Download Implication of novel neurotransmitter systems in the regulation of

Implication of novel neurotransmitter systems in the
regulation of gonadotropin-releasing hormone neurons
Ph.D. Dissertation
Gergely F. Túri
Laboratory of Endocrine Neurobiology
Institute of Experimental Medicine, Hungarian Academy of Sciences
Semmelweis University
János Szentágothai Ph.D. School of Neuroscience
Supervisor: Zsolt Liposits Ph.D., D.Sc.
Chairman of committee: Béla Halász Ph.D., D.Sc.
Members of committee: Katalin Halasy Ph.D., D.Sc.
Opponents: József Kiss Ph.D., D.Sc.
Zoltán Rékási Ph.D.
Budapest
2007
Katalin Köves Ph.D., D.Sc.
Table of contents
1. List of abbreviations...................................................................................................5
2. Introduction ................................................................................................................6
2.1. Regulation of reproduction in mammals...........................................................6
2.2. The anatomy of the GnRH neuronal system in rodents ..................................8
2.2.1. The development, distribution and structural properties of GnRH
neurons in adult rats..................................................................................................8
2.2.2. The efferent pathways and target areas of the GnRH neurons .....................10
2.2.3. The afferent regulation of GnRH neurons....................................................14
2.2.3.1. Innervation of GnRH neurons by afferents containing classical
neurotransmitters ................................................................................................ 14
2.2.3.2. Innervation of GnRH neurons by neuropeptide-containing afferents ... 17
2.2.4. Colocalized neurotransmitters in GnRH neurons.........................................19
2.3. Proposed novel neurotransmitter systems in the regulation of GnRH
neurons ......................................................................................................................22
2.3.1. Involvement of acetylcholine (ACh) in the regulation of the reproduction .22
2.3.2. The role of the NPY in the regulation of reproduction ................................24
2.3.3. Glutamate as a proposed autocrine/paracrine regulator synthesized by
GnRH neurons ........................................................................................................26
3. Specific aims..............................................................................................................27
4. Materials and methods.............................................................................................28
4.1. Direct innervation of GnRH neurons by cholinergic afferent pathways
in male rats ................................................................................................................28
4.1.1. Double-labeling immunocytochemical detection of cholinergic fibers
and GnRH neurons at the light microscopic level..................................................28
4.1.2. Double-labeling immunocytochemical detection of cholinergic fibers
and GnRH neurons at the electron microscopic level ............................................30
4.2. Determining of the origin of NPY-containing afferents to GnRH neurons
in male GnRH-GFP transgenic mice ......................................................................31
4.2.1. Experimental animals ...................................................................................31
4.2.2. Single-labeling immunocytochemical detection of NPY- and AGRPcontaining fibers in the POA of neonatally MSG-treated mice vs. untreated
controls ...................................................................................................................32
4.2.3. Double-labeling immunocytochemical detection of NPY- and AGRPcontaining fibers in contact with GnRH neurons of neonatally MSG-treated
mice vs. untreated controls .....................................................................................33
4.2.4. Immunofluorescent-labeling of AGRP/NPY and DBH/NPY contacts
with GnRH-GFP neurons in intact mice.................................................................34
4.3. Electron microscopic analysis of AGRP afferents to GnRH neurons in
intact male, GnRH-GFP mice .................................................................................36
4.4. Investigation of the putative glutamatergic phenotype of GnRH neurons ..37
4.4.1. Experimental animals ...................................................................................37
4.4.2. Detection of GnRH and Vglut2 mRNAs by dual-label in situ
hybridization histochemistry (ISHH) .....................................................................37
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4.4.3. Dual-immunofluorescent labeling for confocal laser scanning
microscopic analysis of Vglut2- and GnRH-immunoreactive axons in the
OVLT and ME........................................................................................................40
4.4.4. Pre-embedding immuno-gold-labeling of Vglut2 in the hypophysiotropic
nerve terminals of the ME ......................................................................................41
5. Results........................................................................................................................43
5.1. Identification of cholinergic afferents to GnRH neurons of the rat .............43
5.1.1. Light microscopic evidence for cholinergic neuronal contacts on GnRH
neurons ...................................................................................................................43
5.1.2. Electron microscopic evidence for cholinergic afferents to GnRH
neurons ...................................................................................................................45
5.2. Revealing the origin of NPY-containing afferents to GnRH neurons in
male mice ...................................................................................................................47
5.2.1. Comparative studies of NPY- and AGRP-containing fibers in contact
with GnRH neurons of neonatally MSG-treated mice vs. untreated controls ........47
5.2.2. Triple-label fluorescent detection of AGRP/NPY and DBH/NPY fibers
forming neuronal contacts with GnRH-GFP neurons ............................................50
5.3. Electron microscopic detection of AGRP in synaptic afferents to GnRH
neurons ......................................................................................................................53
5.4. Morphological evidence for the glutamatergic phenotype of GnRH
neurons in adult male rat.........................................................................................55
5.4.1. Results of in situ hybridization experiments ................................................55
5.4.2. Results of dual-label immunofluorescent studies.........................................56
5.4.3. Electron microscopic localization of Vglut2 in hypophysiotropic axon
terminals in the ME ................................................................................................58
6. Discussion ..................................................................................................................59
6.1. Proposed nonsynaptic role and receptorial mechanisms of ACh on
GnRH neurons and the possible sites of origin of the direct cholinergic
afferents .....................................................................................................................59
6.1.1. Ach as a proposed nonsynaptic modulator of GnRH neurons......................59
6.1.2. Receptorial mechanisms of ACh actions on GnRH neurons........................60
6.1.3. Possible sites of origin of the cholinergic innervation to GnRH neurons ....61
6.2. The functional roles of the NPYergic afferents of Arc and brainstem in
the regulation of GnRH neurons and the receptorial mechanisms of NPY
action on GnRH neurons .........................................................................................62
6.2.1. The Arc and the brainstem as two major sources of origin of NPY fibers
innervating GnRH neurons.....................................................................................62
6.2.2. Receptorial mechanisms of NPY actions on GnRH neurons .......................67
6.2.3. Methodological considerations.....................................................................68
6.3. Agouti-related peptide as a novel neurotransmitter in the regulation of
the GnRH neurons....................................................................................................68
6.4. Demonstration of glutamatergic phenotype of the GnRH neurons ..............69
6.4.1. Central effects of glutamate on the regulation of reproduction....................70
6.4.2. Glutamatergic phenotype of the GnRH neurons ..........................................70
6.4.3. Subcellular localization of Vglut2 in the axon terminals of the ME ............72
6.4.4. Methodological considerations.....................................................................73
7. Conclusions ...............................................................................................................75
8. Summary ...................................................................................................................77
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9. Összefoglalás .............................................................................................................78
10. Literature cited .......................................................................................................79
11. Bibliography..........................................................................................................101
11.1. List of publications related to the subject of the thesis ...................................101
11.2. List of other publications.................................................................................101
12. Acknowledgements ...............................................................................................103
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1. List of abbreviations
5-HT - Serotonin
ME – Median eminence
ac – Anterior commissure
MPN – Median preoptic nucleus
ACh - Acetylcholine
MPOA – Medial preoptic area
ACTH - Adrenocorticotropic hormone
MSG – Monosodium glutamate
AGRP – Agouti-related protein
NMDA – N-methyl-D-aspartate
AMCA - Aminomethylcoumarin acetate
NMDAR-1 – N-methyl-D-aspartate
Arc – Arcuate nucleus
glutamate receptor subunit
BW - Body weight
NPY – Neuropeptide Y
CCK – Cholecystokinin
NT – Neurotensin
ChAT - Choline acetyltransferase
OVLT – Organum vasculosum laminae
CRH – Corticotropin-releasing hormone
terminalis
Cy3 – Cyanine dye 3
ox – Optic chiasm
DAB – Diaminobenzidine
PBS – Phosphate buffered saline
DBH – Dopamine-β-hydroxylase
PCS – Pericapillary space
DSIP – Delta sleep-inducing peptide
POA – Preoptic area
DV – Dense core vesicle
POMC – Pro-opiomelanocortin
FITC – Fluorescein isothiocyanate
SP – Substance P
FSH – Follicle-stimulating hormone
SSC – Standard saline citrate
GABA - Gamma-aminobutyric acid
SV – Small clear vesicle
GLU - Glutamate
TBS - Tris-buffered saline
GnRH – Gonadotropin-releasing hormone
TH - Tyrosine hydroxylase
HDB - Horizontal limb of the diagonal band
VAChT – Vesicular acetylcholine
of Broca
transporter
HPG – Hypothalamo-pituitary-gonadal axis
Vglut1-3 – Types 1-3 of the vesicular
IGF-1 – Insulin-like growth factor-1
glutamate transporters
INF – Infundibular stalk
αMSH – α-melanocyte-stimulating
ISHH – In situ hybridization histochemistry
hormone
KA-2 - Kainate-2 ionotropic glutamate
β-END – β-endorphin
receptor subunit
LH – Luteinizing hormone
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2. Introduction
2.1. Regulation of reproduction in mammals
The adaptive regulation of the hypothalamo-pituitary-gonadal (HPG) axis is
crucial in the successful reproduction among the vertebrates. The central unit of this
axis is formed by the gonadotropin-releasing hormone (GnRH)-producing neurons
which release their neurohormone content into the portal circulation of the hypophysis
in a pulsatile manner. The episodic hormone release from the GnRH axon terminals
results in a rhythmic discharge of the two gonadotropins, luteinizing hormone (LH) and
follicle stimulating hormone (FSH), by the gonadotroph cells of the anterior pituitary.
The gonadotrop hormones reach the gonads via the systemic circulation and stimulate
their steroid hormone production and gametogenesis. The gonadal steroids, in turn,
exert negative (in females and males) and positive (only in females) feedback actions on
the central components of the axis. (Figure 1.)
The gonadal steroids are crucial hormonal factors in the regulation of the HPG
axis. Gonadectomy results in increased levels of LH/FSH in the systemic circulation
and elevates the level of GnRH in the hypophyseal portal circulation. This confirms the
tonic inhibitory role of the gonadal steroids on the hypothalamo-pituitary unit in intact
animals. The negative feedback effect is a common regulatory mechanism in both
genders. After puberty, the steroid hormone genesis is continuous in males. High levels
of androgens produced by the Leydig cells of the testicles exert a constant inhibitory
action on the hypothalamus and pituitary either via acting on the androgen receptors or
following its aromatization to estradiol, via acting on estrogen receptors (1). In contrast,
in females the gonadal functions show a cyclic activity. In the early phases of ovarian
cycle, the relatively low level of estrogen exerts negative feedback effects on the
hypothalamus and hypophysis. This way estrogen constrains its own follicular synthesis
and constrains the GnRH neurons as well to function in a pulse mode, i.e. to release
their hormone content at regular time intervals. As the follicles mature, they show a
dramatic increase in their secretion of estrogen and a modest increase in their output of
progesterone. These changes in the circulating blood levels of ovarian steroids switch
the function of GnRH neurons to surge mode, which results in a rapid increase of
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hypothalamic GnRH (2) and consequential pituitary gonadotropin secretion. This
phenomenon is referred to as positive feedback and it is a prerequisite for ovulation (3).
In addition to the feedback effects of the steroid hormones on the HPG axis, many
external and internal factors influence GnRH neuronal functions. These include
environmental toxins, stressful stimuli, malnutrition or the overfed status of the animal.
These factors modify the spontaneous electrical (4) and secretory activities (5, 6) of
GnRH neurons either via direct actions or via the regulation of neuronal inputs from
various neurotransmitter systems, which impinge on GnRH nerve cells. The focus of
this thesis is on the neuronal regulation and neurochemical properties of GnRH neurons,
therefore, following the anatomical characterization of the hypophysiotropic GnRH
neurons, their efferent pathways and main target areas, the thesis will discuss the
chemical nature and the possible sources of origin of the regulatory neurotransmitters
within specific neuronal afferents to GnRH neurons. Finally, the co-expressed
neurotransmitters by GnRH neurons will be reviewed briefly.
Figure 1. The schematic drawing depicts
the components of the HPG axis. The
hypophysiotropic GnRH is released by
the hypothalamus and it liberates the
gonadotropins from the hypophysis. LH
and FSH stimulate gonadal functions.
Gonadal steroids, in turn, regulate the
functions of the HPG axis by negative (in
males and females) and positive (only in
females) feedback mechanisms. (FSH:
follicle stimulating hormone, GnRH:
gonadotropin-releasing hormone, LH:
luteinizing hormone)
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2.2. The anatomy of the GnRH neuronal system in rodents
2.2.1. The development, distribution and structural properties of GnRH neurons in
adult rats
In rats, the central regulation of the reproductive axis is carried out by about 10001600 GnRH neurons (7, 8). These neurons ontogenetically originate outside the brain in
the olfactory placodes (9) and migrate to their final location during the embryonic life.
In addition to these data from mice, results of other experiments performed in chicks
(10), rhesus macaques (11), rats (12) and humans (13) indicate that the olfactory origin
of GnRH neurons is a general phenomenon of vertebrate development.
While GnRH neurons in the two genders show functional differences, their
number is equal in the two sexes. Although the hypophysiotropic GnRH neurons serve
the same function, i.e. the stimulation of gonadotropin release from the anterior pituitary,
they are scattered in different anatomical areas. From a topographic viewpoint, GnRH
immunoreactive perikarya can be divided into several subpopulations with the help of
immunocytochemical approaches (7). One group of GnRH neurons can be observed in
the medial septal nucleus, nucleus of the vertical and horizontal limbs of the diagonal
band and in the vicinity of the organum vasculosum laminae terminalis (OVLT).
Another group can be detected in the medial preoptic area with the majority of the
GnRH cells situated in the medial preoptic nucleus (MPN). Fewer GnRH neurons are
found in the periventricular portion of the preoptic nucleus and in the lateral preoptic
area (7). All of these anatomical regions are located rostral to and above the optic
chiasm. Therefore, for the sake of simplicity, in this thesis we will commonly refer to
these sites as the preoptic area (POA).
In addition to hypophysiotropic GnRH neurons in the POA, the detailed light
microscopic mapping of GnRH-immunoreactive perikarya has revealed sparse neurons
in other brain regions as well (7); the function of these non-hypophysiotropic GnRH
neurons needs clarification.
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Figure 2. (A) Distribution of GnRH containing neuronal elements in the rat brain. The
gray dots in the schematic sagittal drawing (A) correspond to GnRH synthesizing
perikarya. The hypophysiotropic GnRH neurons are located in the POA (delineated with
a dashed line) and project to the OVLT (yellow area above the optic chiasm) and to the
ME. (B) Coronal section of a rat brain trough the POA-OVLT region. The immunostained
section illustrates the typical distribution of the hypophysiotropic GnRH neurons (arrows)
and fibers in this area (Scale bar = 50 µm) (III: third ventricle; ac: anterior commissure;
ME: median eminence; OVLT: organum vasculosum lamiae terminalis; ox: optic chiasm;
POA: preoptic area)
The spreading of immunocytochemical methods and highly specific antibodies
against the GnRH decapeptide allowed to characterize the light (7) and electron
microscopic features of GnRH neurons (8, 14, 15). It is worth of note that the
immunocytochemical visualization of GnRH-immunoreactivity limits the microscopic
analysis to the cell body, proximal dendrites, and nerve terminals of GnRH cells, which
contain the neuropeptide abundantly. In contrast, the fluorescent visualization and
subsequent confocal microscopic analysis of biocytin-filled GnRH neurons have
revealed an extensive dendritic arborization (16), which could not be detected reliably
by labeling for the GnRH decapeptide itself.
In most species, the majority of GnRH neurons are oval or fusiform in shape, with
a maximum diameter of 10 to 20 µm. The fine structural properties of these neurons do
not differ from the general neuronal attributes, except that some of the GnRH nerve
cells are ciliated (8, 15). Most of the GnRH neurons show either uni- or bipolar shapes.
The dendritic processes of GnRH cells originate from one or both poles of the cell body.
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The dendrite with the greatest circumference extending from the GnRH soma is
designated as the primary dendrite.
With the aid of validated and highly specific antibodies against GnRH decapeptide,
GnRH neurons can also be divided into two basic morphologic categories: those, which
possess a smooth contour, and those, which have a more ragged surface (8). Somewhat
contrasting this categorization, Campbell and colleagues examined 45 biocytin-filled
cells and observed that all of them exhibited some somal and dendritic spines with a
density up to 1 spine/µm plasma membrane (16). They found that the majority of the
spines were of the simple sessile type, but thin pedunculated and mushroom-like spines
were observed as well.
2.2.2. The efferent pathways and target areas of the GnRH neurons
The GnRH axons follow the septo-preoptico-infundibular tract that emerges from
the GnRH neurons located in the medial septum and the POA (7).The GnRH fibers run
in ventral, caudal and somewhat lateral direction and converge into several fascicles.
The unpaired median fascicle is formed at the rostral level of the optic chiasm. One
bundle of this fascicle is localized immediately beneath the ependymal lining of the
floor of the third ventricle, while the other is widely spread out in a superficial position
along the ventral surface of the optic chiasm. Lateral to the suprachiasmatic nucleus, the
majority of the GnRH fibers forming the septo-preoptico-infundibular tract gather into
two fascicles. The medial fascicle projects to the retrochiasmatic area, arcuate nucleus
(Arc) and inframammillary region, while the lateral fascicle terminates in the median
eminence (ME) (17).
Many fibers originating from the medial septal and preoptic regions pass through
the dorsomedial hypothalamus making up a periventricular subependymal GnRH
network that accounts for most of the extrahypothalamic projections. The scattered,
extrahypothalamic GnRH fibers innervate the following areas: mammillary complex,
raphe nuclei, medial nucleus of the amygdala, medial habenular nuclei, and ventral
tegmental area. In addition to these nuclei, the GnRH neurons compose a dense axonal
network around the aqueductus cerebri in the mesencephalic central gray (7). Moreover,
GnRH neurons that are located in the medial septum, the nucleus of diagonal band, the
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olfactory tubercle and along the medial surface of the hemispheres innervate the medial
layers of the olfactory bulb (7). This implies that the GnRH system may be involved in
the transmission or modulation of olfactory stimuli, many of which are closely related
to reproductive functions and behavior (18).
Nevertheless, the major efferent targets of the GnRH neurons are two
circumventricular organs: the OVLT and the median eminence (ME) (8, 19).
The OVLT is located at the rostral tip of the third ventricle (7, 8, 20) and receives
its innervation from the GnRH cell bodies of the adjacent MPN (21). The OVLT
extends dorsally approximately 1 mm from the optic chiasm and the roof of the optic
recess and it is made up of ependymal cells, neurons and glial cells. The ependymal
cells have tight junctions near their apical surfaces, which prevent the free flow of the
cerebrospinal fluid from the ventricle to the brain parenchyma. Similarly to other
circumventricular organs, the OVLT also contains a rich vascular network with
specialized fenestrated capillaries (20). The highly vascularized outer layer of the
OVLT together with the pia mater composes the external zone, which receives its blood
supply from the preoptic artery. The internal zone contains a complex neuropil with
neurons, fibers, glial elements, capillary loops and perivascular spaces (22). The OVLT
is innervated by axons containing several neuropeptides and neurotransmitters,
including
GnRH,
thyrotropin-releasing
hormone,
somatostatin,
dopamine,
norepinephrine, serotonin, acetylcholine, oxytocin and vasopressin (20). Although the
function of the OVLT is not entirely understood, the presence of estrogen receptors
implies its important role in estrogen feedback to the brain (23). The OVLT also
responds to osmotic challenges (24), which also suggests its important function in
osmotic regulation.
The ME is a rostro-caudally elongated vascular organ on the ventral surface of the
hypothalamus and it is innervated by GnRH axons via the septo-preoptico-infundibular
pathway. In the rat, its extension is over 2.5 mm from the most rostral to the most
caudal part (25). The ME receives an extremely rich blood supply from the superior
hypophyseal artery, which forms a dense, mosaic-like external plexus, and runs along
the ventral surface of the ME. The internal plexus arises from the external plexus and
forms several small, twisted capillary loops spanning between the external and internal
zones of the ME. On the surface or in the close vicinity of this fenestrated capillary
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network terminate the nerve endings of the hypothalamic homeostatic hormone
producing neurons. The capillaries are re-collected to the long portal veins that run on
the surface of the infundibular stalk, the direct continuation of the caudal end of the ME.
The long portal veins then supply for the portal circulation of the pituitary. The ME can
be divided three zones, starting from the bottom of the third ventricle: the ependymal
layer, the internal zone and the external zone (for a review see :(26)).
The main cell type of the ependymal layer is the ependymal cell, which has
microvilli on its apical surface. Adjacent ependymal cells overlying the ME are
interconnected by tight junctions, which limit passive exchange of materials between
the ventricle and the extracellular space of the ME. The other typical cell type of the
ependymal layer is the tanycyte, which has a bipolar shape. Its soma is located in the
ependymal layer, and its basal, column-like process reaches the basal part of the ME.
The internal zone is subdivided into a hypependymal layer and a fiber layer and in
addition to the supportive cells (subependymal cells and pituicytes) it is composed of
axons that originate from the Arc, magnocellular cells of supraoptic and paraventricular
nuclei, and aminergic neurons of the brainstem.
The external zone lies ventral to the internal zone and may be subdivided into a
reticular layer and a palisade layer. The reticular layer contains ependymal and glial
processes and axons of aminergic and parvicellular peptidergic systems. The palisade
layer lies at the surface of the ME. Here one can find nerve terminals of the
hypophysiotropic parvicellular systems, pituicytes, ependymal end-feet and capillaries.
The GnRH fibers preferentially localize in the lateral part of the ME (Figure 3.).
The majority of the GnRH fibers comes from the main septo-preoptico-infundibular
GnRH tracts, but the oral and caudal parts of the ME are also innervated by fibers
originate in the periventricular GnRH network (7). Within the nerve terminals, the
GnRH decapeptide is packed into large granular (also called dense core) vesicles (25).
Although GnRH is released into the portal capillary system, GnRH terminals in direct
contact with the pericapillary space can rarely be observed, except for the day of
proestrus (25). The rest of the time tanycyte processes separate the GnRH nerve endings
from the capillaries (25).
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Figure 3. GnRH immunostained photomicrographs demonstrate the main termination
fields of the GnRH axons. (A) The OVLT is located in the rostral tip of the third ventricle
(III.) above the optic chiasm. The GnRH axons (brown fibers) originating from the medial
preoptic nucleus densely innervate this area. (B) The coronal section illustrates the
topographical distribution of GnRH axons in the ME. GnRH fibers originating from the
POA preferentially localize in the lateral parts of the ME. (C) Parasagittal section through
the ME and its direct caudal continuation, the infundibular stalk (INF). The
photomicrograph demonstrates the rostro-caudal distribution of GnRH fibers. (III.: third
ventricle; INF: infundibular stalk; ME: median eminence; OVLT: organum vasculosum
laminae terminalis; Scale bars: 50 µm)
Besides the hypophysiotropic axons, neurotransmitter-containing fibers also
terminate in the areas of the OVLT and ME and release their peptide content in the
blood stream. For example in the ME, axons containing acetylcholine (27), gammaaminobutyric acid (GABA) (28), galanin (29), substance P, neurotensin (30, 31),
cholecystokinin (31), δ-sleep inducing peptide (32) and catecholamines (33) have been
identified. While the role of these transmitters in the ME is not fully understood, the
expression of their receptors on the glial and neuronal elements supports the view that
these neurotransmitters can exert local paracrine effects. Moreover, their high
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concentration in the hypothalamic portal circulation suggests that they may influence
pituitary functions as well (29, 30).
2.2.3. The afferent regulation of GnRH neurons
Although double immuno-labeling techniques identified various neurotransmitter
and neuropeptide containing synapses along the surface of GnRH neuronal perikarya
and dendrites, GnRH nerve cells receive a relatively sparse synaptic input. While the
interneurons in the hippocampus display 2000 – 16,000 synaptic connections (34, 35),
the ultra structural analysis revealed only few dozen synapses on the cell bodies and
dendrites of GnRH neurons (15, 36). This apparent paucity of synaptic inputs may
partly reflect the previously mentioned technical limits to detect immunocytochemically
the dendritic spines and filopodia of GnRH neurons where excitatory terminals may be
present at higher densities (37). It is also worth noting that the GnRH neurons of female
rats exhibit about a 2-fold greater proportion of plasma membrane occupied by synapses
than do those in males (36). Such a trend cannot be revealed at the level of the dendrites
(36). In addition, Campbell at al. failed to detect any dimorphism in the spine number at
any neuronal segment of biocytin filled GnRH neurons (16).
