and AVT concentration. Double staining immunohistochemistry combined with

confocal laser scanning microscopy was utilized to determine the effect of

dehydration on hypothalamic magnocellular neurons in the supraoptic (SON) and

paraventricular (PVN) nucleus and to verify the extend of co-localization of the two

peptides, AVT and GAL in these two nuclei.

The results indicate that the 48 hours water deprivation caused a moderate

dehydration in the animals resulting in a significant increase in plasma osmolality and

plasma concentrations of AVT and sodium.

The pattern of the immunostaining was different for GAL and AVT. The intensity

of the labelling detected in SON was weaker for GAL than for AVT. In fact, no cell

bodies were marked in SON for GAL. Whereas, there was a dense staining of cell

bodies producing AVT. There was no co-localization of AVT and GAL in SON of

normohydrated animals. Interestingly, there were GAL immunostained cell bodies

visible in SON in response to dehydration. A clear upregulation of AVT cells was also

present in SON during water deprivation period. A co-localization of AVT and GAL

was apparent in SON of dehydrated animals. The magnocellular neurons of PVN

were also heavily stained for AVT in normohydrated chickens. A dense dendritic fiber

network was labelled for GAL. Dehydration upregulated the expression of both GAL

and AVT. Surprisingly, there was no co-localization of GAL and AVT neurons in PVN.

The results show a close functional link between GAL and AVT neurons. The

co-localization and simultaneous upregulation of these peptides in response to water

deprivation may indicate a site specific control of AVT by GAL. The future studies

should clarify, if the co-localization of AVT and GAL and in particular the absence of

any co-localization of these two peptides in PVN is a principle phenomenon or it is

stimuli specific and changes under other stimulatory conditions such as oviposition.

60

Zusammenfassung

8 ZUSAMMENFASSUNG

Secil Cabuk

Co-Lokalisation und gleichzeitige Aufregulation von Arginin-Vasotocin-

und Galanin-Synthese nach dem Wasserentzug beim Geflügel

Im Hypothalamo-neurohypophysären System wird bei aviären Spezies das

Nonapeptid Arginin-Vasotocin (AVT) von neurosekretorischen Nervenzellen

synthetisiert und gelangt über axonalen Transport in die Neurohypophyse. Dort

erfolgt die Freisetzung in das Blut. AVT wirkt beim Geflügel (Gallus domesticus)

antidiuretisch und ist an der Regulation der Oviposition beteiligt. Funktionell

entspricht AVT somit den Nonapeptiden Arginin-Vasopressin und Oxytocin der

Säuger. Neben dieser klassischen endokrinen Funktionsweise sind für AVT auch

neuromodulatorische Wirkungen innerhalb des ZNS (Zentral nerven System)

beschrieben. Galanin (GAL) besteht aus 29 Aminosäuren und wurde im ZNS vor

allem in vorderen Gehirnregion im Septum Region und in der Amygdala bei

Säugetieren nachgewiesen. Galanin hat eine modulatorische Wirkung auf die

Regulation des Neuroendokrinen Systems.

Die in dieser Dissertation dargestellten Untersuchungen hatten zum Ziel, die

Arginin-Vasotocin- und Galanin-Synthese sowie ihre Co-Lokalisation in Folge einer

osmotischen Stimulation aufzuklären. Arginin-Vasotocin spielt eine wesentliche Rolle

bei der Regulation des Wasserhaushaltes and hat eine Wirkung auf die Eiablage

beim weiblichen Tier. In der vorliegenden Arbeit wurden nur männliche Tiere

verwendet, um die Wirkung von AVT im Wasserhaushaltsystem zu beobachten.

Die Fragestellungen waren im einzelnen:

- Ist die Co-Lokalisation von AVT und GAL im Hypothalamus abhängig von

einer osmotischen Stimulation? Und

- Welche Wirkung hat eine osmotische Stimulation auf die GAL-Produktion im

Hypothalamus?

61

Zusammenfassung

Zur Erzeugung einer osmotischen Stimulation wurden adulte (22-35 Wochen

alt) Leghorn-typ Hähne (Lohmann Selected White Leghorn) einem 48 stündigen

Wasserentzug ausgesetzt. Die Plasma Osmolalität-, Natrium-, Kalium- and AVT-

Konzentrationen wurden gemessen, um die Wirkung der Dehydratation zu ermitteln.

Zur Darstellung der AVT- und GAL-Neurone wurde eine immunohistochemische

Methode mit Doppelfärbung kombiniert mit konfokaler Laser-scanning Mikroskopie

eingesetzt.

Die Ergebnisse zeigen, dass der 48 stündige Wasserentzug eine moderate

Dehydration der Tiere verursacht hat. Daraus resultiert ein signifikanter Anstieg in der

Plasma-Osmolalität und der Plasma Konzentrationen von AVT und Natrium.

Das Muster der Immunfärbung war unterschiedlich für GAL und AVT. Die

Intensität der Färbung war im Supraoptic Nucleus (SON) für GAL schwächer als für

AVT im SON. Es wurden keine Zellkörper in SON für GAL markiert. Indessen gab es

eine dichte Färbung von Zellkörpern, die AVT produzierten. Es gab keine Co-

Lokalisation von AVT und GAL im SON bei normohydrierten Tieren.

