Post on 26-Mar-2020
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
Review of Literature
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).
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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.
Review of Literature
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.
Review of Literature
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
Review of Literature
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
Review of Literature
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).
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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
Review of Literature
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
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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).
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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
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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).
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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).
Review of Literature
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.
Review of Literature
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
Materials and Methods
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.
Materials and Methods
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.
Materials and Methods
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
Materials and Methods
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
Materials and Methods
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).
Materials and Methods
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).
Materials and Methods
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%.
Materials and Methods
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
References
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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
94
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
96
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.