2.2.3.1. Innervation of GnRH neurons by afferents containing classical
neurotransmitters
Catecholamines. The effects of catecholamines on GnRH neurons is not regarded
as a conventional inhibitory or excitatory drive (for review see: (38, 39)). Instead,
catecholamines rather play a neuromodulator role that enables interactions within the
network depending upon its local concentrations (38). The primary substrate of
catecholamine biosynthesis is tyrosine. Among the four enzymes that are involved in
the biosynthetic pathway of catecholamines, the following ones are generally used as
marker enzymes of catecholaminergic neurons. Tyrosine hydroxylase (TH), which
produces dopamine from tyrosine, dopamine-β-hydroxylase (DBH), which converts
dopamine to noradrenaline and finally, phenylethanolamine- N-methyltransferase,
which can metabolize further noradrenaline to adrenaline. The ultrastructural analysis of
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the direct interactions between DBH-immunoreactive axons and GnRH dendrites has
demonstrated synaptic connection between noradrenergic fibers and GnRH neurons in
mice, although the number of the synaptic specializations was low (40). In rats, Léránth
and colleagues have also reported that a small number of axons immunopositive for TH
establishes synaptic contacts with GnRH somata and dendrites. The synapses belonged
to the symmetric (Gray II type) category (41). Since the biosynthetic pathways of
dopamine, noradrenaline and adrenaline synthesis commonly require TH, the presence
of TH-immunoreactivity in the catecholaminergic synapses of GnRH neurons (41) does
not show unequivocally the chemical nature and origin of the fibers. The cell bodies of
noradrenaline and adrenaline synthesizing neurons (forming the bulk of the
catecholaminergic nuclei A1–A7 and C1-C3, respectively) make up bilateral cell
clusters in the pons/medulla region of the brainstem, therefore these areas can be
considered as the only sources of the noradrenergic/adrenergic innervation to GnRH
neurons.
Excitatory amino acids. Glutamate and aspartate are important excitatory
neurotransmitters within the neuroendocrine hypothalamus (42) and exert powerful
actions on the neurohormone output of the GnRH network (43). Although a number of
immunoreactive ionotropic and metabotropic glutamate receptor subunits and their
encoding mRNAs could be identified in the hypothalamus and also in GnRH neurons
(44-46), the distribution of glutamatergic fibers and cell bodies in the CNS was
unknown for a long time due to the lack of appropriate glutamatergic neuronal markers.
The detection of the recently discovered vesicular glutamate transporters 1 and 2
(Vglut1, 2) provides a novel tool for neuromorphological studies to reveal glutamatergic
neuronal structures. These transporters show highly specific and saturable ATPdependent transport of glutamate into synaptic vesicles in vitro (47) and show subtypespecific anatomical distributions (48). Recently, Kiss et al. have described the direct
glutamatergic innervations of GnRH neurons (49). According to their report,
specifically the Vglut2-immunoreactive terminals often formed asymmetric synapses
with GnRH dendritic shafts but hardly ever with the somata. It is difficult to estimate
the putative sources of the glutamatergic innervation of GnRH neurons because of the
ubiquity of glutamatergic cell bodies in the CNS. Vglut1 and Vglut2 positive neurons
form two distinct subsets of glutamatergic neurons, with complementary patterns of
15
distribution (50). The lack of Vglut1-immunorective synapses on the surface of GnRH
neurons (49) suggests that the Vglut1 expressing glutamatergic nerves cells do not
contribute to the glutamatergic innervation of GnRH neurons. Moreover, as it has been
revealed by [3H]D-aspartate injection into the medial preoptic area, several
telencephalic (lateral septum, bed nucleus of the stria terminalis and amygdala) and
diencephalic (medial preoptic area, hypothalamic paraventricular, suprachiasmatic,
ventromedial, arcuate, ventral premammillary, supramammillary and thalamic
paraventricular nuclei) areas may give rise to the glutamatergic innervation of the POA,
and possibly, of GnRH neurons (51).
GABA. The main inhibitory neurotransmitter in the CNS as well as in the
hypothalamus is GABA (52). Léránth et al. demonstrated glutamic acid decarboxylase
positive axons forming synaptic contacts with GnRH positive neurons (53). All of these
synapses belonged to the symmetric type, which is a common feature of GABAergic
synapses (54). GnRH neurons express GABAA receptors (55) and results of in vivo
microinfusion experiments indicate that GABA inputs within the vicinity of the GnRH
cell bodies exert a powerful inhibitory influence on the secretory activity of GnRH
neurons in several species (56). Nevertheless, the role of GABAA receptor signaling on
the electrophysiological properties of postnatal GnRH neurons is somewhat
controversial in the literature. Moenter and colleagues have shown that endogenous
synaptic activation of GABAA receptors can be excitatory to mature GnRH neurons due
to their unusual intracellular ionic milieu (57). In contrast, other investigators found
evidence that GABA exerts inhibitory effects on adult GnRH neurons (58). This
controversy has been clarified somewhat by the recent observation that depolarizing
effects of GABA upon adult GnRH neurons only occur when all glutamatergic receptor
signaling is blocked within the brain slice preparation (57). These findings indicate the
importance of the balance between GABA and glutamate neurotransmission in
determining the firing activity of GnRH neurons. The major GABAergic innervation of
GnRH neurons is supposed to arise from the local GABAergic interneurons (38).
Serotonin (5-HT). Serotonin is synthesized by neurons located in the raphe nuclei
and it plays an important role in the generation of LH surge (59). Anatomical studies
have shown that about 5% of nerve terminals apposed to GnRH neurons are
serotonergic (60). Recently, Campbell and co-workers have revealed the median and
16
dorsal raphe nuclei as the sources of serotonergic innervation to a subset of GnRH
neurons that are located in the rostral POA (61). Nevertheless, the pharmacological
studies have provided somewhat conflicting results about the physiological role of
serotonin in the control of LH secretion (59) and some controversy also exists between
the results obtained by dual in situ hybridization and with single-cell microarray
experiments. The former method has failed to detect the mRNA for 5-HT1A, 5-HT1C
and 5-HT2 receptors (62), while the microarray has revealed the presence of 5-HT1A,
5-HT3A and 5-HT4 receptors in a subset of GnRH neurons (63).
2.2.3.2. Innervation of GnRH neurons by neuropeptide-containing afferents
Corticotropin-releasing hormone (CRH). It is well known that prolonged or
repetitive stress inhibits reproductive functions (64). Several neurotransmitters and
hypothalamic nuclei that control reproduction, also take part in the regulation of stress
responses (64). As the paraventricular nuclei of the hypothalamus are activated in all
stressful situations, CRH is one of the candidates that might account for the reduced
GnRH secretion during stress (for a review see: (64)). Its effect on GnRH neurons can
be exerted directly, given that CRH-immunoreactive axon terminals form synaptic
connections with GnRH dendrites (65). Although there are no experimental data
concerning the origins of CRH innervation to GnRH neurons, the possible sources can
be speculated by the comparison of the distribution of CRH neurons (66) and the results
of retrograde tract tracing studies (18, 67). The putative sources of the CRH innervation
to GnRH neurons are the followings: paraventricular nucleus of the hypothalamus,
central amygdala, bed nucleus of stria terminalis, medial and lateral preoptic area,
lateral hypothalamus, central grey, locus coeruleus, parabrachial complex and the area
of the A1 noradrenergic cell group.
GnRH. The light microscopic observations that GnRH neurons often form cell
groups containing two or three neurons led some investigators to examine the nature of
these anatomical associations. They found appositions between GnRH cell bodies in
female rodents and rhesus macaques; the number of the appositions did not correlate
with the stage of the female ovarian cycle (15). The electron microscopic examination
of GnRH-GnRH contacts did not reveal classical types of intercellular communication
17
or membrane specializations, including gap junctions. Nevertheless, in some cases
cytoplasmic bridges were found between GnRH neurons where the cytoplasms of
different neurons were apparently confluent (15). Moreover, several investigators have
observed GnRH processes forming synaptic contacts with other GnRH neurons (15, 68,
69). A putative functional significance of GnRH-GnRH connections may be in the
synchronization of neurohormone release from the network.
Kisspeptin. There is a growing number of reports on the role of kisspeptin in the
regulation of reproduction. Intracerebroventricular administration of kisspeptin, a
recently isolated product of the kiss-1 gene elicits rapid and profound elevation in serum
gonadotropin levels due to the increased activation of the secretory activity of GnRH
neurons (70, 71). This fast effect of kisspeptin is apparently exerted directly on GnRH
neurons because the potent receptor of this peptide (G protein coupled receptor-54)
occurs in the majority of GnRH neurons (70, 71). While the hypothalamus of the female
mouse exhibits more kisspeptin positive neurons and a denser plexus of kisspeptin
positive fibers than it is observable in males (72), the kisspeptin positive fibers establish
direct contacts with GnRH neurons in both genders, as revealed by dual-label
immunfluorescence (72). The possible sources of kisspeptin positive fibers to GnRH
neurons are the anteroventral periventricular nucleus and the Arc (73).
Neuropeptide Y (NPY). Axons immunoreactive for NPY establish synapses with
GnRH neurons (74). The physiological significance of NPY in the regulation of the
HPG axis and the possible sources of NPYergic innervation to GnRH neurons will be
discussed in 2.3.2.
Opioid peptides, β-endorphin (β-END). Substantial literature has been built up
around the role of β-END in the regulation of the gonadal axis (38). β-END results from
the enzymatic cleavage of the precursor pro-opiomelanocortin (POMC) protein. The
intracellular processing of POMC also gives raise to other peptide hormones including
α-melanocyte-stimulating hormone (αMSH) and adrenocorticotropic hormone (ACTH).
Neurons synthesizing β-END have been found in the Arc (75) and in the caudal region
of the nucleus of the solitary tract of the brainstem (76, 77), although only the β-END
synthesizing neurons of the Arc have been implicated in the regulation of GnRH
functions (38, 78). Léránth and colleagues investigated the connection between the
opioid system and GnRH neurons at the ultrastructural level. They used ACTH as a
18
marker peptide for opioid fibers and found that ACTH-immunopositive axons establish
synaptic connections mainly with the GnRH-immunoreactive dendrites in female rats
(78).
Substance P (SP). SP belongs to the peptide family of tachykinins which act as
excitatory neurotransmitters in the CNS (79). Neurons containing SP are widely
distributed throughout the brain (79). Some physiological experiments show the
profound effect of SP on the HPG axis; however, these data are controversial. While
intracerebroventricular administration of SP stimulates the release of LH, it has no
effect on FSH liberation (80). In contrast, SP decreases the elevation of LH as well as
FSH levels when injected subcutaneously to cycling female rats on the day of proestrus
(81). The direct effect of SP on GnRH neurons is supported by the presence of synapses
between SP terminals and GnRH neurons (82). The synapses belonged to the
asymmetric (Gray I type) category (82). As revealed from the use of retrograde tracer
injections into the POA, the possible sources of SP afferents to GnRH neurons may
include the Arc and the ventrolateral areas of the hypothalamus (82).
2.2.4. Colocalized neurotransmitters in GnRH neurons
Cholecystokinin (CCK), neurotensin (NT). Only one paper (31) reported the
presence of CCK and NT in GnRH neurons. Among substances localized within GnRH
neurons, galanin (38), CCK and NT (31) all exhibit pronounced sexual dimorphism and
steroid hormone dependence. In colchicin treated female adult rats quantitative multiple
immunolabeling experiments revealed the presence of CCK in more than half (54.5%)
and NT in one-third (29.4%) of GnRH neurons. The colocalization of both
neuropeptides is unambiguous in the GnRH fibers of the ME without colchicin
treatment. In contrast with females, in male rats NT was undetectable in GnRH cells and
only few (1.1%) GnRH neurons contained CCK. Interestingly, ovariectomy of adult
animals resulted in a complete disappearance of both neurotransmitters from GnRH
neurons, but CCK and NT expression could be restored to the normal level when
applying an estrogen regimen. According to Ciofi’s anatomical characterization, the
“multipeptidergic” GnRH neurons are concentrated immediately around the OVLT and
19
the optic chiasm but the functional significance of this distribution pattern remains
obscure.
GABA. In addition to existing proof for the GABAergic innervation of GnRH
neurons (53), Tobet and colleagues have demonstrated that GABA is transiently present
in a large populations of the migrating GnRH neurons during the embryonic
development of rats and mice (83). Data exist to indicate that GABA is an essential
regulator for the migration of GnRH neurons. The involvement of GABAA receptor in
this process is well established. The migration rate of GnRH neurons out of the
olfactory pit is inhibited in the embryos by the subcutaneous administration of the
GABAA receptor agonist muscimol to pregnant mice (84). Moreover, in the same
paradigm, the antagonist bicuculline leads to a disorganized distribution of GnRH cells
in the forebrain and a concomitant dissociation of GnRH cells from fibers of guidance
(84). The understanding of the role of GABA during development is further
complicated by the presence of GABAB receptors, as well, along the GnRH migratory
route (85). The developmental role of GABA appears to include inhibiting the rate of
GnRH neuronal migration out of the olfactory pit, controlling the association of GnRH
neurons to fibers of guidance, and regulating GnRH fiber extension toward the ME (85).
Galanin. The highest galanin concentrations in the brain are observed in the
hypothalamus, particularly in the ME (86). Galanin producing neurons that project to
the ME, as do classical hypophysiotropic neurons, release their galanin content into the
portal circulation. Beyond these neurohormonal properties, galanin peptide and mRNA
are expressed by a subpopulation of GnRH neurons in a sexual dimorphic manner (29,
38). In female rats with normal gonadal cycle, galanin mRNA expression is the highest
on the afternoon of proestrus (87), suggesting the profound effect of estrogen on the
expression of this gene. Indeed, ovariectomy results in a decrease of galanin mRNA
expression, while estrogen replacement restores the transcript to intact levels. The
positive feedback effect of estrogen stimulates GnRH and galanin mRNA levels
sequentially at the time of the LH surge (38), although the physiological significance of
this differential regulation needs further clarification.
Insulin-like growth factor-1 (IGF-1). The neurotrophic factor IGF-I is thought to
play a role in the onset of reproductive ability at puberty and the control of reproductive
function throughout adult life (88). It is believed that these effects are mediated at least
20
in part by the activation of GnRH neurons by IGF-I, but the interactions of IGF-I with
GnRH neurons in vivo are unknown. Recently, Miller and Gore have shown that more
than two-thirds (78%) (89) of GnRH neurons co-express IGF-1 protein and the majority
of them also possess the receptor for this neurotrophic factor (90). In the GnRH nerve
terminals at the level of ME, only a weak if any IGF-1- immunoreactivity can be
observed (89). The in vivo detected increase in IGF-1 gene expression between different
stages of puberty (89) and the decreasing expression of this substance during the
reproductive senescence (89) suggest that the presence of IGF-1 in GnRH neurons may
indeed play an important role in the regulation of gonadal maturity.
Δ-sleep-inducing-peptide (DSIP). DSIP is presented in different areas of the brain,
including the hypothalamus and has been shown to induce slow-wave (delta) sleep in
various species. The early physiological experiments suggested that DSIP stimulates LH
release at the level of the hypothalamus. Intracerebroventricular injection of DSIP to
ovariectomized and estrogen- and progesterone-treated rats elicits rapid LH release (91).
Moreover, its in vitro administration to hypothalamic ME fragments results in increased
GnRH release (92). In contrast, DSIP has no effect on either basal or GnRH-induced in
vitro LH release when it is applied to hemi pituitaries of ovarian steroid-primed rats
(91). One laboratory have demonstrated the colocalization of DSIP in GnRH neurons
and in GnRH fibers at the level of the ME in several species including cat (93), rat (32),
rabbit (94) and human (95). Interestingly, the consequences of the reported colocalization phenomenon have not been studied further or published.
21
Figure 4. The schematic representation of a GnRH neuron illustrates synaptic inputs and
co-expressed
neurotransmitters.
(5-HT:
serotonin;
β-END:
β-endorphin;
CCK:
cholecystokinin; CRH: corticotropin-releasing hormone; DSIP: delta sleep-inducing
peptide; GABA: gamma-aminobutyric acid, GLU: glutamate, GnRH: gonadotropinreleasing hormone, IGF-1: Insulin-like growth factor-1, NPY: neuropeptide Y; NT:
neurotensin; SP: substance P; TH: tyrosine hydroxylase)
2.3. Proposed novel neurotransmitter systems in the regulation of GnRH
neurons
2.3.1. Involvement of acetylcholine (ACh) in the regulation of the reproduction
The markers of the central cholinergic systems are choline acetyltransferase (ChAT)
which is involved in the synthesis of Ach, and vesicular acetylcholine transporter
(VAChT) which accumulates ACh into secretory vesicles (96). The visualization of
ChAT-positive cell bodies by immunocytochemistry or by in situ hybridization defines
five major cholinergic neuronal groups in the CNS. These include: (i) the efferent
cranial nerve nuclei and motoneurons of the spinal cord; (ii) the parabrachial complex;
(iii the brainstem reticular complex; (iv) the neostriatral complex; and the (v)
cholinergic neurons of the basal forebrain (97, 98). Beyond these principal cholinergic
22
cell groups ChAT-immunoreactivity has been localized within neurons of the medial
habenula, hippocampus, neocortex (97, 98) and of the Arc (99). Retrograde (100), as
well as anterograde tract tracing (18, 67) studies have revealed the connection between
some of the above-delineated cholinergic areas and the POA, suggesting the
contribution of cholinergic neurons to the regulation of GnRH neurons. Indeed, a large
body of evidence exists to indicate the involvement of ACh in the regulation of
reproductive events, including male and female sexual behavior (101-106) and
gonadotropin secretion (107-109). Atropine, the muscarinic acetylcholine receptor
antagonist blocks both spontaneous and reflex ovulation (107). In the spontaneously
ovulating species rat, atropine implants in the anterior lateral hypothalamus disturb the
ovarian cycles and prolong the diestrous phase (108). While the site(s) and
mechanism(s) of the cholinergic actions upon the reproductive axis are not clear, they
appear to be exerted, at least in part, at the hypothalamic level. Simonovic and
colleagues examined the ACh-stimulated FSH release from halved adenohypophyses of
male rats and found enhanced FSH release only if the adenohypophyses were coincubated with hypothalamic fragments (109). On the other hand, nanomolar
concentrations of ACh could markedly stimulate GnRH release from dissected
mediobasal hypothalamic fragments in vitro and this effect could be abolished by the
nicotinic ACh receptor antagonist hexamethonium (110). The presence of both
muscarinic and nicotinic receptors have been reported on at least a subpopulation of
GnRH neurons (63) which suggests a direct effect of ACh on GnRH neurons; however
the anatomical proof for the direct cholinergic innervation is missing from the literature.
In the light of evidence for central cholinergic actions on the reproductive axis,
and considering the presence of muscarinic and nicotinic ACh receptors in GnRH
neurons, in studies under specific aim 1 of this thesis we postulated the direct
innervation of GnRH neurons by cholinergic pathways. Male rats were used as
experimental animals, and immunocytochemical experiments were performed at the
light and electron microscopic levels to investigate the putative cholinergic innervation
of GnRH neurons.
23
2.3.2. The role of the NPY in the regulation of reproduction
Neuropeptide Y has a bimodal effect on the reproductive axis. Depending on the
mode of its administration, further, the gender, the age and/or the hormonal status of the
experimental animal and the time course of the treatment, NPY can exert both
suppressive end excitatory effects on the HPG axis. Chronic increase in NPY tone
inhibits gonadotropin release (111), delays sexual maturation (112) and suppresses
estrous cyclicity (113). In contrast, the direction of acute NPY effects is markedly
influenced by the sexual steroid status of the experimental animals. Likewise, in
castrated rats, rabbits, and monkeys, central administration of NPY decreases
gonadotropin secretion (114-119), whereas, in intact or gonadectomized and steroidprimed rodents, NPY increases serum gonadotropin levels (111, 114, 117, 119-121).
Neuropeptide Y plays important roles in the regulation of reproduction via acting
at multiple levels of the HPG axis (122). The majority of work indicates that NPY
mainly acts centrally to modulate gonadotropin secretion (111-116, 118-121, 123-127)
and the targets of the central NPY action are directly the GnRH neurons of the POA (74,
128-131). However, evidence also exists to support the direct stimulation of pituitary
gonadotrops by NPY (123, 132-134). This multilevel effect of NPY may be due to the
different origins of NPYergic fibers to GnRH neurons. In the rodent brain, NPYimmunoreactive perikarya or fibers were found in most brain areas except the
cerebellum (135). The highest number of NPY-synthesizing neurons can be found in the
hypothalamic Arc, moreover a high number of NPY nerve cells can be detected in the
cerebral cortex, dorsal hypothalamic area, parvicellular part of the ventrolateral
geniculate nucleus, locus coeruleus and the nucleus of the solitary tract (135). The
possible sites of origin of NPY fibers may be diverse and in order to clarify the cause of
the multiple effect of NPY on HPG axis it would be useful to reveal the relative
contribution of the various sources of NPYergic fibers to GnRH neurons. The
determination of possible sources of NPY origin by retrograde tract-tracing studies is
largely complicated by the scattered distribution of GnRH neurons in a relatively large
area. Thus, as an alternative strategy, in studies under specific aim 2, we have selected
topographical markers, which are only co-expressed with NPY in distinct brain regions.
Then we used the co-expression of these markers in NPY fibers as an indication of their
24
sources of origin and for the estimation of the relative contribution of the putative
sources of the NPYergic fibers to GnRH neurons. In our experiments, we used male
GnRH-GFP mice in which a portion of the mouse GnRH promoter directs the selective
expression of GFP to the majority of GnRH neurons (136). We injected four neonatal
animals with a solution of monosodium glutamate (MSG) that caused an extensive
ablation of the neuronal population of the Arc (137-140). Using dual-labeling light
microscopic immunocytochemistry, we compared the frequency of NPY- and agoutirelated protein (AGRP)-immunoreactive (see below) axonal contacts on GnRH neurons
in neonatally MSG-treated mice (with lesioned Arc) with that of the untreated controls.
In this way, we could estimate the ratio of NPY afferents originating in the Arc.
Moreover, we carried out a series of triple-label fluorescent studies in intact male
GnRH-GFP transgenic mice using brain region-specific topographical markers. This
way, we tried to identify the relative importance of NPY neurons located in the Arc and
those corresponding to catecholaminergic nuclei of the brainstem, two possible sources
of NPY afferents to GnRH neurons. AGRP was used as a marker of NPY fibers
originating from the Arc. We used the noradrenaline-synthesizing enzyme DBH, as a
topographic marker of NPY fibers with brainstem origin. The ratios of NPYimmunoreactive neuronal contacts on the surface of GnRH-GFP neurons that contained
DBH or AGRP were determined.
Neuropeptide Y may be one of the essential messenger molecules that serve as a
communication bridge between the neural processes that regulate reproduction and
those that maintain energy homeostasis (141). Chronic central increase in NPY tone has
been implicated in hypogonadism that accompanies malnutrition (111, 113). Moreover,
the significant inhibition of the gonadotropic axis can be observed in genetically obese
mice (142). The Arc is involved in the integration of the satiety signals of the periphery
(143) and contains a high number of NPY neurons (135), that co-express AGRP (137,
144). AGRP also takes part in the regulation of energy metabolism as an orexigenic
signal (143) and beyond this function, its central impact on the HPG axis is suggested as
well (145).
Since NPY afferents with Arc origin contain AGRP, we carried out dual-label
immuno-electron microscopic studies to reveal the synaptic communication between
25
NPY fibers of Arc origin (using AGRP as a neurochemical marker for NPY neurons of
the Arc) and GnRH-immunoreactive neurons.