Interessanterweise waren als Antwort auf die Dehydration GAL-immungefärbte

Zellkörper im SON sichtbar. Die magnozellulären Neurone im PVN waren auch für

AVT in normohydrierten Hähnen stark angefärbt. Galanin-Färbung war nur in

Nervenfasern sichtbar. Dehydration regulierte die Expression von beiden, GAL und

AVT, herauf. Jedoch gab es überraschenderweise keine Co-Lokalisation von GAL-

und AVT- Neuronen im PVN.

Die Ergebnisse zeigen eine enge funktionelle Bindung zwischen GAL und AVT

Neuronen. Die Co-Lokalisation und gleichzeitige Aufregulation dieser Peptide als

Antwort auf den Wasserentzug könnte eine spezifische Kontrolle von AVT durch GAL

bedeuten. Zukünftige Studien sollten klären, ob die Ko-lokalisation von AVT und

GAL, aber vor allem die Abwesenheit der Ko-lokalisation dieses Peptide im PVN, ein

prinzipielles Phänomen ist, oder ob sich dises unter anderen stimulatorischen

Bedingungen wie zum Beispiel die Oviposition ändert.

62

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89

Appendix

10 APPENDIX

10.1 List of Abbreviations

ADH Antidiuretic Hormon

Aq. Dest. Aqua distillata

Arg Arginine

ATP Adenosine triphosphate

AVT Arginine vasotocin

°C Degree celcius

Ca Calcium

cAMP Cyclic adenosine monophosphate

CBS Chromatic Beam Splitter

Cl Chlor

CNS Central Nervous System

et al. et alii

GABA Gamma aminobutyric acid

GAL Galanin

GHRH Growth hormone- releasing hormone

H Hydrogen

HNS Hypothalamo-neurohypophysial System

HPA Hypothalamo-pituitary Adrenal System

LHRH Luteinizing hormone-releasing hormone

-ir Immunoreactivity

K Potassium

Leu Leucine

LHI Low Humidity During Incubation

LSL Lohmann-Selected Leghorn

LVP Lysine Vasopressin

90

Appendix

M Molar

mRNA messenger ribonucleic acid

µl microlitre

mg milligram

ml millilitre

MT mesotocin

Na Sodium

OT oxytocin

PBS phosphate buffer saline

RIA Radioimmunoassay

SEM Standart error mean

TRH Thyrotropin releasing hormone

UV Ultraviolet

PVN Paraventricular Nucleus

SON Supraoptic Nucleus

91

Appendix

Abbreviations of the Brain Nuclei and Structures:

A Archistriatum

AA Archistriatum Anterior

BnST Bed Nucleus of Stria Terminalis

E Ectostriatum

FA Tractus Fronto-Archisriaticus

FDB Fasciculus Diagonalis Brocae

FPL Fasciculus Prosencephali Lateralis

INP Nucleus Intrapedindicularis

LMD Lamina Medullaris Dorsalis

LSO Organum Septi Laterale

LH Lamina Hyperstriatica

LT Lamina terminalis

MPOd Nucleus Magnocellularis preopticus, pars dorsalis

MPOm Nucleus Magnocellularis preopticus, pars medialis

MPOv Nucleus Magnocellularis preopticus, pars ventralis

OVLT Organum Vasculosum Lamina Terminalis

POD Nucleus Preopticus Dorsolateralis

POM Nucleus Preopticus Medialis

POP Nucleus Preopticus Periventricularis

PP Paleostrium Primitivum

PV Nucleus Posterioventralis Talami

QF Tractus Quintofrontalis

SP Nucleus Septalis lateralis

Soe Nucleus Supraopticus, pars externus

Sov Nucleus Supraopticus pars ventralis

TSM Tractus Septomesencephalicus

VIII Ventriculus Tertius

VL Ventriculus Lateralis

92

Appendix

10.2 Tables

Table 1. Plasma Osmolality, Sodium (Na), Potassium (K), and arg-vasotocin (AVT)

concentrations in adult male chickens.