2.3.3. Glutamate as a proposed autocrine/paracrine regulator synthesized by
GnRH neurons
The recent discovery of the three vesicular glutamate transporters which
selectively accumulate glutamate into secretory vesicles has enabled the histochemical
identification of glutamatergic fibers in the central and in the peripheral nervous
systems. The abundance of glutamatergic neurons expressing Vglut2 mRNA in the
hypothalamus (146, 147) and the high density of Vglut2-immunoreactive axon
terminals (146) and ionotropic glutamate receptors (148, 149) in the external zone of the
ME raised the possibility that peptidergic neuroendocrine cells regulating anterior
pituitary functions may secrete glutamate as an autocrine/paracrine modulator of their
neurohormone output. Corroborating this notion, beside the dense core vesicle (DV)containing (peptidergic and catecholaminergic) and electron lucent vesicle containing
(GABAergic, glutamatergic and cholinergic) nerve terminals, “mixed” phenotyped
nerve endings could also be detected by electron microscopy at the level of the ME (26).
Moreover, the POA of postnatal rodents is also populated by high numbers of
glutamatergic neurons containing Vglut2 mRNA (146, 147).
In studies under specific aim 4, we examined the possibility whether mature
GnRH neurons in the POA possess glutamatergic characteristics. We examined the
expression of Vglut2 mRNA in GnRH mRNA-expressing neurons with double-label in
situ hybridization histochemistry. In addition, we addressed the presence of Vglut2immunoreactivity in the axon terminals of GnRH neurons with the aid of confocal laser
scanning microscopy. Finally, we determined the subcellular localization of Vglut2
protein in the hypophysiotropic axon terminals of ME with pre-embedding immunoelectron microscopy.
26
3. Specific aims
Studies in this thesis were aimed at the identification of novel neurotransmitter systems
in the neuronal regulation of the GnRH system.
1. We addressed the direct innervation of GnRH neurons by cholinergic afferent
pathways in male rats at light and electron microscopic levels.
2. We addressed the sources of origin of NPY-containing afferents to GnRH
neurons in male GnRH-GFP transgenic mice.
a. Using neonatally monosodium glutamate treated (with lesioned Arc) and intact
mice we estimated the ratio of NPY afferents originating in the Arc.
b. Using intact mice we carried out a series of triple immuno-fluorescent studies in
GnRH-GFP transgenic mice by brain region-specific topographical markers to
identify the relative importance of NPY neurons located in the Arc and those
corresponding to catecholaminergic nuclei of the brainstem, two putative
sources of NPY afferents to GnRH neurons. AGRP was used as a marker of
NPY fibers originating from the Arc. We used the noradrenaline-synthesizing
enzyme DBH, as a topographic marker of NPY fibers of brainstem origin.
3. We performed ultrastructural studies to reveal the synaptic communication
between NPY/AGRP containing fibers and GnRH neurons.
4. We addressed the putative glutamatergic phenotype of GnRH neurons.
a. We examined the expression of Vglut2 mRNA in GnRH mRNA expressing
neurons with double-label in situ hybridization histochemistry.
b. We addressed the presence of Vglut2-immunoreactivity in the axon terminals of
GnRH neurons with the aid of confocal laser scanning microscopy.
c. We determined the subcellular localization of Vglut2 protein in the
hypophysiotropic axon terminals of ME with immuno-electron microscopy.
27
4. Materials and methods
All of the experiments were carried out in accordance with the European
Communities Council Directive of 24 November 1986 (86/609/EEC) and were
reviewed and approved by the Animal Welfare Committee at the Institute of
Experimental Medicine.
4.1. Direct innervation of GnRH neurons by cholinergic afferent pathways in
male rats
4.1.1. Double-labeling immunocytochemical detection of cholinergic fibers and
GnRH neurons at the light microscopic level
Animals. Adult male Wistar rats [N=3; 260–280 g body weight (BW)] were
purchased from the local breeding colony of the Medical Gene Technology Unit of the
Institute of Experimental Medicine. They were kept under a 12 h light-12 h dark
schedule (lights on at 07:00 h, lights off at 19:00 h), in a temperature (22 ± 2 °C) and
humidity (60 ± 10%) controlled environment with free access to laboratory rat food
(Sniff Spezialdiaeten GmbH, Soest, Germany) and tap water.
Tissue preparation. The animals were deeply anesthetized with sodium
pentobarbital (45 mg/kg BW, i.p.) and perfused via the ascending aorta first, with 20 ml
of 0.1 M phosphate buffered saline (pH 7.4) (PBS) and then with 100 ml fixative
solution containing 4% paraformaldehyde (Sigma Chemical Co., St. Louis, MO) in PBS.
Brains were rapidly removed and immersed into 30% sucrose in PBS overnight
and snap frozen on powdered dry ice. Then 30-μm thick, free-floating coronal sections
were prepared from the POA region with a freezing microtome (Leica Microsystems,
Wetzlar, Germany).
The free-floating sections were collected in anti-freeze solution (30 V/V % ethylene
glycol, 25 V/V % glycerol in 0.05 M phosphate buffer) and stored at minus 20 oC until
use.
Immunocytochemistry. The sections were rinsed in PBS, incubated in 10%
thioglycolic acid for 30 min to suppress tissue argyophilia (150) and then pretreated
28
with a solution of 0.5% H2O2 and 0.2% Triton X-100 in PBS for 30 min. Non-specific
binding of the antibodies was reduced using a 2% normal horse serum blocker in PBS
for 10 min. All primary and secondary antibodies were diluted with 2% normal horse
serum in PBS or Tris buffered saline (TBS; 0.1 M Tris-HCl with 0.9% NaCl; pH 7.8),
which also contained 0.1% sodium azide as a preservative. To detect cholinergic
neuronal elements, the sections were incubated in polyclonal anti-ChAT antiserum
raised in a goat (AB144P; 1:1000; Chemicon, Temecula, CA) for 72h at 4 °C. The
primary antibodies were reacted with donkey, biotin-SP-anti-goat IgG (1:1000; Jackson
ImmunoResearch Laboratories, West Grove, PA) for 2 h. Alternatively, in a second set
of sections the cholinergic system was detected using a rabbit antiserum against VAChT
(V5387; 1:10,000; Sigma), followed by biotin-SP-anti-rabbit secondary antibodies
(Jackson ImmunoResearch Laboratories). The biotinylated secondary antibodies were
reacted with the ABC Elite working solution (Vector Laboratories, Burlingame, CA;
1:1000 dilutions of solutions A and B in 50 mM TBS; pH 7.6) for 1 h. Then the sections
were rinsed in TBS and the color reaction visualized with a developer containing 0.05%
diaminobenzidine (DAB), 0.15% ammonium nickel (II) sulfate and 0.005% H2O2 in
TBS. Finally, the reaction product was silver intensified according to Liposits et al.
(150) to obtain a final black reaction product. Briefly, the sections were rinsed for
several times in 2% sodium acetate and then placed into a silver developer. The
developer consisted of equivalent amount of 5% sodium carbonate and silver
development solution (0.15% formalin-solution, 0.2% ammonium nitrate, 0.2% silver
nitrate, 1% tungstosilicic acid, dissolved in distilled water). The progress of the
intensification reaction was monitored under the light microscope and terminated in
0.5% acetic acid solution when the color of the originally purple nickel-DAB precipitate
turned black. After several rinses in 2% sodium acetate, the sections were dipped into
cold 0.05% of gold-chloride (2x5 min) to stabilize the black silver- ammonium nickel
(II) sulfate -DAB precipitate and then into 3% sodium thiosulfate (2x5 min) solutions to
remove the residual metallic ions.
After the detection of cholinergic neuronal elements, the sections were transferred
into rabbit LR-1 anti-GnRH antibodies (1:10,000 a gift from Dr. R.A. Benoit, Montreal,
Canada) for 2 days at 4 oC. Immunoreactivity for GnRH was visualized with the ABC
method, with the application of biotin-SP-anti-rabbit secondary antibody (1:1000;
29
Jackson ImmunoResearch Laboratories, West Grove, PA) and a developer consisting of
0.025% DAB and 0.0036% H2O2 in TBS which yielded a brown reaction product.
Beyond the use of well-characterized primary antibodies in this study against the
cholinergic system (99, 151), the specificity of labeling was further indicated by the
absence of immunocytochemical signal if the primary or the secondary antibodies were
omitted from the staining procedure.
The dual-immunostained sections were mounted on microscope slides, dehydrated
in ethanol, coverslipped with DePeX mounting medium (Fluka Chemie, Buchs,
Switzerland) and studied with an Axiophot microscope (Zeiss, Göttingen, Germany)
equipped with an RT Spot digital camera (Diagnostic Instruments, Sterling Heights, MI).
4.1.2. Double-labeling immunocytochemical detection of cholinergic fibers and
GnRH neurons at the electron microscopic level
Animals. Adult male Wistar rats (N=3; 260–280 g BW) were purchased from the
local breeding colony of the Medical Gene Technology Unit of the Institute of
Experimental Medicine. The animals were kept under a 12 h light-12 h dark schedule
(lights on at 07:00 h, lights off at 19:00 h), in a temperature (22 ± 2 °C) and humidity
(60 ± 10%) controlled environment with free access to laboratory rat food (Sniff
Spezialdiaeten GmbH, Soest, Germany) and tap water.
Tissue preparation. The deeply anesthetized animals were perfused via the
ascending aorta with 20 ml of 0.1 M PBS followed by 50 ml mixture of 2% freshly
depolymerized paraformaldehyde (Sigma Chemical Co., St. Louis, MO) and 4%
acrolein (Sigma Chemical Co., St. Louis, MO) solution in 0.1 M PBS. The brains were
postfixed in 4% paraformaldehyde for 24 h. Preoptic sections were prepared from the
tissue blocks at 50 µm with a Vibratome (Technical Products International, St. Louis,
Mo., USA).
Immunocytochemistry. Vibratome sections were pretreated with 0.5% sodium
borohydride in PBS for 30 min to eliminate residual aldehydes. Then they were
infiltrated with sucrose for cryoprotection (15% for 1 h and then, 30% overnight) and
permeabilized by three repeated freeze-thaw cycles on liquid nitrogen. The
immunocytochemical dual-labeling procedures were carried out as described for light
30
microscopy. The dual-labeled sections were treated with 1% osmium tetroxide in 0.1 M
PBS for 30 min and dehydrated in an ascending series of ethanol. A 30-min contrasting
step using 1% uranyl acetate in 70% ethanol was inserted in this procedure and the fully
dehydrated sections were immersed in propylene oxide. Finally, the sections were
infiltrated with TAAB 812 Embedding resin (TAAB Laboratory Equipments Ltd.,
Aldermaston, Berks, UK) and flat embedded on liquid release agent (Electron
Microscopy Sciences) coated slides. The coverslipped sections were polymerized at 60
o
C for 24 h. Ultra thin (50-60 nm) sections were cut with Leica Ultracut UCT
ultramicrotome (Leica Microsystems, Wetzlar, Germany), collected onto Formvarcoated single-slot grids and contrasted with a stabilized solution of lead citrate. Digital
images were captured with a cooled CCD camera.
The electron microscopic digital images were processed with the Adobe Photoshop
7.0 software.
4.2. Determining of the origin of NPY-containing afferents to GnRH neurons
in male GnRH-GFP transgenic mice
4.2.1. Experimental animals
To examine the origin of NPY afferents to GnRH neurons, studies were carried
out in a transgenic mouse strain in which a portion of the mouse GnRH promoter directs
the selective expression of GFP to the majority of GnRH neurons (136). Adult (8 wk
old) male GnRH-GFP transgenic mice (N= 16) were bred and housed at the Institute of
Experimental Medicine under conditions of 12 h day-12 h night schedule (lights on at
07:00 h, lights off at 19:00 h), in a temperature (22 ± 2 °C) and humidity (60 ± 10%)
controlled environment, with free access to laboratory food (Sniff Spezialdiaeten GmbH,
Soest, Germany) and tap water.
GnRH-GFP mice treated neonatally with monosodium glutamate. To eliminate
NPY fibers arising from the Arc, the chemical lesion of this region was performed by
MSG treatment of four neonatal mice. The neonatal animals were injected s.c. with
increasing volumes of 8% MSG solution (dissolved in water), using a treatment
paradigm adapted from Légrádi et al. (139): 4 mg/g body weight MSG solution on
31
postnatal days 1 and 3; followed by 8 mg/g body weight MSG solution on postnatal
days 5, 7, and 9. The treated animals and four age-matched untreated mice were allowed
to reach postnatal wk 8 and then killed by transcardiac perfusion. Brain tissues from the
two groups were processed in parallel for comparative histological studies of the Arc
and POA.
Tissue preparation. The animals were deeply anesthetized with sodium
pentobarbital (45 mg/kg BW, i.p.) and perfused via the ascending aorta first, with 20 ml
of 0.1 M PBS and then with 100 ml fixative solution containing 4% paraformaldehyde
(Sigma Chemical Co., St. Louis, MO) in PBS.
Brains were rapidly removed, cryoprotected in 30% sucrose in PBS overnight and
snap frozen on powdered dry ice. Then 30-μm thick, free-floating coronal sections were
prepared from the POA and Arc regions with a freezing microtome (Leica
Microsystems, Wetzlar, Germany).
The free-floating sections were collected in anti-freeze solution (30 V/V % ethylene
glycol, 25 V/V % glycerol in 0.05 M phosphate buffer) and stored at minus 20 oC until
use.
4.2.2. Single-labeling immunocytochemical detection of NPY- and AGRPcontaining fibers in the POA of neonatally MSG-treated mice vs. untreated controls
Coronal sections through the Arc were obtained from neonatally MSG-treated
mice as well as untreated controls and stained with cresyl violet to verify the chemical
lesion in the former group. Sections containing the POA were used for comparative
immunocytochemical studies of NPY- and AGRP-immunoreactivities in these two
groups, as outlined below. Sections were rinsed in PBS and pretreated with 10%
thioglycolic acid (Sigma) for 30 min to suppress tissue argyophilia (150) and with 0.5%
H2O2 in PBS for 15 min to eliminate endogenous peroxidase activity. Nonspecific
antibody binding sites were blocked with 2% normal horse serum then tissues
permeabilized in 0.4% Triton X-100 (Sigma) in PBS for 20 min. Every other preoptic
section was transferred into a 1:100,000 dilution of a polyclonal sheep antiserum to
NPY (FJL no. 14/3A; diluted with Triton X-100-free blocking reagent), which was
generously provided by Dr. István Merchenthaler (Wyeth Research, Collegeville, PA),
32
and applied to the sections for 48 h at 4 oC. The remaining sections were incubated in a
1:8000 dilution of a polyclonal rabbit antiserum to human AGRP (H-003-053; Phoenix
Pharmaceuticals, Inc., Mountain View, CA). Immunoreactivities were detected after
tissue incubations in species-specific biotinylated secondary antibodies (Jackson
ImmunoResearch Laboratories, West Grove, PA; 1:1000), then in ABC-Elite reagent
(Vector Laboratories, Burlingame, CA; 1:1000 dilutions of solutions A and B in TBS),
for 1 h each. The peroxidase developer contained 10 mg DAB, 30 mg ammonium nickel
(II) sulfate, and 0.002% H2O2 in 24 ml TBS. Then silver intensification of the
peroxidase reaction product was carried out as described in section 4.1.1.
4.2.3. Double-labeling immunocytochemical detection of NPY- and AGRPcontaining fibers in contact with GnRH neurons of neonatally MSG-treated mice vs.
untreated controls
The majority of single-labeled sections immunostained for AGRP or NPY were
processed further for the immunocytochemical identification of GnRH neurons, using
sequential incubations in rabbit polyclonal antibodies to GnRH (LR-1; 1:25,000; gift
from Dr. R. Benoit, Montreal, Canada) for 48 h, biotinylated secondary antibodies
(Jackson ImmunoResearch Laboratories; 1:1000) for 1 h, and then ABC Elite working
solution for 1 h. The developer solution contained 10 mg DAB and 0.001% H2O2 in 50
ml TBS. The immunostained sections were mounted on microscope slides, dehydrated
with ethanol, coverslipped with DPX mounting medium (Fluka Chemie, Buchs,
Switzerland), and studied with an Axiophot microscope (Zeiss, Göttingen, Germany)
equipped with an RT Spot digital camera (Diagnostic Instrument, Sterling Heights, MI).
Quantitative microscopic analysis of dual-labeled sections. Sections dualimmunostained for NPY and GnRH from MSG-treated and untreated control animals,
as well as sections dual-labeled for AGRP and GnRH from control animals were
mounted on microscope slides in a random sequence. The sections were individually
labeled with a code, and in each, the number of axonal contacts along the outlines of
GnRH neurons was determined. The analysis was carried out using a x100 objective
lens with immersion oil by an investigator who was blind to the experimental
procedures and the sequence of section mounting. Studies of sections in random
33
sequence ensured the homogenous analysis of groups to compare. A case was
considered as a “contact” based only on highly stringent criteria that were applied
consistently. The axon and GnRH neuron had to occur in the same focal plane without
the presence of an intervening gap, and instances of partial overlap were excluded from
the counting. Two different approaches were used in parallel to estimate the ratio of
NPY afferents of Arc origin to GnRH neurons. First, the average number of AGRPimmunoreactive neuronal appositions to GnRH cells was calculated in intact animals
(after the analysis of 561 GnRH neurons at high power) and compared with the mean of
NPY-immunoreactive contacts on individual GnRH cells (with 546 GnRH neurons
analyzed). Second, the average number of NPY-containing juxtapositions to single
GnRH cells was calculated for neonatally MSG-treated mice (analysis included 408
GnRH neurons) and related to the number determined for the untreated controls. The
summarized data from the light microscopic double-labelings were analyzed with oneway ANOVA (p<0.05 was considered significant) and the results of the analysis were
expressed as mean ± SEM.
4.2.4. Immunofluorescent-labeling of AGRP/NPY and DBH/NPY contacts with
GnRH-GFP neurons in intact mice
To determine the relative contributions of the Arc and brainstem to the innervation
of GnRH neurons, a triple-fluorescence strategy was used, enabling the detection of
NPY in axonal contacts to GnRH neurons simultaneously with the immunofluorescent
visualization of a site-of-origin-specific topographic marker. The presence of AGRP in
NPY axons showed their origin in the Arc, whereas the identification of the
adrenergic/noradrenergic biosynthetic enzyme DBH in NPY fibers served as a selective
marker for NPY axons arising from catecholamine cell groups of the brainstem.
Although DBH is present exclusively in adrenergic/noradrenergic cells of the brainstem,
the fine distinction among various catecholamine cell groups that express NPY
differentially (152, 153) was not possible via the use of this marker. First, a cocktail of
two primary antibodies (sheep anti-NPY, 1:12,000 and rabbit anti-AGRP, 1:4000; sheep
anti-NPY, 1:12,000, and rabbit anti- DBH, 1:8000) (154) was applied to the sections for
48 h at 4 oC. This was followed by sequential incubations in a mixture of secondary
34
antibodies,
donkey
biotin-conjugated
anti-rabbit
IgG
(1:1,000;
Jackson
ImmunoResearch Laboratories), and antisheep-Cy3 (1:250; Jackson ImmunoResearch
Laboratories), and then in the ABC-Elite working solution (Vector Laboratories,
Burlingame, CA; 1:1000 dilutions of solutions A and B in 50 mM TBS; pH 7.6) for 1 h
each. The detection of AGRP and DBH fibers was completed by treating sections with
biotin tyramide working solution (described below) for 30 min and finally with avidinconjugated AMCA fluorochrome (1:200; Vector Laboratories).
To prepare the biotin tyramide solution, a stock solution was first synthesized
according to instruction by Adams (155). This stock was stored in frozen aliquots and
diluted at 1:1000 in TBS/0.005% H2O2 immediately before use.
The GFP expression in GnRH neurons unequivocally revealed the cell bodies and
primary dendrites of the GnRH nerve cells, thus we did not have to apply a third
immunolabeling against the GnRH decapeptide.
Following steps of the dual-immunofluorescent labeling procedure, sections were
mounted on microscope slides and coverslipped with Vectashield mounting medium
(Vector Laboratories). Fibers immunoreactive for NPY appeared in red, AGRP-, or
DBH-containing axons appeared in blue, and GnRH-GFP neurons appeared in green
using the following epifluorescent filter sets:
Fluorochrome
Excitation filter (nm)
Band pass filter (nm)
Emission (nm)
Cy3
540–590
595
600–660
AMCA
320–400
400
430–490
GFP
460–500
505
510–560
Quantitative microscopic analysis of the fluorescent specimen. To determine the
percent ratios of dual-labeled NPY fibers in each set of experiment, individual NPY
juxtapositions were analyzed at high power and categorized as being either positive or
negative for the topographic markers, AGRP or DBH. Each study included the analysis
of more than 200 NPY-containing neuronal contacts with GnRH neuronal cell bodies
and proximal dendrites and percent data were expressed as mean from four animals
±SEM. The fluorescent specimen was analyzed using an x100 objective lens with
35
immersion oil. First, NPY contacts apposed to GnRH profiles were identified using the
same stringent criteria as for bright-field microscopy. Then in each contact identified,
we determined whether the NPY axon contained AGRP (or DBH) by switching
between the fluorescent filter sets.
4.3. Electron microscopic analysis of AGRP afferents to GnRH neurons in
intact male, GnRH-GFP mice
Animals. For electron microscopic experiments we used male mice from the same
GnRH-GFP mouse strain (N=3) as for the light microscopic experiments (section 4.2.1.).
Tissue preparation. The deeply anesthetized animals were perfused via the
ascending aorta with 20 ml of PBS followed by 50 ml mixture of 2% freshly
depolymerized paraformaldehyde (Sigma Chemical Co., St. Louis, MO) and 4%
acrolein (Sigma) solution in PBS. The brains were postfixed in 4% paraformaldehyde
for 24 h. Preoptic sections were prepared from the tissue blocks at 50 µm with a
Vibratome (Technical Products International, St. Louis, Mo., USA).
Immunocytochemistry. A combination of a preembedding immuno-peroxidase
histochemistry and immuno-gold reaction was used for the ultrastructural analysis of
neuronal contacts between AGRP-immunoreactive axons and GnRH cells of the POA.
Sections were treated with 0.5% sodium borohydride in PBS for 30 min to eliminate
residual aldehydes, infiltrated with sucrose for cryoprotection (15% for 1 h and then,
30% overnight), and permeabilized by three repeated freeze-thaw cycles on liquid
nitrogen. The immunocytochemical detection of AGRP used sequential incubations in
primary antibodies to AGRP (Phoenix; 1:2000) for 4 days, biotinylated anti-rabbit
antibodies (Jackson ImmunoResearch Laboratories; 1: 1000) for 2 h, and ABC working
solution (Vector Laboratories, Burlingame, CA; 1:1000 dilutions of solutions A and B
in 50 mM Tris buffer; pH 7.6) for 1 h. Triton X-100 was omitted from all solutions and
immunoreactivity to AGRP was visualized with a DAB in the peroxidase developer.
The detection of AGRP axons was followed by a 4-day incubation (4 °C) of sections in
rabbit polyclonal antibodies to GnRH (LR-1; 1:25,000), a 30-min blocking step using
0.1% cold-water fish gelatin (Electron Microscopy Sciences, Fort Washington, PA) and
1% BSA (fraction V; Sigma) in PBS, and then, a 1-h incubation in goat anti-rabbit IgG
36
conjugated with ultra small colloidal gold (Electron Microscopy Sciences; diluted at
1:100 with the blocking reagent). The sections were rinsed briefly with the same
blocking reagent then with PBS and treated for 10 min with 1.25% glutaraldehyde in
PBS, and rinsed in 0.2 M sodium citrate (pH 7.5). The silver intensification of gold
particles was carried out according to instructions provided with the IntenSE kit
(Amersham, Arlington Heights, IL). The dual-labeled sections were osmicated (1%
osmium tetroxide in 0.1 M PB; 30 min) and dehydrated in serial dilutions of ethanol. A
30-min contrasting step using 1% uranyl acetate in 70% ethanol was inserted in this
procedure and the fully dehydrated sections were finally infiltrated with propylene
oxide and flat embedded in Durcupan ACM epoxy resin (Fluka, Ronkonkoma, NY) on
liquid release agent (Electron Microscopy Sciences) coated microscope slides at 56 oC.
Ultrathin section (50–60 nm) were cut from the resin blocks with an Ultracut UCT
Ultramicrotome (Leica Microsystems AG, Wetzlar, Germany), collected onto Formvarcoated single-slot grids, and examined with a Jeol-100C transmission electron
microscope (JEOL, Tokyo, Japan).
4.4. Investigation of the putative glutamatergic phenotype of GnRH neurons
4.4.1. Experimental animals
Adult male Wistar rats (N=11; 260–280 g BW) were purchased from Charles
River Hungary Ltd. (Isaszeg, Hungary). The animals were kept under a 12 h light-12 h
dark schedule (lights on at 07:00 h, lights off at 19:00 h), in a temperature (22 ± 2 °C)
and humidity (60 ± 10%) controlled environment with free access to laboratory rat food
(Sniff Spezialdiaeten GmbH, Soest, Germany) and tap water.
4.4.2. Detection of GnRH and Vglut2 mRNAs by dual-label in situ hybridization
histochemistry (ISHH)
Tissue preparation for in situ hybridization. Four adult rats were decapitated, and
their brains were snap-frozen on powdered dry ice. Twelve-micrometer thick coronal
37
sections through the POA were cut with a CM 3050 S Cryostat (Leica, Deerfield, IL)
and collected serially on gelatin-coated microscope slides.
ISHH. For dual-label ISHH detection of the GnRH and Vglut2 mRNAs, every
sixth section was processed for the following prehybridization steps. Sections were
fixed for 30 min with 4% solution of freshly depolymerized paraformaldehyde in PBS.
After several step of washing in 2x standard saline citrate solution (SSC; 1x SSC = 0.15
M NaCl/0.015 M sodium citrate, pH 7.0) the sections were placed into 0.25% acetic
anhydride/0.9% NaCl/0.1 M triethanolamine (pH 8.0; Sigma) solution for 10 min.
Following a brief rinse in 2x SSC, sections were dehydrated in a serial dilution of
ethanol (2 min each); delipidated in chloroform (5 min) then partially rehydrated in
100% followed by 96% ethanol (2 min each). The slides were finally air-dried and
processed further for the hybridization procedure.
For the dual-label ISHH, we used a digoxigenin-labeled GnRH probe (transcribed
from a 330-bp long cDNA template; a gift from Dr. J. P. Adelman, Vollume Institute,
Oregon Health Science University, Portland, OR) and one of two non-overlapping 35SUTP-labeled Vglut2 probes (Vglut2-879 or Vglut2-734).
The dried slides were placed into plastic boxes in which the air was humified
using caps of 50 ml centrifuge tubes filled with distilled water. For hybridization, we
dissolved both the
35
S-labeled and the digoxigenin-labeled probes in a SSC-buffered
hybridization solution, which consisted of 50% formamide (Fisher BP228-100), 10%
dextran sulfate (Sigma D8906), 1x Denhardt’s solution, 0.5 mg/ml yeast tRNA (Roche
Diagnostics, St. Louis, MO), 500μg/ml heparin sodium salt (1760 USP units, Sigma
H3391) and 1 M dithiothreitol (Sigma D9779). The hybridization solution was pipetted
onto each slides and covered with glass coverslips. According to a recently introduced
novel approach to enhance hybridization sensitivity (156), we applied unusually high
radioisotopic probe (80,000 cpm/μl) and dithiothreitol (1 M) concentrations in the
hybridization solution and extended the time of hybridization from 16 to 40 h.
After the hybridization process, the slides were subjected to post hybridization
treatments. The coverslips were floated off the slides in 1x SSC solution and the excess
of hybridization solution was rinsed off in several changes of 1x SSC solution. Then the
slides were loaded into metal racks and the following incubation steps were carried out
under agitation. Slides were rinsed in 1x SSC solution twice for 20 min, which was
38
followed by 1 hour incubation in a 50 μg/ml RNase A solution (Roche Diagnostics, St.
Louis, MO) prepared with RNase buffer (500 mM NaCl/10 mM Tris-HCl/1 mM EDTA,
pH 7.8, 37 oC). The RNase A-treated sections were rinsed in 1x SSC several times,
followed by an incubation in 0.1x SSC buffer at 60 oC for 1 hour. Finally, the slides
were rinsed in 2x SSC buffer and placed into PBS before the detection of the
digoxigenin-labeled GnRH probe.
The digoxigenin-labeled cRNA probe to GnRH mRNA was detected by
immunocytochemistry as follows. The slides were pretreated with a solution that
contained 0.2% Triton-X 100 and 0.5% H2O2 in PBS in order to permeabilize the tissue
and to eliminate the endogenous peroxidase. After extensive rinsing in PBS, the slides
were dipped into a blocker solution (Roche Diagnostics, St. Louis, MO) for 30 min. The
sections were incubated overnight with anti-digoxigenin antibodies conjugated to
horseradish peroxidase (1:200; anti-digoxigenin-POD, Roche, St. Louis, MO). After
several rinses in PBS, the biotin tyramide signal amplification procedure was applied to
the sections to amplify the non-isotopic hybridization signal for GnRH mRNA. This
included a 1-hour incubation in a 1:1000 dilution of biotin tyramide stock solution (155)
in TBS/0.005% H2O2, followed by a 1 hour incubation in ABC Elite working solution
(Vector Laboratories, Burlingame, CA; 1:1000 dilutions of solutions A and B in 50 mM
Tris buffer; pH 7.6). The final peroxidase reaction was visualized with DAB chromogen
in the peroxidase developer (0.025% DAB and 0.0036% H2O2 in TBS). Subsequently,
the
35
S-labeled cRNA probes to Vglut2 mRNA were detected on autoradiographic
emulsion (NTB-3; Eastman Kodak Co., Rochester, NY) after a 2-wk exposure. Finally,
the sections were coverslipped and examined with an Axiophot microscope (Zeiss,
Göttingen, Germany) equipped with an RT Spot digital camera (Diagnostic Instruments,
Sterling Heights, MI), under bright field and dark field illumination.
Hybridization controls. The
35
S-labeled Vglut2–879 probe corresponded to bases
522-1400 of Vglut2 mRNA (GenBank accession no. NM_053427). As a positive
control for hybridization specificity, a second series of dual-label ISHH experiment was
carried out with a distinct Vglut2-734 probe that was complementary to bases 1704–
2437 (The template was a gift from Dr. J. P. Herman, University of Cincinnati Medical
Center, Cincinnati, OH). Negative control experiments were performed with the
combined use of the GnRH probe and the sense strand Vglut2 RNA transcripts.
39
4.4.3. Dual-immunofluorescent labeling for confocal laser scanning microscopic
analysis of Vglut2- and GnRH-immunoreactive axons in the OVLT and ME
Tissue preparation. The animals (N=4) were deeply anesthetized with sodium
pentobarbital (35 mg/kg BW, i.p.) and perfused via the ascending aorta, with 150 ml
mixture of 2% freshly depolymerized paraformaldehyde (Sigma Chemical Co., St.
Louis, MO) and 4% acrolein (Sigma) in PBS.
Section preparation and storage. Brains were rapidly removed, immersed into
30% sucrose in PBS overnight and snap frozen on powdered dry ice. Then 20-μm thick,
free-floating coronal sections were prepared from the regions of interest (POA, ME and
Arc) with a Leica CM 3050 S cryostat (Meyer Instruments, Houston, TX).
The free-floating sections were collected in anti-freeze solution (30 V/V % ethylene
glycol, 25 V/V % glycerol in 0.05 M phosphate buffer) and stored at minus 20 oC until
use.
Double-labeling immunocytochemistry. The sections were rinsed in TBS, treated
with 0.5% sodium borohydride (Sigma-Aldrich Corp.; 30 min) and 0.5% H2O2 (15 min),
and finally treated with a mixture of 0.2% Triton X-100 (Sigma-Aldrich Corp.) and 2%
normal horse serum in TBS (30 min). After these pretreatments, the sections were
incubated (72 h; 4 oC) in anti-Vglut2 primary antibodies raised in a guinea pig (AB
5907; Chemicon International, Temecula, CA; 1:1000). Steps to enhance the fluorescent
signal included sequential incubations with biotinylated anti-guinea pig IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA; 1:1000; 2 h), ABC Elite solution
(Vector Laboratories, Burlingame, CA; 1:1000 dilutions of solutions A and B in 50 mM
TBS; pH 7.6; 1 h), biotin tyramide working solution prepared and used as described
previously (section 4.2.4.), and streptavidin-conjugated FITC fluorochrome (Jackson
ImmunoResearch Laboratories; 1:200; 12 h). Immunoreactivity for GnRH was detected
with rabbit LR-1 primary antibodies (1:30,000; gift from Dr. R. Benoit, Montreal,
Canada). This antiserum was applied to the sections for 48 h (4 oC) and then reacted
with Cy3- conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories; 1:200;
12 h). The colocalization of Vglut2 and GnRH immunoreactivities was examined with a
40
Radiance 2100 confocal microscope (Bio-Rad Laboratories, Hemel Hempstead, UK)
using the following parameters:
Fluorochrome
Laser excitation line (nm)
Dicroic mirror/ Emission
filter (nm)
FITC
488
560 /500–530
Cy3
543
- /565–625
To distinguish axonal colocalizations from cases of overlap, individual optical
slices of minimal thickness (<0.7 µm) were obtained using a x60 objective lens (with
immersion oil) and an optimized pinhole size.
Control experiments. Parallel control experiments used Vglut2 antibodies
preabsorbed with 10 µM immunization antigen (AG209, Chemicon International). As a
positive control for the specificity of Vglut2 immunostaining, dual-immunofluorescent
experiments were carried out with the combined use of guinea pig anti- Vglut2 primary
antibodies (AB 5907, Chemicon) and rabbit anti- Vglut2 primary antibodies (1:5000;
AB 135103; SYnaptic SYstems, Göttingen, Germany). The primary antibodies were
detected with anti- guinea pig-FITC (Jackson ImmunoResearch Laboratories; 1:200)
and anti-rabbit-Cy3 (Jackson ImmunoResearch Laboratories; 1:200) conjugates,
respectively, and were analyzed by confocal microscopy.
4.4.4. Pre-embedding immuno-gold-labeling of Vglut2 in the hypophysiotropic
nerve terminals of the ME
Tissue preparation. For electron microscopic localization of Vglut2 in
hypophysiotropic axon terminals in the ME immersion fixation was used. Three adult
male rats were anesthetized with pentobarbital (35 mg/kg BW, i.p.) and killed by
decapitation. The hypothalami were removed rapidly and fixed for 24 h by immersion
into a freshly made fixative solution containing 4% paraformaldehyde, 0.3%
glutaraldehyde and 15% (V/V %) saturated picric acid in 0.1 M PBS (pH 7.4). The
tissues were fixed further for 3 days in the same fixative, with the omission of
glutaraldehyde. The basal hypothalami were sliced at 50 µm with a Vibratome
(Technical Products International, St. Louis, MO, USA).
41
Immunogold-labeling. Pre-embedding immuno- electron microscopy was used for
the ultrastructural analysis of Vglut2-immunoreactivity in the ME. The sections were
first treated with 0.5% sodium borohydride in 0.1 M PBS for 20 min to eliminate
residual aldehydes and then infiltrated with increasing concentrations of sucrose in 0.1
M PBS for cryoprotection (7.5%, 15% and 30%; 20 min for each). Subsequently the
tissues were permeabilized by three repeated freeze-thaw cycles on liquid nitrogen. For
the immunocytochemical detection of Vglut2, the sections were incubated for four days
in a 1:1000 dilution of the rabbit primary antibodies against Vglut2 (AB 135103;
SYnaptic SYstems). These antibodies performed better than the guinea-pig anti-Vglut2
antibodies following the use of glutaraldehyde in the tissue fixative. The primary
antibody incubation was followed by a 30-min blocking step using 0.1% cold-water fish
gelatin (Electron Microscopy Sciences, Fort Washington, PA, USA) and 1% BSA
(fraction V; Sigma) in 0.1 M PBS, and then, an overnight incubation in goat anti-rabbit
IgG conjugated with ultra small gold particles (Electron Microscopy Sciences; diluted at
1:100 with the blocking reagent). The sections were rinsed briefly with the same
blocking reagent, followed by PBS. Then they were treated with 2% glutaraldehyde in
PBS for 10 min and rinsed in distilled water (3x5 min). The silver-intensification of
gold particles was carried out according to the instructions provided with the Aurion RGent SE-LM silver enhancing kit (AURION ImmunoGold Reagents & Accessories,
Wageningen, The Netherlands). Hypothalamic sections were osmicated (0.5% osmium
tetroxide in 0.1 M PBS, 20 min) and dehydrated in serial dilutions of ethanol. A 30-min
contrasting step using 1% uranyl acetate in 70% ethanol was inserted in this procedure
and the fully dehydrated sections were finally infiltrated with propylene oxide and flat
embedded at 60 oC in TAAB 812 Embedding resin (TAAB Laboratory Equipments Ltd.,
Aldermaston, Berks, UK) on microscope slides that were pre-coated with liquid release
agent (Electron Microscopy Sciences). Ultra thin (50–60 nm) sections were cut from the
resin blocks with a Leica Ultracut UCT Ultramicrotome (Leica Microsystems AG,
Wetzlar, Germany), collected onto Formvar-coated single- slot grids and examined with
a Hitachi 7100 (Yokohama, Japan) transmission electron microscope.
42
5. Results
5.1. Identification of cholinergic afferents to GnRH neurons of the rat
5.1.1. Light microscopic evidence for cholinergic neuronal contacts on GnRH
neurons
The cell bodies of GnRH neurons, immunostained with brown DAB, were
scattered around the OVLT and in the MPOA (Figs. 5A, B), and their regional
distribution followed the characteristic patterns described earlier by others (7). The
same regions also contained a fine network of varicose cholinergic fibers that were
immunoreactive for ChAT (Fig. 5A) and VAChT (Fig. 5B) and contained gray-to-black
silver-intensified ammonium nickel (II) sulfate-DAB chromogen. Immunostaining with
the VAChT antiserum resulted in a somewhat higher fiber density compared to that
obtained with the ChAT antiserum, corroborating previous observations with the same
antisera by others (99). High-power analysis of dual-labeled sections revealed that the
cholinergic axons were apposed to GnRH-immunoreactive neuronal elements.
Cholinergic axons contacted the cell bodies (Fig. 5C) as well as the dendrites (Figs. 5DF) of GnRH neurons.
43
Figure 5. Cholinergic afferents to gonadotropin-releasing hormone (GnRH) producing
neurons in the male rat. A, B: The cell bodies of GnRH neurons (arrowheads),
immunostained with brown diaminobenzidine chromogen, are scattered within the
organum vasculosum laminae terminalis (OVLT) and the medial preoptic area (MPOA).
GnRH-immunoreactive fibers abundantly innervate the OVLT. Cholinergic neuronal cell
bodies (gray-to-black immunostaining with silver-intensified ammonium nickel (II)
sulfate-diaminobenzidine), immunoreactive for choline acetyltransferase (ChAT; A) and
vesicular acetylcholine transporter (VAChT; B) occur in high numbers in the horizontal
limb of the diagonal band of Broca (HDB). C, D: Thin and varicose cholinergic axons
(arrows) immunoreactive for ChAT show an overlapping distribution with that of GnRH
neurons and form appositions (arrowheads) to GnRH-immunoreactive cell bodies and
dendrites. E, F: the cholinergic afferent contacts (arrowheads) on GnRH neurons are also
detectable using VAChT as the cholinergic marker. Scale bars: A, B: 100 µm; C, D: 15 µm
E, F: 20 µm.
44
5.1.2. Electron microscopic evidence for cholinergic afferents to GnRH neurons
At the ultrastructural level, the cholinergic axons were labeled with highly
electron dense silver-gold-intensified ammonium nickel (II) sulfate – DAB precipitate.
Some of the cholinergic axonal profiles established asymmetric synapses with unlabeled
dendrites (Fig. 6A). Gonadotropin-releasing hormone neurons, labeled with electron
dense DAB, were contacted by the cholinergic fibers on their somata as well as
dendrites (Figs. 6B-D). While the appositions were devoid of glial intercalations (Figs.
6C, D), state-of-the-art synaptic specializations occurred rarely (Figs. 6E, F).
45
46
Figure 6. Ultrastructural evidence for cholinergic innervation of gonadotropin-releasing
hormone (GnRH) neurons in the preoptic area (POA). A: Cholinergic axons
immunoreactive for choline acetyltransferase (ChAT) exhibit high electron density due to
the deposition of silver-gold-intensified ammonium nickel (II) sulfate – diaminobenzidine
precipitate. Occasionally, they establish asymmetric synapses (arrowheads) with unlabeled
dendrites. Gonadotropin-releasing hormone-immunoreactive neurons contain dense
diaminobenzidine chromogen. B: The ChAT-immunoreactive cholinergic axons are
occasionally apposed (arrows) to GnRH-immunoreactive neuronal perikarya. C, D:
Intercalation of glial elements is absent at the apposition sites (arrows) between ChAT- (C)
or vesicular acetylcholine transporter- (VAChT; D) immunoreactive cholinergic axons
and the GnRH-immunoreactive neuronal elements. The typical appositions do not exhibit
any synaptic specialization. E, F: State-of-the-art synapses between the cholinergic axons
and GnRH neurons were rare. Arrowheads in F (framed region from E) point to the
postsynaptic density of an axo-dendritic synapse. Nu, cell nucleus. Scale bars: A: 500 nm;
B, C: 50 nm; D: 400 nm; E: 800 nm F: 500 nm.
5.2. Revealing the origin of NPY-containing afferents to GnRH neurons in
male mice
5.2.1. Comparative studies of NPY- and AGRP-containing fibers in contact with
GnRH neurons of neonatally MSG-treated mice vs. untreated controls
Comparison of cresyl-violet-stained sections from MSG-treated mice (Fig. 7B) vs.
untreated controls (Fig. 7A) showed a marked reduction in the size of the Arc, whereas
neurons of the ventromedial nuclei remained intact.
Results of immunocytochemical experiments showed that the density of NPYimmunoreactive fibers was largely reduced in the POA of Arc-lesioned animals (Fig.
7D), in comparison with untreated controls (Fig. 7C). This observation indicated that a
major, albeit not the sole, source of NPY fibers to the POA is the Arc. Axons containing
AGRP-immunoreactivity (Fig. 7E) formed a somewhat less dense plexus in the POA
than did NPY fibers (Fig. 7C) in control animals. As opposed to NPY immunostaining
(Fig. 7D), AGRP-immunoreactivity was almost completely absent from the POA (Fig.
47
7F) in MSG-treated animals, corroborating previous data that the Arc is the only brain
region in which this neuropeptide is synthesized (137).
The analysis of dual-labeled sections from intact mice demonstrated numerous
contacts between NPY-immunoreactive axons and the perikarya and proximal dendrites
of GnRH neurons (Fig. 8A). The frequency of NPY-containing axonal appositions to
individual GnRH cells was significantly higher by ANOVA (P < 0.05) than the
frequency at which AGRP-immunoreactive neuronal contacts occurred on GnRH
neurons (Fig. 8B). The number of AGRP-immunopositive contacts was 56.5 ± 9.8% of
that of NPY immunoreactive contacts. Furthermore, significantly less (P < 0.05) NPYcontaining neuronal contacts were visible in neonatally MSG-treated animals (Fig. 8C
vs. 8A) vs. controls. The lesion of the Arc caused a 63.7 ± 5.0% loss of NPYimmunopositive
juxtapositions.
Altogether,
the
two
different
methodological
approaches suggested that about 56.5–63.7% of NPY axons to GnRH cells arise from
NPY neurons of the Arc.
48
Figure 7. Effects of neonatal monosodium glutamate (MSG) treatment on the morphology
of the Arc and innervation pattern of the medial POA (MPOA) by NPY and AGRP fibers.
A, Cresyl violet-stained section shows the normal structure of the Arc. B, Neonatal MSG
treatment of mice results in a marked shrinkage and cell paucity of this region (dashed
line) due to a major neuronal cell loss. Note that the lesion is region specific, and neurons
outside the Arc escape the effects of treatment. C and D, Innervation of the MPOA by
NPY-immunoreactive axons is largely reduced in the Arc-lesioned animal (D) vs. the
control (C). The source of the NPY network that survives the treatment is likely outside
the Arc. E and F, Neuronal fibers immunoreactive to AGRP densely innervate the MPOA
in untreated animals (E). Because the Arc is the only brain region synthesizing AGRP,
immunoreactive axons almost completely disappear after neonatal lesioning of the Arc by
MSG treatment (F). Note that the loss of NPY fibers in (D) and AGRP fibers in (F) reflects
necrosis of neurons that synthesize both AGRP and NPY in the Arc. Scale bar, 100 µm
(A–F).
49
Figure 8. Innervation of GnRH neurons by NPY and AGRP fibers. Effects of neonatal
monosodium glutamate (MSG) treatment. (A), Axons immunoreactive to NPY form
juxtapositions (arrowheads) with the cell bodies and proximal dendrites of GnRH neurons.
(B), Neuronal contacts (arrowheads) between AGRP-immunoreactive axons and GnRH
cells are less frequent. (C), GnRH neurons of neonatally MSG-treated mice are contacted
(arrowheads) by significantly less NPY axons than GnRH cells of untreated controls (A).
Scale bar, 10 µm (A–C).
5.2.2. Triple-label fluorescent detection of AGRP/NPY and DBH/NPY fibers
forming neuronal contacts with GnRH-GFP neurons
The distributions of NPY- (Fig. 9A) and AGRP-containing (Fig. 9B) fibers largely
overlapped (Fig. 9C) in the POA, and the heaviest immunolabeling for both was
observed in the ventral aspect of this region. Axons double labeled for NPY and AGRP
often formed serial contacts with the cell bodies and proximal dendrites (Fig. 9 D–F) of
GnRH-GFP neurons that exhibited bright green fluorescence. High-power analysis of
the fluorescent specimen established that 49.1 ± 7.3% of NPY-containing neuronal
50
contacts also contained AGRP (Figs. 9 D–F). In addition, nearly all AGRP fibers were
immunoreactive to NPY.
Similar to AGRP-immunoreactive axons, fibers containing DBH (Fig. 9H) showed
overlapping distribution (Fig. 9I) with that of NPY axons in the POA (Fig. 9G). Highpower analysis of immunostained axons established the presence of DBH in 25.4 ±
3.3% of NPY-containing fibers in contact with GnRH neurons (Fig. 9 J–L). Other types
of fibers observed in the POA contained NPY without DBH or DBH without NPY (Figs.
9 J–L), and they were apposed to GnRH neurons.
51
Figure 9. Immunofluorescent identification of AGRP and DBH in NPY afferents to GnRH neurons.
(A–C), Both NPY (A; red Cy-3 fluorochrome) and AGRP-immunoreactive (B; blue AMCA
fluorochrome) fibers form dense plexus in the medial POA (MPOA). The purple-to-white color that
dominates over red in the merged figure (C) indicates that many NPY-immunoreactive axons cocontain AGRP. Note the bright green fluorescence in GnRH neurons (A–C) due to the presence of
the GnRH-GFP transgene product. D–F, High-power photomicrographs reveal an NPY axon (D)
that also contains AGRP (E). Arrows indicate the same points of contact between a dual-labeled
NPY/AGRP axon and a GnRH neuron in unmerged (D, E) and merged (F) digital images. G–I, The
distribution of NPY-immunoreactive axons (G; red Cy-3 fluorochrome) also overlaps with that of
DBH-containing fibers (H; blue AMCA fluorochrome) in the MPOA. Note that this match (purpleto-white color in the merged panel; I) is of lower degree than in case of NPY- and AGRPimmunoreactive fibers (C). J–L, A noradrenergic/adrenergic axon that contains NPY (J) as well as
DBH (K) establishes contact (arrows) with the soma of a GnRH neurons. The merged image in L
clearly shows that many NPY axons (red color) are devoid of DBH, and vice versa, DBHimmunoreactive axons (blue color) often remain immunonegative for NPY. The scale bar for A-C
and G-I is presented in I (100 µm). Sale bar for D–F and J–L is presented in L (10 µm)
52
5.3. Electron microscopic detection of AGRP in synaptic afferents to GnRH
neurons
At the ultrastructural level, AGRP-immunoreactive fibers contained electrondense DAB deposits, and they were frequently apposed to GnRH neurons accumulating
highly electron-dense silver-gold particles (Fig. 10 A and B). Synapses of symmetric
morphology were observed between AGRP axons and GnRH neurons as well as nonGnRH structures (Fig. 10B). The absence of metal particles over AGRP fibers indicated
that antibody cross-reaction did not arise from the species identity of the two primary
antibodies.