Chicken No. Status

Osmol. Mmol/l

Na Mmol/l

K Mmol/l

AVT pg/ml

14350

Control

314

160.1

2.7

18.2

14254

Control

288

151.9

3.6

10.9

18

Control

300

158.8

3.3

17.8

32

Control

299

156.6

3.9

9.1

43

Control

293

157.9

3.6

8.9

48

Control

288

152.1

3.3

27.2

121

Control

290

151.4

3.9

____

123

Control

288

151.6

4.0

____

14153

Dehydrated

320

163.5

2.7

59.4

14123

Dehydrated

315

161.6

4.3

32.3

24

Dehydrated

320

172.5

3.9

38.3

26

Dehydrated

309

169.7

3.4

33.4

28

Dehydrated

315

172.5

3.0

61.1

46

Dehtdrated

316

165.0

3.4

40.5

122

Dehydrated

303

161.5

3.7

____

124

Dehydrated

300

163.5

4.4

____

93

Appendix

10.3 Chemical Reagents

Albumin SIGMA, Deisenhofen, Germany

Alexa Flour 555 (ab) MOLECULAR PROBES,

EUROPE BV, Netherlands

Calciumiodid MERCK, Darmstadt, Germany

Chloramine T MERCK, Darmstadt, Germany

Di-Natriumhydrogenphosphatdi-hydrate MERCK, Darmstadt, Germany

Fluorescein Anti-guinea Pig IgG VECTOR LABORATORIES,

California, USA

Goat Serum (Normal) DAKO, Denmark

Natruimdihydrogenphosphat-1-hydrat MERCK, Darmstadt, Germany

Neomycine SIGMA, Deisenhofen, Germany

Paraformaldehyd SIGMA, Deisenhofen, Germany

Picric acid MERCK, Darmstadt, Germany

Saccarosi MERCK, Darmstadt, Germany

Syntetic AVT SIGMA, Deisenhofen, Germany

Sodium chloride MERCK, Darmstadt, Germany

Sodium nitrit SIGMA, Deisenhofen, Germany

Sodium sulfate MERCK, Darmstadt, Germany

Tris MERCK, Darmstadt, Germany

Vectashield Mounting Medium VECTOR LABORATORIES,

California, USA

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Appendix

10.4 Instruments

Automatic Gamma Master Millipore GmbH, Düsseldorf, Germany

(1277 Gamma Master)

CCD Blutgas System Ciba Corning Diagnostics,Cheshire, England

Cryostat Frigocut Model 2700 Reichert Jung GmbH, Germany

Magnetic stir Heidolph, Düsseldorf, Germany

Laser Scanning Confocal Microscope Carl Zeiss, Jena, Germany

(LSM 510)

Reaction glass Eppendorf, Mannheim, Germany

Ria CalcProgramm Pharmacia, Dreieich, LKB Wallac

pH meter Knick, Karlsruhe, Germany

Spectophotometer SLT-Spectra SLT Labinstrument Deutschland GmbH,

Heidelberg

Spectophotometer UV/visible Pharmacia Biotech, Hannover, Germany

Ultraspec 2000 (Ultra Turpax)

Tissue culture plate Gainer, Neu-Ulm, Germany

Vacuum centrifuge SpeedVac SVC200H, Savant, Instruments,

Farmingdale, NY, USA

Vacuum manifold and pump Millipore GmbH, Freiburg, Germany

Vapour Pressure Osmometry Wescor Inc. Utah, USA

Vortex Heidolph, Düsseldorf, Germany

95

Appendix

10.5 Composition of Solutions and Buffer

5* PBS Buffer

Composition

NaCl 39,74 g

Na2HPO4 5,745 g

KCl 1,005 g

KH2PO4 1,02 g

Aq.Dest. To 1000,0 ml

pH 6,8

Tris buffer for AVT analysis

Composition

Tris 12,11 g

Neomycin sulfate 2 g

Bovine serum albumin 3 g

Aq. Dest. To 1000,0 ml

pH 7,4

Zamboni Solution for Perfussion

Composition

Paraformaldehyd 40 g

Picric acid 150 ml

0.1 M PBS To 1000,0 ml

pH 7,4

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Appendix

25% Saccarose Solution

Composition

Saccarose 250 g

0.1 M PBS To 1000,0 ml

pH 7,4

PBS + Heparin + 0,1% Natrium Nitrit Solution

Composition

Natrium nitrit 1 g

Heparin 25000/5ml 2 ml

0.1 M PBS To 1000,0 ml

pH 7,4

97

Acknowledgment

98

11 Acknowledgment I wish to express my gratitude to Prof. Dr. Dr. Nahid Parvizi, for giving me the oppotunity to study this interesting, for her advice and guidance. I express sincere thanks to Prof. Dr. Roland Grossmann who supported in laboratory work and gave valuable advice on performing the experiments. His helpful advice and nice support were excellent. Special thanks and appreciation are expressed to Prof. Dr. Folkmar Elsaesser and Prof. Dr. Behic Serpek who provided scientific and personal comments. Special thanks and are expressed to Prof. Dr. Silke Rautenschlein who provided scientific and other comments for the final thesis I am grateful to and Prof. Dr. Dr. Franz Ellendorff and Dr. Lars Schrader, head of institut, for giving me the opportunity to work in the federal research centre of Agricultural, Institute for animal Science I would like to thank Dr. Alexandr Sirotkin, Dr. V. Lebedev and Dr. I Lebedeva, who advice on thesis. Special thanks go out to Mrs Maritta Ledwoch ( Akademisches Auslandsamt) for her kind efforts I would like to thank Corinna Klein, Geovanna Brenner and Monika Nowak, who advice on writing the thesis. My immense thanks also go to the laboratory technician and colleages of department of functional genetic and bioregulation, especially Gudrun Neuhaus and Dr Sabine Klein.

Upregulation and changes in expression pattern of Arg ... · 2.2 Vasotocin (AVT) Gene Structure The...

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