53
Figure 10. Electron microscopic evidence for synaptic communication between AGRPcontaining axons and GnRH neurons. A and B, an AGRP-immunoreactive axon terminal
(at) labeled by electron-dense DAB deposits is closely apposed to the perikaryon of a
GnRH-immunoreactive neuron (GnRH), which contains highly electron-dense silverintensified gold particles (arrows). High-power image in B reveals axosomatic synaptic
communication between the labeled structures. Arrowheads point to the postsynaptic
density of a symmetrical synapse. Also note the presence of a symmetric synapse between
the same AGRP-immunoreactive terminal and an unlabeled dendrite (d). Scale bars, 0.8
µm in A and 0.5 µm in B.
54
5.4. Morphological evidence for the glutamatergic phenotype of GnRH neurons in
adult male rat
5.4.1. Results of in situ hybridization experiments
Development of emulsion autoradiographs exposed for 2 wk resulted in strong
hybridization signal for Vglut2 mRNA in the POA (Fig. 11A). Heavy accumulation of
silver grains was observed frequently above glutamatergic cells in the vicinity of GnRH
neurons (Fig. 11 A1, A2, and B). Virtually all (99.5 ± 0.2%) of the total 438 GnRH
neurons analyzed (Fig. 11 A1–A3) also contained Vglut2 hybridization signal, usually
at moderate levels. Confirmative results obtained with a distinct Vglut2 probe (Fig.
11B) and the lack of Vglut2 signal using the sense strand Vglut2–879 RNA transcript
(Fig. 11C) provided support for hybridization specificity.
Figure 11. Dual-label ISHH experiments reveal high expression levels of Vglut2 mRNA
(autoradiographic grain clusters in (A) in regions also populated by GnRH neurons (brown
histochemical staining in (A1–A3), including the medial preoptic area (MPOA) and the median
preoptic nucleus (MPN). High power insets (A1–A3) illustrate moderate levels of Vglut2
mRNA in GnRH neurons (arrows) from frames in (A) after use of the Vglut2–879 probe.
Demonstration of Vglut2 mRNA-containing GnRH neurons with a distinct antisense probe
(Vglut2–734; B) and lack of such dual-labeled GnRH neurons after use of the sense strand
Vglut2–879 RNA transcript (C) serve as controls for hybridization specificity.
55
5.4.2. Results of dual-label immunofluorescent studies
Both glutamatergic and GnRH-containing fibers formed dense plexus in the
OVLT (Fig. 12A) and the external zone of the ME (Fig. 12C). High power confocal
images demonstrated extensive terminal co-expression of Vglut2 with GnRH
immunoreactivities in both circumventricular organs (Fig. 12B, and D). Although most
dual-labeled axons occurred laterally in the ME, a dense Vglut2-immunoreactive plexus
was also observed medially (Fig. 12C). Vglut2-immunoreactive axons (Fig. 12E) were
eliminated (Figs. 12F) from the ME when using primary antibodies preabsorbed with 10
µM immunization antigen. In addition, simultaneous use of the two different primary
antisera against Vglut2 labeled identical axons throughout the hypothalamus, in further
support of labeling specificity (Fig. 12 G–I).
56
Figure 12. Dual-immunofluorescent images (A and C) illustrate the overlapping
distribution of fibers immunoreactive for Vglut2 (green fluorochrome) and GnRH (red) in
the OVLT (A) and the ME (C). Although sites of overlap (yellow) occur mostly in the
lateral part of the ME, the dense Vglut2-immunoreactive plexus is also present medially
(C). Arrows in high power confocal images (B and D) point to dual-labeled axon
varicosities (yellow) immunoreactive for both GnRH and Vglut2. The specificity of Vglut2
immunostaining in the ME (E) is indicated by the lack of labeled axons after adding the
immunization antigen (10 µM) to the working dilution of AB 5907 (F). In addition, dualimmunofluorescent labeling of hypothalamic axons (arrows in confocal images G, H, and
merged panel I) using two distinct primary antibodies for Vglut2 further supports the
authenticity of Vglut2-immunoreactivity. Scale bars, 200 µm in A, 30 µm in D and F, and
10 µm in the other panels.
57
5.4.3. Electron microscopic localization of Vglut2 in hypophysiotropic axon
terminals in the ME
Pre-embedding colloidal gold labeling for Vglut2 identified numerous
glutamatergic axon terminals in the external layer of the ME. Many of these axons
established direct contact with the external limiting membrane of portal vessels (Fig. 13
A, B), indicating that they represented neurosecretory terminals. The labeled structures
contained small clear as well as DV at a highly variable ratio. The immunocytochemical
labeling clearly tended to occur at subcellular domains occupied primarily by small
clear vesicles (Fig. 13A, B).
Figure
13.
Electron
microscopic localization of
Vglut2-immunoreactivity
in the external zone of the
ME.
(A)
Three
neuroendocrine
axon
terminals (AT1-3) freely
communicate
with
the
pericapillary space (PCS)
of portal blood vessels.
Arrowheads delineate the
basal lamina. AT1 and
AT2 contain both dense
core vesicles (DV; arrows)
and small clear vesicles
(SV), whereas AT3 comprises SVs only. The silver-intensified gold particles identify the
Vglut2 content of the terminals. Note that the immunocytochemical label is preferentially
associated with subcellular sites densely populated by SVs, indicating that Vglut2 is
contained in SVs. (B) A glutamatergic axon terminal (AT) terminates on the external
limiting membrane (arrowheads) of a portal capillary. Arrowheads delineate the basal
lamina. Note the presence of DVs (arrows) as well as SVs in the same terminal profile and
the association of the Vglut2 immune signal with SVs. Scale bars=0.5 µm.
58
6. Discussion
6.1. Proposed nonsynaptic role and receptorial mechanisms of ACh on GnRH
neurons and the possible sites of origin of the direct cholinergic afferents
6.1.1. Ach as a proposed nonsynaptic modulator of GnRH neurons
We present neuromorphological evidence for a direct cholinergic afferent input to
the GnRH neuronal system of the rat. These results reveal a previously unknown
neurotransmitter system, which directly regulates GnRH neurons and suggest a
dominantly nonsynaptic signaling mechanism in this interaction.
Our results indicate that the cholinergic axons often formed direct contacts but
rarely established classical synapses with the GnRH neurons. This is highly reminiscent
to the previous morphological findings by several other investigators. The observations
that out of cholinergic axon varicosities only 7 % in the hippocampus (CA1, stratum
radiatum) (157), 10 % in the neostriatum (158) and 14 % in the parietal cortex (159)
were engaged in synapses may indicate nonsynaptic mechanisms (160) whereby
cholinergic axons influence their target neurons, including GnRH cells. The main
properties of nonsynaptic communication include the long lasting and tonic action (s,
min, h), axonal varicosities without synaptic contacts, the high affinity receptors and the
high concentration of neurotransmitters in the extracellular space (160). The
nonsynaptic localization of cholinergic heteroreceptors allows the modulation of
GABAergic, glutamatergic, noradrenergic and serotonergic neurotransmission by ACh
in the hippocampus (160, 161). These neurotransmitter systems also innervate the
GnRH neurons (see in section 2.2.3.1.), therefore, the proposed nonsynaptic cholinergic
interactions on GnRH neurons may involve the modulation of the above-mentioned
neurotransmitter-containing neuronal afferents of the GnRH nerve cells. Further
neuropharmacological experiments and the anatomical localization of cholinergic
receptors on GnRH nerve cells and their surrounding neural elements will be needed to
clarify this hypothesis.
Our anatomical observations showed that GnRH neurons receive cholinergic
innervation on their cell bodies as well as dendrites. Campbell and colleagues who used
59
biocytin-filled GnRH neurons of mice detected a surprisingly rich dendritic arborization,
which was unknown previously (16). While the number of spines showed a trend to
decrease with increasing distance from the cell body, they remained detectable even on
the most distal elements of the primary dendrite (16). Considering that cholinergic
axons preferentially form contacts and synapses with dendritic branches and spines in
other brain regions (157, 159, 162, 163), our study likely left a significant proportion of
cholinergic contacts undetected at the distal dendrites and spines of GnRH neurons,
which might remain unlabeled for GnRH.
Finally worth of note, that the external zone of the ME exhibits a high density of
VAChT- and ChAT-immunoreactivities (99, 164). This observation suggests the
possible paracrine regulatory role of ACh at the level of the GnRH nerve terminals as
well.
6.1.2. Receptorial mechanisms of ACh actions on GnRH neurons
To estimate the exact functional consequences of ACh release on GnRH neurons
the determination of the receptor subtypes and their site of locations (i.e. non-, pre- or
postsynaptic) would be crucial. The receptorial mechanisms of cholinergic actions on
GnRH neurons appear to be multiple. Krsmanovic and coworkers studied perifused
hypothalamic primary cell cultures and immortalized GnRH producing GT 1-7 cells and
concluded a dual receptorial effect of ACh on GnRH neurons. While the cholinergic
activation of M2 muscarinic receptors reduced basal GnRH release via inhibiting G
protein-coupled intracellular cAMP elevation, the activation of M1 receptors led to
stimulation of phosphoinositide hydrolysis, with the result of a rapid and transient
increase in GnRH neurosecretion (165). In addition, ACh also increased GnRH
secretion via causing increased Na+ and Ca2+ influx due to nicotinic receptor activation
(165). The Ca2+ delivery from intracellular Ca2+ stores also forms an important
component of cholinergic signaling as shown by Morales and coworkers. The AChinduced [Ca2+]i elevation from the endoplasmic reticulum of GT 1-7 cells could be
blocked by atropine. Moreover, the cholinergic stimulation of [Ca2+]i delivery showed a
strong modulation by estrogen and was reduced rapidly by 50-60% in the presence of
10 nM 17β-estradiol. The fast onset of this effect and the similar action of a membrane
60
impermeant estradiol conjugate implicated a membrane associated estrogen receptor in
this modulatory action (166). Since the M1 muscarinic and several nicotinic receptors
(Chrnb1-2, Chrnbg) are represented in a subpopulation of GnRH neurons in vivo (63),
the direct cholinergic afferents on GnRH neurons revealed by us may stimulate (109,
110) the functional properties of the GnRH neurons via the above-described second
messenger systems. In contrast, GnRH neurons in vivo appear to lack the M2 receptors,
which would suggest the presynaptic role of this cholinergic receptor, although further
pharmacological and/or anatomical experiments would need to clarify this question.
6.1.3. Possible sites of origin of the cholinergic innervation to GnRH neurons
Cholinergic neurons of the basal forebrain (167) may represent one possible
source for the cholinergic innervation of GnRH neurons. Retrograde tact tracing studies
in rat (67) and mouse (18) revealed retrogradely labeled nerve cells in the following
parts of the basal forebrain: nucleus of the horizontal and vertical limbs of the diagonal
band (18, 67) and medial septal nucleus (18). Although the chemotype of retrogradely
labeled neurons has not been determined. The main projection field of the cholinergic
neurons of the basal forebrain are neocortical areas (167), however the cholinergic
neurons in the septum and diagonal band indeed possess extensive local collaterals
(168) from which they are capable of releasing ACh locally (for a review see: (168)).
This observation suggests that ChAT- and VAChT-immunoreactive fibers in the POA
revealed by us may arise from particular parts of basal forebrain.
The parabrachial complex also contains a major cholinergic cell population (98).
The idea that these cells may contribute to the cholinergic innervation of the GnRH
system gains support from the observation that a large number of neurons in the central
and a somewhat lower number of cells in the dorsal and superior lateral parabrachial
nuclei could be retrogradely labeled from the POA (67, 100).
Both of the basal forebrain and the parabrachial complex belong to the ascending
arousal system (169) that contributes to the generalized arousal state of the organism.
Sexual arousal, which is a subcategory of the former (170), is a state of the animal that
is requisite of the activation for those complex cognitive and physiologic processes that
result in sexual behavior (106, 170). Cholinergic activation of the POA is involved in
61
the normal pattern of the sexual behavior (104, 105). Although for the activation effect
of ACh, the presence of the gonadal steroids appears to be essential components (101).
These observations emphasize the importance of both the adaptive functioning of
arousal systems and the appropriate level of the gonadal steroids in the governing of the
reproductive behavior. Thus, the basal forebrain and/or the parabrachial nuclei as a
proposed source of cholinergic afferents to the GnRH neurons may provide a novel
relation between the neural processes of sexual behavior and its accompanying
hormonal adaptations.
Aside from tract tracing studies (18, 67, 129), several pieces of indirect evidence
indicate that the Arc as one primary site of origin of the cholinergic afferents, to the
GnRH neurons. About a third (37%) of the POMC neurons in the Arc was found to
contain the cholinergic marker VAChT (99). The POMC neurons establish synaptic
contacts with GnRH neurons (78) and the proposed source of the opioid innervation to
GnRH neurons is the Arc (56, 78). The presence of cholinergic/αMSH positive fibers in
the POA (99) also raises the possibility that the Arc is a potential source of cholinergic
innervation to GnRH neurons. The POMC neurons in the Arc are an important neuron
population in the regulation of food intake and in energy homeostasis (143), which
would indicate the role of POMC/cholinergic neurons as a possible linkage between the
regulation of body weight and the HPG axis. Nevertheless, the functional role of the
possible co-release of ACh and one of the POMC derived peptides on the GnRH
neurons would need further clarifications, because the GnRH neurons do not express
either the melanocortin receptors (receptors of αMSH and ACTH) or the opioid
receptors (receptors of β-END) (171) in rats.
6.2. The functional roles of the NPYergic afferents of Arc and brainstem in
the regulation of GnRH neurons and the receptorial mechanisms of NPY action on
GnRH neurons
6.2.1. The Arc and the brainstem as two major sources of origin of NPY fibers
innervating GnRH neurons
62
In the first set of our experiments, the possible origins of NPY-containing
afferents to GnRH cells were investigated. Our results indicate that NPY neurons of the
Arc give rise to 49–64% of the NPY-immunoreactive axonal contacts on the somata and
proximal dendrites of GnRH neurons (depending on the calculation approach we used),
and an additional 25% of juxtapositions originate in adrenergic/noradrenergic cell
groups of the brainstem (Figure 14.).
Figure 14. The scheme illustrates the relative contribution of the hypothalamic Arc and
the catecholaminergic nuclei of the brainstem to the NPY innervation of GnRH neurons.
Our results indicate that NPY/AGRP neurons of the Arc give rise to 49–64% of the NPYimmunoreactive axonal contacts on the somata and proximal dendrites of GnRH neurons
(depending on the calculation approach we used), and an additional 25% of juxtapositions
originate in adrenergic/noradrenergic cell groups of the brainstem. (Arc: arcuate nucleus;
A1, C1, C2, C2: catecholaminergic nuclei of the brainstem; POA: preoptic area)
These quantitative data supplement results of a previous report by Simonian et al.
(131), who described retrograde labeling of NPY neurons in both the Arc and
ventrolateral medulla after tracer injection around GnRH neurons. The use of AGRPimmunoreactivity as a topographic marker for NPY axons of Arc origin was introduced
to our studies based on previous evidence that AGRP and NPY neurons of the Arc are
essentially identical (137). Results of our triple-fluorescence studies indicate that
NPY/AGRP neurons of the Arc give rise to 49% of NPY axons that form contacts with
the somata and proximal dendrites of GnRH nerve cells. A similar ratio for NPY axons
of Arc origin (56%) was calculated based on the results of our light microscopic doublelabeling experiment from the comparison of AGRP-immunoreactive vs. all NPY-
63
immunoreactive contacts on GnRH neurons in intact animals. A somewhat heavier
innervation appears to originate from the Arc (64%) if the lost fraction of NPY contacts
in MSG-treated animals is considered.
The intense NPYergic innervation, which arises from the Arc is involved in the
mediation of the nutritional imbalance and metabolic disturbances of the organism
toward the reproductive axis (141), since the Arc is one among the main target areas of
the metabolic signals from the periphery (143, 172). The classic example in this context
is the adipocyte-derived hormone leptin, a satiety factor that not only reduces food
intake and energy consumption but also influences the HPG axis (173). The obese leptin
deficient ob/ob mouse is known to harbor serious reproductive effects including
variable degrees of hypogonadotropic hypogonadism and infertility (142). Most of the
effects of leptin in the control of food intake are exerted within the brain, predominantly
in the hypothalamus, where leptin is known to modulate both orexigenic (mostly those
co-expressing NPY and AGRP) and anorexigenic neurons (mostly those co-expressing
POMC and cocaine- and amphetamine-regulated transcript) (174). A subpopulation of
NPY neurons express the long form of leptin receptors (175, 176) and exhibit a
pronounced inhibition in their electrophysiological activities in response to leptin
administration (177). Leptin concentration falls during starvation, which accompanied
by the increased firing rate of the NPY-synthesizing cells (177), a concomitant increase
of NPY mRNA in the Arc (176) and increased NPY peptide release (178). There is
strong evidence to indicate that the hyperfunction of NPY neurons in the ob/ob mouse
inhibits the reproduction axis: leptin replacement therapy to these mice restores normal
LH secretion and fertility (142) with a concomitant suppression of hypothalamic NPY
gene expression. Moreover, crossbreeding of ob/ob mice with NPY-knockout animals
results in a significant improvement of the reproductive phenotype (179). Given that a
subpopulation of NPY neurons express GAD-65 a biosynthetic enzyme of GABA (180),
the inhibition of HPG axis in fasting and leptin deficient animals might be due to the
inhibitory effect of increased electrophysiological and/or secretory activity of the leptin
sensitive NPY/GABA neurons on the GnRH cells. Further support for the inhibitory
influence of NPY afferents is the observed symmetric morphology (i.e. inhibitory) of
NPY synapses on GnRH neurons (74).
64
In addition to AGRP/NPY contacts of Arc origin, our analysis found DBH
immunfluorescence within 25% of NPY immunoreactive axonal contacts on GnRH
neurons. This observation indicates that noradrenergic/adrenergic pathways directly
regulate GnRH neurons and is also in accordance with previous results by others (131,
152, 181) showing noradrenergic cell groups projecting to the immediate vicinity of
GnRH neurons in rats and mice (61). The present work did not address the
ultrastructural characteristics of DBH-immunoreactive juxtapositions to GnRH neurons.
One previous study (40) suggested that noradrenergic neurons could communicate with
GnRH cells through classical synaptic mechanisms, whereas other investigators (41)
debated the abundance of such synapses. Potential difficulties to reveal synaptic
specializations between adrenergic/noradrenergic terminals and GnRH neurons may
indicate the involvement of nonsynaptic routes in the catecholamine-GnRH
communication. Although this concept will require formal support by the immunoelectron microscopic analysis of DBH contacts on GnRH cells, there is little doubt that
the proposed nonsynaptic mechanisms often play a role in noradrenergic
neurotransmission (160). For example, only a small fraction of DBH-immunoreactive
juxtapositions establishes classical synapses with neurons of the cerebral cortex and the
subcellular distribution of α2A adrenoceptors is not restricted to the postsynaptic
membranes (182). It is also worth noting that a large subset of DBH-immunopositive
axons in contact with GnRH cells was devoid of NPY-immunoreactivity, in concert
with
the
finding
that
NPY
is
expressed
differentially
among
distinct
noradrenergic/adrenergic cell groups (131, 153, 183). Likewise, high percentages of
C1–3 adrenergic neurons and A1 noradrenergic neurons were found to contain NPY,
whereas A2 and A6 noradrenergic neurons often lacked any NPY immunostaining (131,
153). The adrenergic/noradrenergic input to GnRH cells is consistent with a large body
of evidence in the literature indicating the important role of noradrenergic stimuli in the
regulation of the ovarian cycle and the steroid-induced gonadotropin surge (38, 39). A
prominent increase can be observed in the turnover rate of noradrenaline within the
vicinity of the GnRH nerve bodies and terminals before and at the time of the steroid
induced or proestrus LH surge. The increased noradrenaline concentration is assigned to
the estrogen receptor positive A2 neurons, which were found to express the immediate
early gene Fos on the morning of proestrus. Moreover, the noradrenaline concentration
65
in the POA also exhibits a circadian periodicity, which is probably maintained by A1
neurons exhibiting a diurnal pattern of Fos expression. Thus, it is possible that the A1
input is liable for the circadian rhythm of noradrenaline in the POA, while the A2
neurons provide a steroid dependent component to the noradrenaline release. (for a
review see: (39))
Finally, while it is likely that at least some of the AGRP/NPY input that reaches
GnRH neurons from the Arc is GABAergic (180, 184), the recent demonstration of
Vglut2 in C1 adrenergic and other catecholamine neurons of the brainstem (185), which
also synthesize NPY (153), raises the possibility that the excitatory neurotransmitter
glutamate is co-released with NPY and epinephrine/norepinephrine from afferents to
GnRH neurons.
Although the Arc and brainstem can be viewed as the most important sources of
central NPY, neuronal perikarya synthesizing NPY in other brain areas that are
interconnected with the POA may also contribute to the NPYergic innervation of GnRH
nerve cells. The putative sources can be speculated by the comparison of the distribution
of NPY neurons (135) and the results of retrograde tract tracing studies (18, 67). The
possible brain regions are the followings: dorsomedial hypothalamic nucleus, bed
nucleus of the stria terminalis, anterior horn of the anterior commissure, lateral preoptic
area, dorsal hypothalamic area and the mesencephalic central gray (18, 67). Among
these areas, the neurons in the dorsomedial hypothalamic nucleus, bed nucleus of the
stria terminalis and lateral preoptic area establish synaptic connection with GnRH
neurons in the mouse (18). The calculated percentage of these alternative afferents can
be up to 20–26% of NPY fibers form the Arc if identified by their AGRP content but
somewhat lower (11%) if the percent loss of NPY afferents in MSG-treated animals is
considered. Several explanations for this discrepancy may exist. It is possible that MSG
treatment altered other NPY systems, in addition to induce the lesion of Arc neurons.
Alternatively, the ratio of NPY afferents reaching GnRH neurons from the Arc may be
somewhat higher than we estimated based on their AGRP content. A subset of NPY
neurons in the Arc (5%) seem to be devoid of AGRP (137), and these cells may
contribute to the innervation of GnRH neurons. Finally, we cannot rule out the
possibility that the immunocytochemical assay conditions were suboptimal to reveal
66
low levels of DBH or AGRP in subsets of NPY axons, despite the high detection
sensitivity provided by the tyramide signal amplification technique (155).
Finally, we would note that, the NPYergic innervation of GnRH neurons is known
in human brain as well (186). It would be interesting to find out whether the sources of
this NPY innervation are similar to those we revealed in the present studies in mice.
6.2.2. Receptorial mechanisms of NPY actions on GnRH neurons
Neuropeptide Y receptors are signed with a Y character and belong to the family
of the seven-transmembrane domain, G protein-coupled receptors, which act primarily
via inhibiting adenylate cyclase (187). In the CNS, six types of Y receptors have been
characterized up to now, and except for Y6, all appear to play role in the regulation of
reproduction. Pharmacological studies using selective ligands to distinct Y receptor
subtypes for intracerebroventricular acute injections into castrated rats (118) and
chronic infusions to intact male rats and mice (111) provided evidence that the Y5
receptor plays a crucial role in the inhibitory control of gonadotropin release by NPY.
Given that more than half (55%) of GnRH neuronal perikarya bear this receptor subtype
(128), the Y5 receptor-mediated reproductive actions of NPY may be exerted, at least
partly, on GnRH cells. In pentobarbital-blocked, proestrus rats, i.v. pulses of Y1
receptor antagonist blocked the endogenous LH surges and prevented the amplification
of the GnRH-induced LH surges by NPY, implicating Y1 receptor activation in the
stimulatory control of LH secretion (188). Morphological evidence supports the
presence of the Y1 receptor subtype in axon terminals of GnRH neurons in the median
eminence as well as in NPY fibers that innervate GnRH neurons from the Arc (129). It
is reasonable to speculate that Y1 receptors presynaptic to GnRH neurons may
modulate the synaptic release of NPY, AGRP, or a putative classic neurotransmitter of
these neurons, GABA (184). Accordingly, NPY agonism on presynaptic Y1 and Y2
receptors has been shown to inhibit glutamate (189) GABA (190) and norepinephrine
(191) release. Beyond the Y1 and the Y2 receptor subtypes, a recently published paper
(192) has provided evidence for the role of Y4 in the regulation of the HPG axis. The
authors compared GnRH mRNA levels in Y2 and Y4 knock out and Y2/Y4 doubleknock out animals. They found significantly higher GnRH mRNA in the Y4- and
67
double-knock out vs. Y2-knock out or wild type mice. In addition, after 24h fasting
GnRH mRNA decreased in Y2-knock out mice similarly to wild type animals, while the
measured GnRH mRNA levels in Y4 and double-knock out animals did not show any
decline. The lack of Y4 receptor expression in the POA (193) appears to suggest that
the Y4 receptor does not act directly on GnRH neurons.
6.2.3. Methodological considerations
The AGRP and DBH contents of NPY afferents to GnRH neurons were analyzed
in GnRH-GFP transgenic mice (136) in which green fluorescence is detectable in the
vast majority of GnRH neurons, without a significant ectopic expression of the
transgene (136). This approach alleviated the need of using three primary antibodies, all
of which should be generated in different species to avoid cross-reactions. A second
technical consideration is that the innervation pattern we observed reflects only the
situation on the somata and proximal dendrites of GnRH neurons. Because distal
dendrites of GnRH neurons may not contain GFP fluorescence, our studies do not allow
us to conclude about their putative regulation by NPY afferents.
6.3. Agouti-related peptide as a novel neurotransmitter in the regulation of
the GnRH neurons
In order to analyze the nature of the putative neuronal communication between
AGRP axons and GnRH neurons, we carried out electron microscopic studies and
demonstrated that AGRP-immunoreactive axons establish symmetric synapses with
GnRH neurons.
From a functional viewpoint, it is important to note that AGRP-immunoreactive
axons formed only symmetric-type synapses with GnRH as well as non-GnRH neurons
of the POA. This observation is in concert with the previous findings of symmetric
synapses between NPY axons and GnRH neurons of the POA (74). Furthermore, this
synaptic morphology also characterizes GABAergic synapses (54). Therefore, the
identification of the biosynthetic enzyme of GABA, GAD-65, in 30% of NPY neurons
in the Arc (180) along with the light and electron microscopic demonstration of GABAimmunoreactivity in subsets of NPY axons in the POA (184) highly indicate that at least
68
some AGRP/NPY terminals that innervate GnRH neurons may co-contain GABA.
Although this hypothesis awaits confirmation, recent evidence supports the
physiological importance of a GABA/NPY interplay in the regulation of gonadotropin
secretion (184).
Little is currently known about the role of AGRP, an endogenous antagonist of
melanocortin 3 and 4 receptors in the regulation of the reproductive axis. It is likely that
AGRP primarily stimulates gonadotropin secretion via acting at hypothalamic sites.
Increased gonadotropin secretion was observed 40 min after intracerebroventricular
injection of AGRP to male rats, whereas AGRP was unable to alter either basal- or
GnRH-stimulated gonadotropin secretion from dispersed pituitary cells (194).
Furthermore, AGRP also stimulated GnRH release from mediobasal hypothalamic
explants in vitro, and this effect was prevented by the presence of αMSH in the medium
(194), indicating that AGRP partially acts via antagonizing melanocortin receptors in
the Arc-ME region. Somewhat contrasting the finding that in vivo chronic infusion of
AGRP to ovariectomized female rhesus monkey suppresses pulsatile LH release (115,
145). The above-mentioned observations concerning the GABAergic phenotype of a
subpopulation of the NPY neurons (180, 184) emphasize the possible inhibitory role of
AGRP. The symmetric, i.e. inhibitory properties of the synapses between AGRP- and
GnRH-immunoreactive profiles revealed by us also confirm the hypothesis that at least
a subpopulation of AGRP neurons transmits inhibitory effects toward the GnRH
neurons. We can conclude that the effect of AGRP may depend on the gender and/or the
hormonal status of the animal or whether or not the innervating AGRP/NPY neurons
possess GABAergic chemotype. Identification of the type and cellular location of NPY
and AGRP receptors will provide a better insight into mechanism whereby NPY/AGRP
neurons modify GnRH neuronal functions. Future research will also need to address any
difference of chronic vs. acute AGRP effects on gonadotropin secretion as well as the
potential sexual steroid dependence of AGRP actions, features well-characterized for
the regulation of the reproductive axis by NPY.
6.4. Demonstration of glutamatergic phenotype of the GnRH neurons
69
We have provided several pieces of anatomical evidence for the novel
glutamatergic phenotype of the GnRH neurons. Our ISHH experiments have shown that
nearly all GnRH neurons (~99%) in male rats express the glutamatergic marker Vglut2
mRNA. The confocal microscopic observations have confirmed the presence of Vglut2
protein in the GnRH axon terminals of the OVLT and the ME. Finally, our
preembedding electron microscopic examinations have verified the presence of Vglut2
in the axon terminals of the ME and established the association of Vglut2immunoreactivity with small clear vesicles.
6.4.1. Central effects of glutamate on the regulation of reproduction
Glutamate is a critically important neurotransmitter in the regulation of the GnRH
neuronal system. Intravenous N-methyl-D,L-aspartate infusion can induce precocious
puberty in immature rats (195) and activation of ionotropic glutamate receptors plays a
crucial role in both pulse (5) and surge (196) modes of GnRH secretion. Administration
of glutamate can enhance circulating LH levels and this effect is exerted centrally, since
exposure of pituitary slices to glutamate or glutamate injection directly into the
hypophysis do not alter LH release (44). Indeed, glutamate can regulate GnRH neurons
at the level of GnRH cell bodies and dendrites, which receive Vglut2-immunoreative
synapses (49, 146) and exhibit immunoreactivity for ionotropic glutamate receptors (45,
46). Moreover, compelling evidence indicates that an additional major site of action for
glutamate is the ME. GnRH terminals in the ME are apposed to glutamatergic axons
(146, 149) and express immunoreactivity for the kainite-2 (KA2) and N-methyl-Daspartate-1 ionotropic glutamate receptor subunits (NMDAR-1) (149). Further,
glutamate and agonists of ionotropic glutamate receptors can induce Ca2+-dependent
release of GnRH from superfused ME fragments (148).
6.4.2. Glutamatergic phenotype of the GnRH neurons
Our ISHH finding that virtually all GnRH neurons expressed Vglut2 mRNA in the
adult male rat strongly suggests that the glutamatergic chemotype is a critically
important feature of the GnRH neuronal system. It is important to note that the ME also
70
contained a large number of Vglut2-immunoreactive terminals that were devoid of
immunostaining for GnRH. A likely source of some of these fibers is the parvicellular
part of the hypothalamic paraventricular nucleus, where Vglut2-expressing neurons
occur in high numbers (146, 147). Indeed, according to our subsequent studies, the
parvicellular hypophysiotropic neurons including thyrotropin-releasing hormone, CRH
(197) and somatostatin-synthesizing neurons (198) express both the mRNA and the
transporter protein of Vglut2.
A large subset of GnRH neurons in rats, mice, and humans exhibit GABAimmunoreactivity (83, 85) during fetal migration from the olfactory placode to the
forebrain (9). Tobet and co-workers (83) found no GABA-immunoreactivity in GnRH
neurons that migrated further caudal to the olfactory bulbs, suggesting that GnRH
neurons may switch from the GABAergic to the glutamatergic phenotype perinatally.
Nevertheless, the image about the GABA/glutamate switch in the GnRH neurons during
the prenatal migration may be more shaded. Interestingly, Vglut2-immunoreactive cell
groups also occur in the olfactory placode at E11.5 (199), around the time when the first
juvenile GnRH neurons can be detected there and begin their migration toward the POA
(200). Moreover, Honma and coworkers were able to reveal GnRH-associated protein
immunoreactivity in a few migrating Vglut2-immunoreactive nerve cells between E14.5
- E16.5 (199). Considering that the peak in the number of GABA positive neurons in the
peripheral olfactory sites (i.e. vomeronasal organ and around the vomeronasal and
olfactory nerve) is around E15 (83), the juvenile GnRH neurons or at least a
subpopulation of them may use both GABA and glutamate signaling. The question
whether or not the adult GnRH neurons also have such a double GABA/glutamatergic
phenotype remains opens. We showed that virtually all GnRH neuron in adult male rats
possessed Vglut2 mRNA. This finding does not exclude the possibility that the GABA
synthetic enzymes and the vesicular GABA transporter may continue to be expressed
together with Vglut2 in a subset of adult GnRH neurons. In this context, it is interesting
to note that Ottem et al. observed neurons in the anteroventral periventricular nucleus
that possess such a GABA/glutamate double phenotype (201). Similarly, prenatal
GnRH neurons may also express a dual GABA/glutamate phenotype.
The putative existence of autocrine/paracrine glutamatergic mechanisms in the
terminal regulation of GnRH neurosecretion receives substantial support from i) our
71
present observation that GnRH terminals co-contain Vglut2; ii) the identification of
immunoreactivities for the KA-2 and the NMDAR-1 ionotropic glutamate receptor
subunits on GnRH-immunoreactive terminals (148, 149, 202); and iii) the ability of
ionotropic glutamate receptor agonists to elicit GnRH release from the mediobasal
hypothalamus (44). Glutamate receptors may also be present on glial and vascular
elements of the ME and posterior pituitary to influence their functions, possibly
including morphological plasticity. Tanycytes lining the ventral wall of the third
ventricle and astrocytes of the ME were, indeed, found to contain mRNAs and
immunoreactivity for kainate receptors (203-205) and to also express the activity
marker c-Fos immunoreactivity in response to stimulation by kainate (204).
6.4.3. Subcellular localization of Vglut2 in the axon terminals of the ME
Our electron microscopic observations have demonstrated the presence of Vglut2
in the hypophysiotropic nerve terminals of the ME and the close association of Vglut2immunoreactivity to the small clear vesicles.
It is worth noting that neuroendocrine axons were particularly rich in small clear
vesicles, close to their termination around the pericapillary space in both the ME and the
posterior pituitary. Inversely, DVs were often decreased in number as the
neuroendocrine axons approached the basal lamina. Previous studies have revealed a
similar change in the vesicular composition of CRH-containing axons in the ME in that
DVs dominated the preterminals, whereas small clear vesicles became more
characteristic to terminals reaching the pericapillary space (206). Glutamateimmunoreactive small clear vesicles also had a tendency to accumulate at the terminal
segment of magnocellular axons in the posterior pituitary (207), which is in line with
our recent finding of Vglut2-immunoreactive small clear vesicles in magnocellular
terminals (208). To some extent, the differential distribution of the two types of vesicles
is also reminiscent to their relationship at chemical synapses of various species. The
presynaptic specialization contains primarily small clear vesicles, whereas dense core
granules do not tend to occur close to the synaptic specializations (209, 210). Although
the astrocytes in vitro possess all types of Vgluts (211) we could not observed
significant in situ Vglut2-immunolabeling above the glial elements of the ME with our
72
pre-embedding electron microscopic labeling. In contrast, the observable ultrastructural
distribution of Vglut2 above small clear and not dense core, vesicles agrees with the
presumed site of location of this classical neurotransmitter in neurons. This location is
also in agreement with the association of Vglut1- and Vglut2- immunoreactivities with
small clear vesicles in excitatory synapses (47, 212, 213) in other brain areas.
To elucidate the role of glutamate co-secretion in neuroendocrine regulation, it is
of prime importance to first find and characterize glutamate receptors in the ME and
hypothalamus. Theoretically, such receptors may be located on the neuroendocrine
terminals to inhibit or facilitate peptide neurosecretion. In a paper published recently
(202), Yin and coworkers compared the distribution of the ionotropic NMDAR-1
subunit with GnRH-immunoreactivity by a sensitive double-labeling postembedding
electron microscopy in the axon terminals of the ME. Their analysis showed that
NMDAR-1 subunits frequently colocalized with GnRH decapeptide even within the
same DV, while the plasma membrane exhibited low immunoreactivity for this receptor.
The authors concluded that since the sparse occurrence of synaptic connections in the
ME, the liberating glutamate from the nerve terminals might triggers the release of other
dense core granules, which are located either in the neighboring nerve terminals or even
within the same nerve ending.
From the occurrence of Vglut2 in functionally and neurochemically diverse
neurosecretory endings, it is tempting to speculate that intrinsic L-glutamate fulfills
similar regulatory functions in several neuroendocrine systems and may contribute to
the generation of the pulsatile patterns of neurohormone output. Nevertheless, this
hypothesis would require experimental support. The molecular mechanisms by which
glutamate endogenous to GnRH neurons regulates reproduction require future
clarification.
6.4.4. Methodological considerations
The reason why previous confocal and electron microscopic studies (49, 146, 148,
149) failed to reveal the glutamatergic phenotype of the GnRH neuronal system is not
clear. In view of the moderate Vglut2 mRNA and protein levels we found in GnRH
cells, the use of amplification methods could be essential for our colocalization studies
73
to succeed. Furthermore, although Vglut2 mRNA was present in virtually all GnRH
neurons, Vglut2-immunoreactivity often remained undetectable in GnRH terminals.
This discrepancy may be attributable to a general limitation of the immunocytochemical
detection method due to the low amount of Vglut2 protein. Another possibility is the
heterogeneity of GnRH axon varicosities, in that some may mostly contain DVs with
GnRH and only few small clear vesicles with Vglut2.
74
7. Conclusions
1. We
have
provided
anatomical
proof for the direct innervation of
hypophysiotropic GnRH neurons by the cholinergic neurotransmitter system in
male rats. We have suggested that this neuronal communication takes place
mostly via nonsynaptic mechanisms.
2. We have determined that in male mice 49-64% of the NPY innervation of GnRH
neurons arises from the Arc of the hypothalamus, while 25% of NPY contacts
originate from the catecholaminergic nuclei of the brainstem.
3. We have revealed synaptic contacts between AGRP-immunoreactive terminals,
which originate from the Arc and the hypophysiotropic GnRH neurons in male
mice. The symmetric morphology we observed is typical of inhibitory synapses.
4. We have provided evidence for the presence of Vglut2 mRNA and protein in the
hypophysiotropic GnRH neurons of male rats. Moreover, we have verified the
presence of Vglut2 in the axon terminals of the ME and the close association of
Vglut2-immunoreactivity to small clear vesicles. These data suggest a novel
glutamatergic phenotype of GnRH neurons and can serve as the basis of a novel
autocrine/paracrine regulation of neurohormone release.
75
Figure 15. The schematic drawing represents the known synaptic inputs to GnRH neurons
(orange color). The terminals indicated by orange color correspond to the novel
transmitter systems that directly innervate GnRH neurons and were revealed by our
studies
in
this
thesis.
Inside
the
GnRH
neuron
can
be
found
neurotransmitters/neuromodulators co-expressed in GnRH cells. The presence of Vglut2
in GnRH neurons, which was revealed in our studies, suggests the previously unknown
glutamatergic phenotype of GnRH neurons. (5-HT: serotonin; β-END: β-endorphin; ACh:
acetylcholine; AGRP: agouti-related peptide; DSIP: delta sleep-inducing peptide; CCK:
cholecystokinin; CRH: corticotropin-releasing hormone; DBH: dopamine-β-hydroxylase;
GABA: gamma-aminobutyric acid; GnRH: gonadotropin-releasing hormone; GLU:
glutamate; NT: neurotensin; SP: substance P; TH: tyrosine hydroxylase; Vglut2: vesicular
glutamate transporter-2)
76
8. Summary
The hypothalamo-pituitary-gonadal (HPG) axis regulates the reproductive
functions. The central unit of the axis is the gonadotropin-releasing hormone (GnRH)producing neuron population located in the preoptic area (POA). The neurosecretory
activity of GnRH neurons is under the influence of hormonal factors and several
neurotransmitter systems. The aim of this dissertation is the identification of novel
neurotransmitter systems in the neuronal regulation of the GnRH neurons. (1)
Acetylcholine (ACh) is able to modulate the activity of GnRH neurosecretion and
sexual behavior. We have provided neuroanatomical evidence for the direct innervation
of GnRH neurons by cholinergic axons in male rats. Our results indicate that the
cholinergic axons often formed direct contacts but rarely established classical synapses
with the GnRH neurons. Therefore, similarly to other brain areas, we propose
nonsynaptic mechanisms whereby ACh modulates the functions of GnRH neurons in
the POA. (2) Despite the profound central effect of neuropeptide Y (NPY) on the HPG
axis, the sources of NPY afferent to GnRH neurons were unidentified. Therefore, we
addressed the sources of origin of NPY-containing afferents to GnRH neurons in male
GnRH-GFP transgenic mice. Our results indicate that NPY/AGRP neurons of the Arc
gave rise to 49–64% of the NPY-immunoreactive axonal contacts on the somata and
proximal dendrites of GnRH neurons (depending on the calculation approach we used),
and an additional 25% of juxtapositions originated in adrenergic/noradrenergic cell
groups of the brainstem. (3) Moreover, we have revealed with electron microscopy that
the AGRP-immunoreactive terminals establish symmetric synapses with GnRH neurons.
(4) The neurotransmitter glutamate is packed into synaptic vesicles by the three recently
discovered vesicular glutamate transporters (Vglut1-3). Using the Vglut2 as a marker of
glutamatergic neuronal phenotype, we have provided conclusive evidence for a marked
glutamatergic phenotype of GnRH neurons in the adult male rat by demonstrating
Vglut2 mRNA expression in the perikarya and Vglut2-immunoreactivity in the axons of
these cells. Using immuno-electron microscopy, we have demonstrated that the Vglut2immunoreactivity is localized to small clear vesicles in the neurosecretory endings of
the ME. The physiological significance of endogenous glutamate in the regulation of
GnRH secretion requires clarification.
77
9. Összefoglalás
A szaporodást és az azzal összefüggő élettani folyamatokat a hypothalamohypophyseo-gonadális
tengely szabályozza. Rágcsálókban
a
tengely
centrális
szabályozó elemei a preoptikus area területén elhelyezkedő gonadotropin-releasing
hormon (GnRH)-t termelő idegsejtek, melyek neuroszekréciós működését humorális
faktorok és neurotranszmitter rendszerek együttesen modulálják. A doktori értekezés
témája a GnRH idegsejtek neurális szabályozásban részt vevő új neurotranszmitterek
azonosítása volt. (1) Az acetilkolin (ACh) in vitro már nanomoláris koncentrációban is
képes
serkenteni
a
GnRH
neuroszekréciót.
Fény-
és
elektronmikroszkópos
vizsgálatainkkal kimutattuk, hogy hím patkányokban a kolinerg axonok közvetlenül
beidegzik a GnRH neuronokat, azonban a klasszikus morfológiájú szinapszisok a
kolinerg idegnyúlványok valamint a GnRH idegsejtek és dendritjeik között ritkák voltak.
Megfigyeléseink alapján azt feltételeztük, hogy - hasonlóan a központi idegrendszer
egyéb területeihez -, az ACh a hypothalamusban is főként nem-szinaptikus módon hat
az idegsejtek, így a GnRH neuronok működésére. (2) A centrálisan adott neuropeptid Y
(NPY) reprodukció szabályozására gyakorolt élettani hatásai jól ismertek. A NPY
tartalmú idegnyúlványok beidegzik a GnRH idegsejteket, azonban az afferensek pontos
eredete nem ismert. A GnRH idegsejtek NPY afferenseinek kvantitatív analízisével
megállapítottuk, hogy hím egerekben az NPY beidegzés két fő forrása a hypothalamus
acuatus (Arc) idegmagja (~49-64%), valamint az agytörzsi katekolaminerg idegmagok
(~25%). (3) További immun-elektronmikroszkópos vizsgálatainkkal bebizonyítottuk,
hogy az Arc eredetű NPY-t és agouti-releated peptidet egyaránt tartalmazó
idegnyúlványok szimmetrikus szinapszisokat létesítenek a GnRH neuronokkal. (4) A
glutamátot a központi idegrendszer területén a vezikuláris glutamát transzporterek 3
típusa (Vglut1-3) csomagolja szinaptikus vezikulákba. Igazoltuk, hogy a hím patkányok
GnRH idegsejtjeinek döntő többsége (~99%) glutamáterg kemotípusú is, mivel a GnRH
sejttestekben Vglut2 mRNS in situ hibridizációs jelet, az axonokban pedig Vglut2immunreaktivitást tudtunk kimutatni. Vizsgálatainkkal megállapítottuk, hogy a Vglut2immunreaktivitás ultrastruktúrális szinten a neuroszekretoros idegvégződésekben
található szinaptikus vezikulákhoz kötődik. A GnRH neuronhálózat újonnan leírt
glutamáterg kemotípusának funkcionális jelentőségére egyelőre nincsen magyarázat.
78
10. Literature cited
1
Tilbrook AJ, Clarke IJ (2001) Negative feedback regulation of the secretion and
actions of gonadotropin-releasing hormone in males. Biol Reprod, 64:735-742
2
Moenter SM, Caraty A, Locatelli A, Karsch FJ (1991) Pattern of gonadotropinreleasing hormone (GnRH) secretion leading up to ovulation in the ewe:
existence of a preovulatory GnRH surge. Endocrinology, 129:1175-1182
3
Freeman M. The neuroendocrine control of ovarian cycle of the rat. In: Knobil E,
Neill JD (eds.), The Physiology of reproduction. 2nd ed. Raven Press, New York,
1994: 613-658
4
Nunemaker CS, DeFazio RA, Geusz ME, Herzog ED, Pitts GR, Moenter SM
(2001) Long-term recordings of networks of immortalized GnRH neurons reveal
episodic patterns of electrical activity. J Neurophysiol, 86:86-93
5
Bourguignon JP, Gerard A, Mathieu J, Simons J, Franchimont P (1989) Pulsatile
release of gonadotropin-releasing hormone from hypothalamic explants is
restrained by blockade of N-methyl-D,L-aspartate receptors. Endocrinology,
125:1090-1096
6
Martinez de la Escalera G, Choi AL, Weiner RI (1992) Generation and
synchronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic
properties of the GT1-1 GnRH neuronal cell line. Proc Natl Acad Sci U S A,
89:1852-1855
7
Merchenthaler I, Gorcs T, Setalo G, Petrusz P, Flerko B (1984) Gonadotropinreleasing hormone (GnRH) neurons and pathways in the rat brain. Cell Tissue
Res, 237:15-29
8
Silverman A-J, Livne I, Witkin JW. The gonadortophin-releasing hormone
(GnRH) neural systems: immunocytochemistry and in situ hybridization. In:
Knobil E ed. The Physiology of Reproduction. 2nd ed. Raven Press, New York,
1994: 1683-1709
9
Schwanzel-Fukuda M, Pfaff DW (1989) Origin of luteinizing hormone-releasing
hormone neurons. Nature, 338:161-164
10
Sullivan KA, Silverman AJ (1993) The ontogeny of gonadotropin-releasing
hormone neurons in the chick. Neuroendocrinology, 58:597-608
79
11
Ronnekleiv OK, Resko JA (1990) Ontogeny of gonadotropin-releasing
hormone-containing neurons in early fetal development of rhesus macaques.
Endocrinology, 126:498-511
12
Daikoku-Ishido H, Okamura Y, Yanaihara N, Daikoku S (1990) Development of
the hypothalamic luteinizing hormone-releasing hormone-containing neuron
system in the rat: in vivo and in transplantation studies. Developmental biology,
140:374-387
13
Schwanzel-Fukuda M, Crossin KL, Pfaff DW, Bouloux PM, Hardelin JP, Petit
C (1996) Migration of luteinizing hormone-releasing hormone (LHRH) neurons
in early human embryos. The Journal of comparative neurology, 366:547-557
14
Witkin JW, Ferin M, Popilskis SJ, Silverman AJ (1991) Effects of gonadal
steroids on the ultrastructure of GnRH neurons in the rhesus monkey: synaptic
input and glial apposition. Endocrinology, 129:1083-1092
15
Witkin JW, O'Sullivan H, Silverman AJ (1995) Novel associations among
gonadotropin-releasing hormone neurons. Endocrinology, 136:4323-4330
16
Campbell RE, Han SK, Herbison AE (2005) Biocytin filling of adult
gonadotropin-releasing hormone neurons in situ reveals extensive, spiny,
dendritic processes. Endocrinology, 146:1163-1169
17
Rethelyi M, Vigh S, Setalo G, Merchenthaler I, Flerko B, Petrusz P (1981) The
luteinizing hormone releasing hormone-containing pathways and their cotermination with tanycyte processes in and around the median eminence and in
the pituitary stalk of the rat. Acta morphologica Academiae Scientiarum
Hungaricae, 29:259-283
18
Boehm U, Zou Z, Buck LB (2005) Feedback loops link odor and pheromone
signaling with reproduction. Cell, 123:683-695
19
Merchenthaler I (1991) Neurons with access to the general circulation in the
central nervous system of the rat: a retrograde tracing study with fluoro-gold.
Neuroscience, 44:655-662
20
Cone R, Low JM, Elmquist KJ, Cameron LJ. Neuroendocrinology. In:
Kronenberg H, Williams RH (eds.), Williams textbook of endocrinology. 10th
ed. Saunders, Philadelphia, 2003: 81-176
80
21
Palkovits M. Neural and vascular connections between the organum vasculosum
lamiae terminalis and preoptic nuclei. In: Scott DE, Kozlowski GP, Weinl A
(eds.), Brain-Endocrine Interaction: Neuronal Hormones and Reproduction.
Karger, Basel, 1978: 302-312
22
Oldfield BJ, McKinley MJ. Circumventricular Organs. In: Paxinos G ed. The
Rat Nervous System. Second Edition ed. Academic Press, San Diego, 1995:
391-403
23
Langub MC, Jr., Watson RE, Jr. (1992) Estrogen receptor-immunoreactive glia,
endothelia, and ependyma in guinea pig preoptic area and median eminence:
electron microscopy. Endocrinology, 130:364-372
24
McKinley MJ, Mathai ML, McAllen RM, McClear RC, Miselis RR, Pennington
GL, Vivas L, Wade JD, Oldfield BJ (2004) Vasopressin secretion: osmotic and
hormonal regulation by the lamina terminalis. J Neuroendocrinol, 16:340-347
25
Prevot V, Dutoit S, Croix D, Tramu G, Beauvillain JC (1998) Semi-quantitative
ultrastructural analysis of the localization and neuropeptide content of
gonadotropin releasing hormone nerve terminals in the median eminence
throughout the estrous cycle of the rat. Neuroscience, 84:177-191
26
Page
RB
(1986)
The
Pituitary
Portal
System.
Current
Topics
in
Neuroendocrinology, 7:1-48
27
Carson KA, Nemeroff CB, Rone MS, Young-Blood WW, Prange AJ, Jr.,
Hanker JS, Kizer JS (1977) Biochemical and histochemical evidence for the
existence of a tuberoinfundibular cholinergic pathway in the rat. Brain Res,
129:169-173
28
Vincent SR, Hokfelt T, Wu JY (1982) GABA neuron systems in hypothalamus
and the pituitary gland. Immunohistochemical demonstration using antibodies
against glutamate decarboxylase. Neuroendocrinology, 34:117-125
29
Lopez FJ, Merchenthaler I, Ching M, Wisniewski MG, Negro-Vilar A (1991)
Galanin: a hypothalamic-hypophysiotropic hormone modulating reproductive
functions. Proc Natl Acad Sci U S A, 88:4508-4512
30
Kahn D, Abrams GM, Zimmerman EA, Carraway R, Leeman SE (1980)
Neurotensin neurons in the rat hypothalamus: an immunocytochemical study.
Endocrinology, 107:47-54
81
31
Ciofi P (2000) Phenotypical segregation among female rat hypothalamic
gonadotropin-releasing hormone neurons as revealed by the sexually dimorphic
coexpression of cholecystokinin and neurotensin. Neuroscience, 99:133-147
32
Vallet PG, Charnay Y, Boura C, Kiss JZ (1991) Colocalization of delta sleep
inducing peptide and luteinizing hormone releasing hormone in neurosecretory
vesicles in rat median eminence. Neuroendocrinology, 53:103-106
33
Fuxe K (1963) Cellular Localization of Monoamines in the Median Eminence
and in the Infundibular Stem of Some Mammals. Acta physiologica
Scandinavica, 58:383-384
34
Gulyas AI, Megias M, Emri Z, Freund TF (1999) Total number and ratio of
excitatory and inhibitory synapses converging onto single interneurons of
different types in the CA1 area of the rat hippocampus. J Neurosci, 19:1008210097
35
Matyas F, Freund TF, Gulyas AI (2004) Convergence of excitatory and
inhibitory inputs onto CCK-containing basket cells in the CA1 area of the rat
hippocampus. Eur J Neurosci, 19:1243-1256
36
Chen WP, Witkin JW, Silverman AJ (1990) Sexual dimorphism in the synaptic
input to gonadotropin releasing hormone neurons. Endocrinology, 126:695-702
37
Nimchinsky EA, Sabatini BL, Svoboda K (2002) Structure and function of
dendritic spines. Annual review of physiology, 64:313-353
38
Herbison AE (1998) Multimodal influence of estrogen upon gonadotropinreleasing hormone neurons. Endocr Rev, 19:302-330
39
Herbison AE (1997) Noradrenergic regulation of cyclic GnRH secretion. Rev
Reprod, 2:1-6.
40
Miller MM, Zhu L (1995) Ovariectomy and age alter gonadotropin hormone
releasing hormone-noradrenergic interactions. Neurobiol Aging, 16:613-625.
41
Leranth
C,
MacLusky
NJ,
Shanabrough
M,
Naftolin
F
(1988)
Catecholaminergic innervation of luteinizing hormone-releasing hormone and
glutamic acid decarboxylase immunopositive neurons in the rat medial preoptic
area. An electron-microscopic double immunostaining and degeneration study.
Neuroendocrinology, 48:591-602
82
42
van den Pol AN, Wuarin JP, Dudek FE (1990) Glutamate, the dominant
excitatory transmitter in neuroendocrine regulation. Science, 250:1276-1278
43
Brann DW, Mahesh VB (1995) Glutamate: a major neuroendocrine excitatory
signal mediating steroid effects on gonadotropin secretion. The Journal of
steroid biochemistry and molecular biology, 53:325-329
44
Jennes L, Lin W, Lakhlani S (2002) Glutamatergic regulation of gonadotropinreleasing hormone neurons. Prog Brain Res, 141:183-192
45
Eyigor O, Jennes L (2000) Kainate receptor subunit-positive gonadotropinreleasing hormone neurons express c-Fos during the steroid-induced luteinizing
hormone surge in the female rat. Endocrinology, 141:779-786
46
Gore AC, Wu TJ, Rosenberg JJ, Roberts JL (1996) Gonadotropin-releasing
hormone and NMDA receptor gene expression and colocalization change during
puberty in female rats. J Neurosci, 16:5281-5289
47
Fremeau RT, Jr., Troyer MD, Pahner I, Nygaard GO, Tran CH, Reimer RJ,
Bellocchio EE, Fortin D, Storm-Mathisen J, Edwards RH (2001) The expression
of vesicular glutamate transporters defines two classes of excitatory synapse.
Neuron, 31:247-260
48
Kaneko T, Fujiyama F, Hioki H (2002) Immunohistochemical localization of
candidates for vesicular glutamate transporters in the rat brain. The Journal of
comparative neurology, 444:39-62
49
Kiss J, Kocsis K, Csaki A, Halasz B (2003) Evidence for vesicular glutamate
transporter synapses onto gonadotropin-releasing hormone and other neurons in
the rat medial preoptic area. Eur J Neurosci, 18:3267-3278
50
Fremeau RT, Jr., Voglmaier S, Seal RP, Edwards RH (2004) VGLUTs define
subsets of excitatory neurons and suggest novel roles for glutamate. Trends
Neurosci, 27:98-103
51
Kocsis K, Kiss J, Csaki A, Halasz B (2003) Location of putative glutamatergic
neurons projecting to the medial preoptic area of the rat hypothalamus. Brain
Res Bull, 61:459-468
52
Decavel C, Van den Pol AN (1990) GABA: a dominant neurotransmitter in the
hypothalamus. The Journal of comparative neurology, 302:1019-1037
83
53
Leranth C, MacLusky NJ, Sakamoto H, Shanabrough M, Naftolin F (1985)
Glutamic acid decarboxylase-containing axons synapse on LHRH neurons in the
rat medial preoptic area. Neuroendocrinology, 40:536-539
54
Peters A, Palay SL, deF Webster H. The fine structure of the nervous system.
Neurons and their supporting cells. Third Edition ed. Oxford University Press,
Oxford, 1991
55
Petersen SL, McCrone S, Coy D, Adelman JP, LC. M (1993) GABAA receptor
subunit mRNAs in cells of the preoptic area: colocalization with LHRH mRNA
using dual-label in situ hybridization histochemistry. Endocrine:29-34
56
Herbison AE, Chapman C, Dyer RG (1991) Role of medial preoptic GABA
neurones in regulating luteinising hormone secretion in the ovariectomised rat.
Experimental brain research Experimentelle Hirnforschung, 87:345-352
57
Moenter SM, DeFazio RA (2005) Endogenous gamma-aminobutyric acid can
excite gonadotropin-releasing hormone neurons. Endocrinology, 146:5374-5379
58
Han SK, Todman MG, Herbison AE (2004) Endogenous GABA release inhibits
the firing of adult gonadotropin-releasing hormone neurons. Endocrinology,
145:495-499
59
Kordon C, Drouva S, Martinez de la Escalera G, Weiner R, Knobil E, Neill JD.
The Physiology of reproduction. In: Knobil E ed. 2nd ed. Raven Press, New
York, 1994: 1621-1681
60
Kiss J, Halasz B (1985) Demonstration of serotoninergic axons terminating on
luteinizing hormone-releasing hormone neurons in the preoptic area of the rat
using
a
combination
of
immunocytochemistry
and
high
resolution
autoradiography. Neuroscience, 14:69-78
61
Campbell RE, Herbison AE (2007) Definition of brainstem afferents to
gonadotropin-releasing hormone (GnRH) neurons in the mouse using
conditional viral tract tracing. Endocrinology,
62
Wright DE, Jennes L (1993) Lack of expression of serotonin receptor subtype 1a, 1c, and -2 mRNAs in gonadotropin-releasing hormone producing neurons of
the rat. Neurosci Lett, 163:1-4
63
Todman MG, Han SK, Herbison AE (2005) Profiling neurotransmitter receptor
expression in mouse gonadotropin-releasing hormone neurons using green
84
fluorescent protein-promoter transgenics and microarrays. Neuroscience,
132:703-712
64
Dobson H, Ghuman S, Prabhakar S, Smith R (2003) A conceptual model of the
influence of stress on female reproduction. Reproduction (Cambridge, England),
125:151-163
65
MacLusky NJ, Naftolin F, Leranth C (1988) Immunocytochemical evidence for
direct synaptic connections between corticotrophin-releasing factor (CRF) and
gonadotrophin-releasing hormone (GnRH)-containing neurons in the preoptic
area of the rat. Brain Res, 439:391-395
66
Swanson LW, Sawchenko PE, Rivier J, Vale WW (1983) Organization of ovine
corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an
immunohistochemical study. Neuroendocrinology, 36:165-186
67
Hahn JD, Coen CW (2006) Comparative study of the sources of neuronal
projections to the site of gonadotrophin-releasing hormone perikarya and to the
anteroventral periventricular nucleus in female rats. The Journal of comparative
neurology, 494:190-214
68
Leranth C, Segura LM, Palkovits M, MacLusky NJ, Shanabrough M, Naftolin F
(1985) The LH-RH-containing neuronal network in the preoptic area of the rat:
demonstration of LH-RH-containing nerve terminals in synaptic contact with
LH-RH neurons. Brain Res, 345:332-336
69
Pelletier G (1987) Demonstration of contacts between neurons staining for
LHRH in the preoptic area of the rat brain. Neuroendocrinology, 46:457-459
70
Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ,
Gottsch ML, Clifton DK, Steiner RA (2004) Kisspeptin activation of
gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the
male rat. Neuroendocrinology, 80:264-272
71
Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, Thresher
RR, Malinge I, Lomet D, Carlton MB, Colledge WH, Caraty A, Aparicio SA
(2005) Kisspeptin directly stimulates gonadotropin-releasing hormone release
via G protein-coupled receptor 54. Proc Natl Acad Sci U S A, 102:1761-1766
85
72
Clarkson J, Herbison AE (2006) Postnatal development of kisspeptin neurons in
mouse hypothalamus; sexual dimorphism and projections to gonadotropinreleasing hormone neurons. Endocrinology, 147:5817-5825
73
Dungan HM, Clifton DK, Steiner RA (2006) Minireview: kisspeptin neurons as
central processors in the regulation of gonadotropin-releasing hormone secretion.
Endocrinology, 147:1154-1158
74
Tsuruo Y, Kawano H, Kagotani Y, Hisano S, Daikoku S, Chihara K, Zhang T,
Yanaihara N (1990) Morphological evidence for neuronal regulation of
luteinizing hormone-releasing hormone-containing neurons by neuropeptide Y
in the rat septo-preoptic area. Neurosci Lett, 110:261-266
75
Mezey E, Kiss JZ, Mueller GP, Eskay R, O'Donohue TL, Palkovits M (1985)
Distribution of the pro-opiomelanocortin derived peptides, adrenocorticotrope
hormone, alpha-melanocyte-stimulating hormone and beta-endorphin (ACTH,
alpha-MSH, beta-END) in the rat hypothalamus. Brain Res, 328:341-347
76
Ellacott KL, Halatchev IG, Cone RD (2006) Characterization of leptinresponsive neurons in the caudal brainstem. Endocrinology, 147:3190-3195
77
Palkovits M, Eskay RL (1987) Distribution and possible origin of betaendorphin and ACTH in discrete brainstem nuclei of rats. Neuropeptides, 9:123137
78
Leranth
C,
MacLusky
NJ,
Shanabrough
M,
Naftolin
F
(1988)
Immunohistochemical evidence for synaptic connections between proopiomelanocortin-immunoreactive axons and LH-RH neurons in the preoptic
area of the rat. Brain Res, 449:167-176
79
Mantyh PW (2002) Neurobiology of substance P and the NK1 receptor. The
Journal of clinical psychiatry, 63 Suppl 11:6-10
80
Vijayan E, McCann SM (1979) In vivo and in vitro effects of substance P and
neurotensin on gonadotropin and prolactin release. Endocrinology, 105:64-68
81
Duval P, Lenoir V, Garret C, Kerdelhue B (1996) Reduction of the amplitude of
preovulatory LH and FSH surges and of the amplitude of the in vitro GnRHinduced LH release by substance P. Reversal of the effect by RP 67580.
Neuropharmacology, 35:1805-1810
86
82
Tsuruo Y, Kawano H, Hisano S, Kagotani Y, Daikoku S, Zhang T, Yanaihara N
(1991) Substance P-containing neurons innervating LHRH-containing neurons
in the septo-preoptic area of rats. Neuroendocrinology, 53:236-245.
83
Tobet SA, Chickering TW, King JC, Stopa EG, Kim K, Kuo-Leblank V,
Schwarting GA (1996) Expression of gamma-aminobutyric acid and
gonadotropin-releasing hormone during neuronal migration through the
olfactory system. Endocrinology, 137:5415-5420
84
Bless EP, Westaway WA, Schwarting GA, Tobet SA (2000) Effects of gammaaminobutyric acid(A) receptor manipulation on migrating gonadotropinreleasing hormone neurons through the entire migratory route in vivo and in
vitro. Endocrinology, 141:1254-1262
85
Tobet SA, Bless EP, Schwarting GA (2001) Developmental aspect of the
gonadotropin-releasing hormone system. Mol Cell Endocrinol, 185:173-184
86
Skofitsch G, Jacobowitz DM (1985) Immunohistochemical mapping of galaninlike neurons in the rat central nervous system. Peptides, 6:509-546
87
Marks DL, Smith MS, Vrontakis M, Clifton DK, Steiner RA (1993) Regulation
of galanin gene expression in gonadotropin-releasing hormone neurons during
the estrous cycle of the rat. Endocrinology, 132:1836-1844
88
Daftary SS, Gore AC (2005) IGF-1 in the brain as a regulator of reproductive
neuroendocrine function. Experimental biology and medicine (Maywood, NJ,
230:292-306
89
Miller BH, Gore AC (2001) Alterations in hypothalamic insulin-like growth
factor-I and its associations with gonadotropin releasing hormone neurones
during reproductive development and ageing. J Neuroendocrinol, 13:728-736
90
Daftary SS, Gore AC (2004) The hypothalamic insulin-like growth factor-1
receptor and its relationship to gonadotropin-releasing hormones neurones
during postnatal development. J Neuroendocrinol, 16:160-169
91
Sahu A, Kalra SP (1987) Delta sleep-inducing peptide (DSIP) stimulates LH
release in steroid-primed ovariectomized rats. Life Sci, 40:1201-1206
92
Iyer KS, McCann SM (1987) Delta sleep inducing peptide (DSIP) stimulates the
release of LH but not FSH via a hypothalamic site of action in the rat. Brain Res
Bull, 19:535-538
87
93
Charnay Y, Leger L, Golaz J, Sallanon M, Vallet PG, Guntern R, Bouras C,
Constantinidis J, Jouvet M, Tissot R (1990) Immunohistochemical mapping of
delta sleep-inducing peptide in the cat brain and hypophysis. Relationships with
the LHRH system and corticotropes. Journal of chemical neuroanatomy, 3:397412
94
Charnay Y, Bouras C, Vallet PG, Golaz J, Guntern R, Constantinidis J (1989)
Immunohistochemical colocalization of delta sleep-inducing peptide and
luteinizing hormone-releasing hormone in rabbit brain neurons. Neuroscience,
31:495-505
95
Vallet PG, Charnay Y, Bouras C (1990) Distribution and colocalization of delta
sleep-inducing peptide and luteinizing hormone-releasing hormone in the aged
human brain: an immunohistochemical study. Journal of chemical neuroanatomy,
3:207-214
96
Arvidsson U, Riedl M, Elde R, Meister B (1997) Vesicular acetylcholine
transporter (VAChT) protein: a novel and unique marker for cholinergic neurons
in the central and peripheral nervous systems. The Journal of comparative
neurology, 378:454-467
97
Gilmor ML, Nash NR, Roghani A, Edwards RH, Yi H, Hersch SM, Levey AI
(1996) Expression of the putative vesicular acetylcholine transporter in rat brain
and localization in cholinergic synaptic vesicles. J Neurosci, 16:2179-2190
98
Lauterborn JC, Isackson PJ, Montalvo R, Gall CM (1993) In situ hybridization
localization of choline acetyltransferase mRNA in adult rat brain and spinal cord.
Brain Res Mol Brain Res, 17:59-69
99
Meister B, Gomuc B, Suarez E, Ishii Y, Durr K, Gillberg L (2006)
Hypothalamic proopiomelanocortin (POMC) neurons have a cholinergic
phenotype. Eur J Neurosci, 24:2731-2740
100
Castaneyra-Perdomo A, Perez-Delgado MM, Montagnese C, Coen CW (1992)
Brainstem projections to the medial preoptic region containing the luteinizing
hormone-releasing hormone perikarya in the rat. An immunohistochemical and
retrograde transport study. Neurosci Lett, 139:135-139
88
101
Retana-Marquez S, Velazquez-Moctezuma J (1997) Cholinergic-androgenic
interaction in the regulation of male sexual behavior in rats. Pharmacol Biochem
Behav, 56:373-378
102
Singer JJ (1968) Hypothalamic control of male and female sexual behavior in
female rats. J Comp Physiol Psychol, 66:738-742
103
Kow LM, Pfaff DW (1988) Transmitter and peptide actions on hypothalamic
neurons in vitro: implications for lordosis. Brain Res Bull, 20:857-861
104
Hull EM, Bitran D, Pehek EA, Holmes GM, Warner RK, Band LC, Clemens LG
(1988) Brain localization of cholinergic influence on male sex behavior in rats:
agonists. Pharmacol Biochem Behav, 31:169-174
105
Hull EM, Pehek EA, Bitran D, Holmes GM, Warner RK, Band LC, Bazzett T,
Clemens LG (1988) Brain localization of cholinergic influence on male sex
behavior in rats: antagonists. Pharmacol Biochem Behav, 31:175-178
106
Pfaff D, Frohlich J, Morgan M (2002) Hormonal and genetic influences on
arousal--sexual and otherwise. Trends Neurosci, 25:45-50
107
Everett JW (1964) Central Neural Control of Reproductive Functions of the
Adenohypophysis. Physiol Rev, 44:373-431
108
Benedetti WL, Lozdziejsky R, Sala MA, Monti JM, Grino E (1969) Blockade of
ovulation after atropine implants in the lateral hypothalamus of the rat.
Experientia, 25:1158-1159
109
Simonovic I, Motta M, Martini L (1974) Acetylcholine and the release of the
follicle-stimulating hormone-releasing factor. Endocrinology, 95:1373-1379
110
Richardson SB, Prasad JA, Hollander CS (1982) Acetylcholine, melatonin, and
potassium depolarization stimulate release of luteinizing hormone-releasing
hormone from rat hypothalamus in vitro. Proc Natl Acad Sci U S A, 79:26862689
111
Raposinho PD, Pierroz DD, Broqua P, White RB, Pedrazzini T, Aubert ML
(2001) Chronic administration of neuropeptide Y into the lateral ventricle of
C57BL/6J male mice produces an obesity syndrome including hyperphagia,
hyperleptinemia, insulin resistance, and hypogonadism. Mol Cell Endocrinol,
185:195-204.
89
112
Pierroz DD, Gruaz NM, d'Alieves V, Aubert ML (1995) Chronic administration
of neuropeptide Y into the lateral ventricle starting at 30 days of life delays
sexual maturation in the female rat. Neuroendocrinology, 61:293-300.
113
Catzeflis C, Pierroz DD, Rohner-Jeanrenaud F, Rivier JE, Sizonenko PC, Aubert
ML (1993) Neuropeptide Y administered chronically into the lateral ventricle
profoundly inhibits both the gonadotropic and the somatotropic axis in intact
adult female rats. Endocrinology, 132:224-234.
114
Kalra SP, Crowley WR (1984) Norepinephrine-like effects of neuropeptide Y on
LH release in the rat. Life Sci, 35:1173-1176.
115
Kaynard AH, Pau KY, Hess DL, Spies HG (1990) Third-ventricular infusion of
neuropeptide Y suppresses luteinizing hormone secretion in ovariectomized
rhesus macaques. Endocrinology, 127:2437-2444.
116
Kerkerian L, Guy J, Lefevre G, Pelletier G (1985) Effects of neuropeptide Y
(NPY) on the release of anterior pituitary hormones in the rat. Peptides, 6:12011204.
117
Khorram O, Pau KY, Spies HG (1987) Bimodal effects of neuropeptide Y on
hypothalamic release of gonadotropin-releasing hormone in conscious rabbits.
Neuroendocrinology, 45:290-297.
118
Raposinho PD, Broqua P, Pierroz DD, Hayward A, Dumont Y, Quirion R,
Junien JL, Aubert ML (1999) Evidence that the inhibition of luteinizing
hormone secretion exerted by central administration of neuropeptide Y (NPY) in
the rat is predominantly mediated by the NPY-Y5 receptor subtype.
Endocrinology, 140:4046-4055.
119
Sahu A, Crowley WR, Tatemoto K, Balasubramaniam A, Kalra SP (1987)
Effects of neuropeptide Y, NPY analog (norleucine4-NPY), galanin and
neuropeptide K on LH release in ovariectomized (ovx) and ovx estrogen,
progesterone-treated rats. Peptides, 8:921-926.
120
Allen LG, Kalra PS, Crowley WR, Kalra SP (1985) Comparison of the effects of
neuropeptide Y and adrenergic transmitters on LH release and food intake in
male rats. Life Sci, 37:617-623.
90
121
Berria M, Pau KY, Spies HG (1991) Evidence for alpha 1-adrenergic
involvement in neuropeptide Y-stimulated GnRH release in female rabbits.
Neuroendocrinology, 53:480-486.
122
Kalra SP, Crowley WR (1992) Neuropeptide Y: a novel neuroendocrine peptide
in the control of pituitary hormone secretion, and its relation to luteinizing
hormone. Front Neuroendocrinol, 13:1-46.
123
Crowley WR, Kalra SP (1987) Neuropeptide Y stimulates the release of
luteinizing hormone-releasing hormone from medial basal hypothalamus in
vitro: modulation by ovarian hormones. Neuroendocrinology, 46:97-103.
124
Woller MJ, Terasawa E (1991) Infusion of neuropeptide Y into the stalk-median
eminence stimulates in vivo release of luteinizing hormone-release hormone in
gonadectomized rhesus monkeys. Endocrinology, 128:1144-1150
125
Zarjevski N, Cusin I, Vettor R, Rohner-Jeanrenaud F, Jeanrenaud B (1993)
Chronic intracerebroventricular neuropeptide-Y administration to normal rats
mimics hormonal and metabolic changes of obesity. Endocrinology, 133:17531758.
126
Gore AC, Mitsushima D, Terasawa E (1993) A possible role of neuropeptide Y
in the control of the onset of puberty in female rhesus monkeys.
Neuroendocrinology, 58:23-34
127
Kalra PS, Bonavera JJ, Kalra SP (1995) Central administration of antisense
oligodeoxynucleotides to neuropeptide Y (NPY) mRNA reveals the critical role
of newly synthesized NPY in regulation of LHRH release. Regul Pept, 59:215220
128
Campbell RE, ffrench-Mullen JM, Cowley MA, Smith MS, Grove KL (2001)
Hypothalamic circuitry of neuropeptide Y regulation of neuroendocrine function
and food intake via the Y5 receptor subtype. Neuroendocrinology, 74:106-119.
129
Li C, Chen P, Smith MS (1999) Morphological evidence for direct interaction
between arcuate nucleus neuropeptide Y (NPY) neurons and gonadotropinreleasing hormone neurons and the possible involvement of NPY Y1 receptors.
Endocrinology, 140:5382-5390.
91
130
Urban JH, Das I, Levine JE (1996) Steroid modulation of neuropeptide Yinduced luteinizing hormone releasing hormone release from median eminence
fragments from male rats. Neuroendocrinology, 63:112-119
131
Simonian SX, Spratt DP, Herbison AE (1999) Identification and characterization
of estrogen receptor alpha-containing neurons projecting to the vicinity of the
gonadotropin-releasing hormone perikarya in the rostral preoptic area of the rat.
The Journal of comparative neurology, 411:346-358
132
Bauer-Dantoin AC, McDonald JK, Levine JE (1992) Neuropeptide Y potentiates
luteinizing hormone (LH)-releasing hormone-induced LH secretion only under
conditions leading to preovulatory LH surges. Endocrinology, 131:2946-2952.
133
Crowley WR, Hassid A, Kalra SP (1987) Neuropeptide Y enhances the release
of luteinizing hormone (LH) induced by LH-releasing hormone. Endocrinology,
120:941-945.
134
Parker SL, Kalra SP, Crowley WR (1991) Neuropeptide Y modulates the
binding of a gonadotropin-releasing hormone (GnRH) analog to anterior
pituitary GnRH receptor sites. Endocrinology, 128:2309-2316.
135
Chronwall BM, DiMaggio DA, Massari VJ, Pickel VM, Ruggiero DA,
O'Donohue TL (1985) The anatomy of neuropeptide-Y-containing neurons in rat
brain. Neuroscience, 15:1159-1181.
136
Suter KJ, Song WJ, Sampson TL, Wuarin JP, Saunders JT, Dudek FE, Moenter
SM (2000) Genetic targeting of green fluorescent protein to gonadotropinreleasing hormone neurons: characterization of whole-cell electrophysiological
properties and morphology. Endocrinology, 141:412-419
137
Broberger C, Johansen J, Johansson C, Schalling M, Hokfelt T (1998) The
neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal,
anorectic, and monosodium glutamate-treated mice. Proc Natl Acad Sci U S A,
95:15043-15048.
138
Olney JW (1969) Brain lesions, obesity, and other disturbances in mice treated
with monosodium glutamate. Science, 164:719-721.
139
Legradi G, Lechan RM (1998) The arcuate nucleus is the major source for
neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the
hypothalamic paraventricular nucleus. Endocrinology, 139:3262-3270.
92
140
Meister B, Ceccatelli S, Hokfelt T, Anden NE, Anden M, Theodorsson E (1989)
Neurotransmitters, neuropeptides and binding sites in the rat mediobasal
hypothalamus: effects of monosodium glutamate (MSG) lesions. Experimental
brain research Experimentelle Hirnforschung, 76:343-368.
141
Kalra SP, Kalra PS (1996) Nutritional infertility: the role of the interconnected
hypothalamic neuropeptide Y-galanin-opioid network. Front Neuroendocrinol,
17:371-401.
142
Mounzih K, Lu R, Chehab FF (1997) Leptin treatment rescues the sterility of
genetically obese ob/ob males. Endocrinology, 138:1190-1193.
143
Schwartz MW, Woods SC, Porte D, Jr., Seeley RJ, Baskin DG (2000) Central
nervous system control of food intake. Nature, 404:661-671
144
Hahn TM, Breininger JF, Baskin DG, Schwartz MW (1998) Coexpression of
Agrp and NPY in fasting-activated hypothalamic neurons. Nature neuroscience,
1:271-272
145
Vulliemoz NR, Xiao E, Xia-Zhang L, Wardlaw SL, Ferin M (2005) Central
infusion of agouti-related peptide suppresses pulsatile luteinizing hormone
release in the ovariectomized rhesus monkey. Endocrinology, 146:784-789
146
Lin W, McKinney K, Liu L, Lakhlani S, Jennes L (2003) Distribution of
vesicular glutamate transporter-2 messenger ribonucleic Acid and protein in the
septum-hypothalamus of the rat. Endocrinology, 144:662-670
147
Ziegler DR, Cullinan WE, Herman JP (2002) Distribution of vesicular glutamate
transporter mRNA in rat hypothalamus. The Journal of comparative neurology,
448:217-229
148
Kawakami S, Ichikawa M, Murahashi K, Hirunagi K, Tsukamura H, Maeda K
(1998) Excitatory amino acids act on the median eminence nerve terminals to
induce gonadotropin-releasing hormone release in female rats. General and
comparative endocrinology, 112:372-382
149
Kawakami SI, Hirunagi K, Ichikawa M, Tsukamura H, Maeda KI (1998)
Evidence for terminal regulation of GnRH release by excitatory amino acids in
the median eminence in female rats: a dual immunoelectron microscopic study.
Endocrinology, 139:1458-1461
93
150
Liposits Z, Setalo G, Flerko B (1984) Application of the silver-gold intensified
3,3'-diaminobenzidine chromogen to the light and electron microscopic
detection of the luteinizing hormone-releasing hormone system of the rat brain.
Neuroscience, 13:513-525.
151
Harkany T, Hartig W, Berghuis P, Dobszay MB, Zilberter Y, Edwards RH,
Mackie K, Ernfors P (2003) Complementary distribution of type 1 cannabinoid
receptors and vesicular glutamate transporter 3 in basal forebrain suggests inputspecific retrograde signalling by cholinergic neurons. Eur J Neurosci, 18:19791992
152
Simonian SX, Herbison AE (1997) Differential expression of estrogen receptor
and neuropeptide Y by brainstem A1 and A2 noradrenaline neurons.
Neuroscience, 76:517-529.
153
Sawchenko PE, Swanson LW, Grzanna R, Howe PR, Bloom SR, Polak JM
(1985) Colocalization of neuropeptide Y immunoreactivity in brainstem
catecholaminergic neurons that project to the paraventricular nucleus of the
hypothalamus. The Journal of comparative neurology, 241:138-153.
154
Chen IL, Hansen JT, Yates RD (1985) Dopamine beta-hydroxylase-like
immunoreactivity in the rat and cat carotid body: a light and electron
microscopic study. J Neurocytol, 14:131-144.
155
Adams JC (1992) Biotin amplification of biotin and horseradish peroxidase
signals in histochemical stains. J Histochem Cytochem, 40:1457-1463.
156
Hrabovszky
E,
Petersen
SL
(2002)
Increased
concentrations
of
radioisotopically-labeled complementary ribonucleic acid probe, dextran sulfate,
and dithiothreitol in the hybridization buffer can improve results of in situ
hybridization histochemistry. J Histochem Cytochem, 50:1389-1400
157
Umbriaco D, Garcia S, Beaulieu C, Descarries L (1995) Relational features of
acetylcholine, noradrenaline, serotonin and GABA axon terminals in the stratum
radiatum of adult rat hippocampus (CA1). Hippocampus, 5:605-620
158
Contant C, Umbriaco D, Garcia S, Watkins KC, Descarries L (1996)
Ultrastructural characterization of the acetylcholine innervation in adult rat
neostriatum. Neuroscience, 71:937-947
94
159
Umbriaco D, Watkins KC, Descarries L, Cozzari C, Hartman BK (1994)
Ultrastructural and morphometric features of the acetylcholine innervation in
adult rat parietal cortex: an electron microscopic study in serial sections. The
Journal of comparative neurology, 348:351-373
160
Vizi ES, Kiss JP (1998) Neurochemistry and pharmacology of the major
hippocampal transmitter systems: synaptic and nonsynaptic interactions.
Hippocampus, 8:566-607
161
Lendvai B, Vizi ES (in press) Nonsynaptic Chemical Transmission through
Nicotinic Acetylcholine Receptors. Physiological Reviews,
162
Mechawar N, Cozzari C, Descarries L (2000) Cholinergic innervation in adult
rat cerebral cortex: a quantitative immunocytochemical description. The Journal
of comparative neurology, 428:305-318
163
Mechawar N, Watkins KC, Descarries L (2002) Ultrastructural features of the
acetylcholine innervation in the developing parietal cortex of rat. The Journal of
comparative neurology, 443:250-258
164
Weihe E, Tao-Cheng JH, Schafer MK, Erickson JD, Eiden LE (1996)
Visualization of the vesicular acetylcholine transporter in cholinergic nerve
terminals and its targeting to a specific population of small synaptic vesicles.
Proc Natl Acad Sci U S A, 93:3547-3552
165
Krsmanovic LZ, Mores N, Navarro CE, Saeed SA, Arora KK, Catt KJ (1998)
Muscarinic regulation of intracellular signaling and neurosecretion in
gonadotropin-releasing hormone neurons. Endocrinology, 139:4037-4043
166
Morales A, Diaz M, Ropero AB, Nadal A, Alonso R (2003) Estradiol modulates
acetylcholine-induced Ca2+ signals in LHRH-releasing GT1-7 cells through a
membrane binding site. Eur J Neurosci, 18:2505-2514
167
Zaborszky L, Pang K, Somogyi J, Nadasdy Z, Kallo I (1999) The basal forebrain
corticopetal system revisited. Ann N Y Acad Sci, 877:339-367
168
Zaborszky L, Duque A (2003) Sleep-wake mechanisms and basal forebrain
circuitry. Front Biosci, 8:d1146-1169
169
Saper CB, Chou TC, Scammell TE (2001) The sleep switch: hypothalamic
control of sleep and wakefulness. Trends Neurosci, 24:726-731
95
170
Schober JM, Pfaff D (2007) The neurophysiology of sexual arousal. Best
practice & research, 21:445-461
171
Sannella MI, Petersen SL (1997) Dual label in situ hybridization studies provide
evidence that luteinizing hormone-releasing hormone neurons do not synthesize
messenger ribonucleic acid for mu, kappa, or delta opiate receptors.
Endocrinology, 138:1667-1672
172
Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG (1996)
Identification of targets of leptin action in rat hypothalamus. J Clin Invest,
98:1101-1106.
173
Brann DW, Wade MF, Dhandapani KM, Mahesh VB, Buchanan CD (2002)
Leptin and reproduction. Steroids, 67:95-104.
174
Magni P, Motta M, Martini L (2000) Leptin: a possible link between food intake,
energy expenditure, and reproductive function. Regul Pept, 92:51-56.
175
Hakansson ML, Brown H, Ghilardi N, Skoda RC, Meister B (1998) Leptin
receptor immunoreactivity in chemically defined target neurons of the
hypothalamus. J Neurosci, 18:559-572.
176
Baskin DG, Breininger JF, Schwartz MW (1999) Leptin receptor mRNA
identifies a subpopulation of neuropeptide Y neurons activated by fasting in rat
hypothalamus. Diabetes, 48:828-833.
177
Takahashi KA, Cone RD (2005) Fasting induces a large, leptin-dependent
increase in the intrinsic action potential frequency of orexigenic arcuate nucleus
neuropeptide Y/Agouti-related protein neurons. Endocrinology, 146:1043-1047
178
Kalra SP, Dube MG, Sahu A, Phelps CP, Kalra PS (1991) Neuropeptide Y
secretion increases in the paraventricular nucleus in association with increased
appetite for food. Proc Natl Acad Sci U S A, 88:10931-10935.
179
Erickson JC, Hollopeter G, Palmiter RD (1996) Attenuation of the obesity
syndrome of ob/ob mice by the loss of neuropeptide Y. Science, 274:1704-1707.
180
Horvath TL, Bechmann I, Naftolin F, Kalra SP, Leranth C (1997) Heterogeneity
in the neuropeptide Y-containing neurons of the rat arcuate nucleus: GABAergic
and non-GABAergic subpopulations. Brain Res, 756:283-286.
96
181
Wright DE, Jennes L (1993) Origin of noradrenergic projections to GnRH
perikarya-containing areas in the medial septum-diagonal band and preoptic area.
Brain Res, 621:272-278.
182
Aoki C, Venkatesan C, Go CG, Forman R, Kurose H (1998) Cellular and
subcellular sites for noradrenergic action in the monkey dorsolateral prefrontal
cortex as revealed by the immunocytochemical localization of noradrenergic
receptors and axons. Cereb Cortex, 8:269-277
183
Everitt BJ, Hokfelt T, Terenius L, Tatemoto K, Mutt V, Goldstein M (1984)
Differential co-existence of neuropeptide Y (NPY)-like immunoreactivity with
catecholamines in the central nervous system of the rat. Neuroscience, 11:443462
184
Horvath TL, Pu S, Dube MG, Diano S, Kalra SP (2001) A GABA-neuropeptide
Y (NPY) interplay in LH release. Peptides, 22:473-481.
185
Stornetta RL, Sevigny CP, Guyenet PG (2002) Vesicular glutamate transporter
DNPI/VGLUT2 mRNA is present in C1 and several other groups of brainstem
catecholaminergic neurons. The Journal of comparative neurology, 444:191-206.
186
Dudas B, Mihaly A, Merchenthaler I (2000) Topography and associations of
luteinizing hormone-releasing hormone and neuropeptide Y-immunoreactive
neuronal systems in the human diencephalon. The Journal of comparative
neurology, 427:593-603
187
Blomqvist AG, Herzog H (1997) Y-receptor subtypes--how many more? Trends
Neurosci, 20:294-298.
188
Leupen SM, Besecke LM, Levine JE (1997) Neuropeptide Y Y1-receptor
stimulation is required for physiological amplification of preovulatory
luteinizing hormone surges. Endocrinology, 138:2735-2739
189
van den Pol AN, Obrietan K, Chen G, Belousov AB (1996) Neuropeptide Ymediated long-term depression of excitatory activity in suprachiasmatic nucleus
neurons. J Neurosci, 16:5883-5895.
190
Chen G, van den Pol AN (1996) Multiple NPY receptors coexist in pre- and
postsynaptic sites: inhibition of GABA release in isolated self-innervating SCN
neurons. J Neurosci, 16:7711-7724.
97
191
Simonneaux V, Ouichou A, Craft C, Pevet P (1994) Presynaptic and
postsynaptic effects of neuropeptide Y in the rat pineal gland. J Neurochem,
62:2464-2471.
192
Lin S, Lin EJ, Boey D, Lee NJ, Slack K, During MJ, Sainsbury A, Herzog H
(2007) Fasting inhibits the growth and reproductive axes via distinct Y2 and Y4
receptor-mediated pathways. Endocrinology, 148:2056-2065
193
Larsen PJ, Kristensen P (2000) Central Y4 receptor distribution. Radioactive
ribonucleotide probe in situ hybridization with in vitro receptor autoradiography.
Methods in molecular biology (Clifton, NJ, 153:185-198
194
Stanley SA, Small CJ, Kim MS, Heath MM, Seal LJ, Russell SH, Ghatei MA,
Bloom SR (1999) Agouti related peptide (Agrp) stimulates the hypothalamo
pituitary gonadal axis in vivo & in vitro in male rats. Endocrinology, 140:54595462.
195
Urbanski HF, Ojeda SR (1987) Activation of luteinizing hormone-releasing
hormone release advances the onset of female puberty. Neuroendocrinology,
46:273-276
196
Ping L, Mahesh VB, Bhat GK, Brann DW (1997) Regulation of gonadotropinreleasing hormone and luteinizing hormone secretion by AMPA receptors.
Evidence for a physiological role of AMPA receptors in the steroid-induced
luteinizing hormone surge. Neuroendocrinology, 66:246-253
197
Hrabovszky E, Wittmann G, Turi GF, Liposits Z, Fekete C (2005)
Hypophysiotropic thyrotropin-releasing hormone and corticotropin-releasing
hormone neurons of the rat contain vesicular glutamate transporter-2.
Endocrinology, 146:341-347
198
Hrabovszky E, Turi GF, Liposits Z (2005) Presence of vesicular glutamate
transporter-2 in hypophysiotropic somatostatin but not growth hormonereleasing hormone neurons of the male rat. Eur J Neurosci, 21:2120-2126
199
Honma S, Kawano M, Hayashi S, Kawano H, Hisano S (2004) Expression and
immunohistochemical localization of vesicular glutamate transporter 2 in the
migratory pathway from the rat olfactory placode. Eur J Neurosci, 20:923-936
98
200
Schwanzel-Fukuda M, Jorgenson KL, Bergen HT, Weesner GD, Pfaff DW
(1992) Biology of normal luteinizing hormone-releasing hormone neurons
during and after their migration from olfactory placode. Endocr Rev, 13:623-634
201
Ottem EN, Godwin JG, Krishnan S, Petersen SL (2004) Dual-phenotype
GABA/glutamate neurons in adult preoptic area: sexual dimorphism and
function. J Neurosci, 24:8097-8105
202
Yin W, Mendenhall JM, Bratton SB, Oung T, Janssen WG, Morrison JH, Gore
AC (2007) Novel localization of NMDA receptors within neuroendocrine
gonadotropin-releasing hormone terminals. Experimental biology and medicine
(Maywood, NJ, 232:662-673
203
Diano S, Naftolin F, Horvath TL (1998) Kainate glutamate receptors (GluR5-7)
in the rat arcuate nucleus: relationship to tanycytes, astrocytes, neurons and
gonadal steroid receptors. J Neuroendocrinol, 10:239-247
204
Eyigor O, Jennes L (1998) Identification of kainate-preferring glutamate
receptor subunit GluR7 mRNA and protein in the rat median eminence. Brain
Res, 814:231-235
205
Kawakami S (2000) Glial and neuronal localization of ionotropic glutamate
receptor subunit-immunoreactivities in the median eminence of female rats:
GluR2/3 and GluR6/7 colocalize with vimentin, not with glial fibrillary acidic
protein (GFAP). Brain Res, 858:198-204
206
Liposits Z, Paull WK (1985) Ultrastructural alterations of the paraventriculoinfundibular corticotropin releasing factor (CRF)-immunoreactive neuronal
system in long term adrenalectomized rats. Peptides, 6:1021-1036
207
Meeker RB, Swanson DJ, Greenwood RS, Hayward JN (1991) Ultrastructural
distribution of glutamate immunoreactivity within neurosecretory endings and
pituicytes of the rat neurohypophysis. Brain Res, 564:181-193
208
Hrabovszky E, Deli L, Turi GF, Kallo I, Liposits Z (2007) Glutamatergic
innervation of the hypothalamic median eminence and posterior pituitary of the
rat. Neuroscience, 144:1383-1392
209
Buma P, Roubos EW (1986) Ultrastructural demonstration of nonsynaptic
release sites in the central nervous system of the snail Lymnaea stagnalis, the
insect Periplaneta americana, and the rat. Neuroscience, 17:867-879
99
210
Zhu PC, Thureson-Klein A, Klein RL (1986) Exocytosis from large dense cored
vesicles outside the active synaptic zones of terminals within the trigeminal
subnucleus caudalis: a possible mechanism for neuropeptide release.
Neuroscience, 19:43-54
211
Montana V, Ni Y, Sunjara V, Hua X, Parpura V (2004) Vesicular glutamate
transporter-dependent glutamate release from astrocytes. J Neurosci, 24:26332642
212
Fujiyama F, Furuta T, Kaneko T (2001) Immunocytochemical localization of
candidates for vesicular glutamate transporters in the rat cerebral cortex. The
Journal of comparative neurology, 435:379-387
213
Herzog E, Bellenchi GC, Gras C, Bernard V, Ravassard P, Bedet C, Gasnier B,
Giros B, El Mestikawy S (2001) The existence of a second vesicular glutamate
transporter specifies subpopulations of glutamatergic neurons. J Neurosci,
21:RC181
100
11. Bibliography
11.1. List of publications related to the subject of the thesis
1.
Turi GF, Liposits Z, Hrabovszky E.
Cholinergic afferents to gonadotropin-releasing hormone neurons of the rat
Neurochemistry International 2007 Sep 8; [Epub ahead of print]
2.
Hrabovszky E, Deli L, Turi GF, Kallo I, Liposits Z.
Glutamatergic innervation of the hypothalamic median eminence and posterior pituitary
of the rat
Neuroscience 2007 Feb 23; 144(4):1383-92.
3.
Hrabovszky E, Turi GF, Kallo I, Liposits Z.
Expression of vesicular glutamate transporter-2 in gonadotropin-releasing hormone
neurons of the adult male rat
Endocrinology 2004 Sep; 145(9):4018-21.
4.
Turi GF, Liposits Z, Moenter SM, Fekete C, Hrabovszky E.
Origin of neuropeptide Y-containing afferents to gonadotropin-releasing hormone
neurons in male mice
Endocrinology 2003 Nov; 144(11):4967-74.
11.2. List of other publications
5.
Hrabovszky E, Kallo I, Turi GF, May K, Wittmann G, Fekete C, Liposits Z.
Expression of vesicular glutamate transporter-2 in gonadotrope and thyrotrope cells of
the rat pituitary. Regulation by estrogen and thyroid hormone status
Endocrinology 2006 Aug; 147(8):3818-25.
6.
Hrabovszky E, Csapo AK, Kallo I, Wilheim T, Turi GF, Liposits Z.
101
Localization and osmotic regulation of vesicular glutamate transporter-2 in
magnocellular neurons of the rat hypothalamus
Neurochem Int. 2006 Jun; 48(8):753-61.
7.
Hrabovszky E, Turi GF, Liposits Z.
Presence of vesicular glutamate transporter-2 in hypophysiotropic somatostatin but not
growth hormone-releasing hormone neurons of the male rat
Eur J Neurosci. 2005 Apr; 21(8):2120-6.
8.
Hrabovszky E, Wittmann G, Turi GF, Liposits Z, Fekete C.
Hypophysiotropic thyrotropin-releasing hormone and corticotropin-releasing hormone
neurons of the rat contain vesicular glutamate transporter-2
Endocrinology 2005 Jan; 146(1):341-7.
9.
Threlfell S, Cragg SJ, Kallo I, Turi GF, Coen CW, Greenfield SA.
Histamine H3 receptors inhibit serotonin release in substantia nigra pars reticulata
J Neurosci. 2004 Oct 6; 24(40):8704-10.
102
12. Acknowledgements
First, I would like to thank my supervisor Professor Zsolt Liposits for giving me
the opportunity to work in his research group in a very stimulating atmosphere. I also
would like to thank Dr. Erik Hrabovszky who was my tutor during the undergraduate
and Ph.D. years and helped my Ph.D. work with valuable ideas and practical advices.
Further, I am grateful to all of my colleagues in the Laboratory of Endocrine
Neurobiology, Bekó Norbertné Hajni, Dr. Imre Farkas, Dr. Csaba Fekete, Dr. Balázs
Gereben and Dr. Imre Kalló. I could always turn to them with my questions and
problems and they helped my work with invaluable advices.
I would like to express my heartfelt gratitude for my closest colleagues Levente
Deli, Tamás Füzesi, Vivien Hársfalvi, Edit Juhász, Andrea Kádár, Judit Menyhért,
Ágnes Simon, Dr. Patrícia Varjú, Barbara Vida, Gábor Wittmann and Dr. Anikó Zeöld
for the joyful atmosphere in the laboratory and the great hours that we spent together.
Finally, I will be grateful forever to my beloved parents and my sister for their
encouragement and support during my studies and my life.
103