Chem. Senses 26: 1193-1202,
2001
© Oxford University Press 2001
The Prairie Vole Vomeronasal Organ is a Target for Gonadotropin-releasing Hormone
1 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA 2 Department of Zoology, University of Oklahoma, Norman, OK 73019, USA 3 Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
Correspondence to be sent to: C.R. Wirsig-Wiechmann, Department of Cell Biology, University of Oklahoma Health Sciences Center, PO Box 26901, Oklahoma City, OK 79190, USA. e-mail: celeste-wirsig{at}ouhsc.edu
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Abstract |
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Gonadotropin-releasing hormone (GnRH) is present in nervus terminalis neurons in chemosensory nerve fascicles in vertebrates. In rodents, the majority of GnRH fibers are located within vomeronasal nerves. We have shown that GnRH can alter vomeronasal receptor neuron responses to odors. In this study, using prairie voles, we tested the hypotheses that (i) GnRH-immunoreactive (-ir) neurons project to the vomeronasal organ and accessory olfactory bulb; (ii) a radioactive-labeled GnRH agonist, buserelin, binds to vomeronasal sensory neurons; and (iii) vomeronasal receptor cells express GnRH receptor mRNA as evidenced by reverse transcription—polymerase chain reaction (RT-PCR) combined with Southern blotting. In neonatal voles, GnRH-ir cell bodies and fibers were observed within the vomeronasal epithelium, vomeronasal nerves and accessory olfactory bulbs. In adult voles, GnRH-ir fibers were observed not only in the lamina propria of the vomeronasal mucosa, but also along vomeronasal nerves and in the accessory olfactory bulb. Binding of [125I]buserelin was observed specifically over the vomeronasal sensory epithelium, and RT-PCR/Southern blotting demonstrated GnRH receptor expression in the vomeronasal mucosa, as well as in olfactory epithelium and pterygopalatine ganglion, two additional structures containing GnRH-ir neurons of the nervus terminalis. This study supports the hypothesis that GnRH is released from nervus terminalis fibers to modulate chemosensory processes, especially those involving chemoreception in the vomeronasal organ.
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Introduction |
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TopAbstractIntroductionMaterials and methodsRT-PCRResultsDiscussionReferences |
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Gonadotropin-releasing hormone (GnRH) is a neurohormone involved in a number of reproductive processes, including release of gonadotropin hormones from the pituitary gland and stimulation of brain centers that initiate or facilitate sexual behavior. The neurons that synthesize and release GnRH are present within various regions of the forebrain, midbrain and several peripheral neural structures related to olfaction, such as nasal mucosa (Schwanzel-Fukuda and Silverman, 1980
) and cranial autonomic ganglia
(Wirsig-Wiechmann and Lepri,
1991; Wirsig-Wiechmann,
1993). Within the rostral forebrain and the peripheral olfactory
structures, the GnRH neurons are considered to be part of the nervus
terminalis system (Figure 1).
This neural system is present in all vertebrates and is thought to act as a
neuromodulatory system for both peripheral
(Eisthen et al., 2000;
Wirsig-Wiechmann et al.,
2000) and central brain regions
(Oka, 1992).
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GnRH receptors have been visualized by in vitro autoradiography
(Badr and Pelletier, 1987;
Jennes et al., 1988;
Jennes and Conn, 1994) and
in situ hybridization (Jennes
et al., 1997) in rodent brain, demonstrating the sites of
central GnRH action. The presence of GnRH in peripheral chemosensory-related
structures suggests that GnRH modulates chemoreception at the level of
transduction of chemosensory signals or modulates autonomic inputs to
chemosensory structures. We have previously shown that a GnRH agonist,
buserelin, binds to tiger salamander olfactory neurons
(Wirsig-Wiechmann and Jennes,
1993) and that GnRH modulates olfactory neuron voltage-gated
responses in mudpuppies (Eisthen et
al., 2000). Our recent findings indicate that GnRH can
specifically modulate olfactory and vomeronasal neuron responses to odors in
rodents (Wirsig-Wiechmann et al.,
2000). In rodents, the majority of peripheral nervus terminalis
fibers projects to the vomeronasal organ, suggesting that the main focus of
GnRH action in the periphery is to control vomeronasal detection of
chemosensory stimuli.
In the present study, we used prairie voles to visualize the projections of
the peripheral nervus terminalis and determine whether a GnRH agonist binds to
vomeronasal neurons and whether vomeronasal mucosa expresses the GnRH
receptor. We used prairie voles because their nervus terminalis system
demonstrates the most robust labeling in immunocytochemical studies designed
to study the projections of the GnRH-immunoreactive (-ir) neurons in the nasal
cavity. We have confirmed that GnRH-ir neurons of the nervus terminalis
project directly to the vomeronasal organ, that binding of the GnRH agonist,
buserelin, occurs specifically to vomeronasal chemosensory neurons and that
the vomeronasal cells express the GnRH receptor. These findings support our
previous data that GnRH influences the processing of chemical stimuli at the
sensory receptor level (Eisthen et
al., 2000;
Wirsig-Wiechmann et al.,
2000).
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsRT-PCRResultsDiscussionReferences |
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Animals
Ten neonatal and six adult prairie voles were used in this study for
immunocytochemical analysis of GnRH projections in the nasal cavity. Two adult
voles were used for in vitro GnRH agonist binding
(Wirsig-Wiechmann and Jennes,
1993) and two adult male voles were used for determination of GnRH
receptor expression by reverse transcription—polymerase chain reaction
(RT-PCR) and Southern blotting. Voles were obtained from breeding colonies in
the laboratories of Dr John Lepri (University of North Carolina, Greensboro,
NC) and Dr Thomas Insel (Emory University, Atlanta, GA). Animals were
maintained on a diet of rabbit chow and sunflower seeds and were housed in
plastic cages with wood chips. Animals were kept on a 14 h light/10 h dark
illumination schedule. The animals were cared for in accordance with the NIH
Guide for the Care and Use of Laboratory Animals.
Immunocytochemistry
Voles were anesthetized by halothane inhalation and perfused transcardially with saline followed by Zamboni's fixative (2% paraformaldehyde-15% picric acid in 0.1 M sodium phosphate buffer, pH 7.4). Adult heads were decalcified in DeCal (Omega Chemical Corp., Cold Spring, NY) and all tissues were cryoprotected in 30% sucrose-phosphate buffer. Tissue was sectioned (20 µm) in the sagittal plane on a cryostat microtome. Tissue was placed directly onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), air-dried and stored at -80°C until immunocytochemical processing.
For immunocytochemistry, tissue was rinsed in six 5 min rinses of phosphate buffered saline (PBS), pH 7.4, between incubations. Tissue was incubated in rabbit anti-GnRH antisera (1:4000; Lot No. 635479; DiaSorin, Stillwater, MN) or guinea pig anti-GnRH antisera 6-4 (1:4000; kindly supplied by Dr Lothar Jennes) for 3 days at 4°C, followed by a 30 min incubation in each of the following: biotinylated goat anti-rabbit IgG (or biotinylated goat anti-guinea pig IgG) and avidin-conjugated horseradish peroxidase (Vector Laboratories, Burlingame, CA). Labeling was conducted with either diaminobenzidine (DAB) or Chromo-red (3-amino-9-ethylcarbazole; Sigma, St Louis, MO). For DAB processing, tissue was rinsed in PBS and then immersed in 0.05 M Tris, pH 7.4, for 5 min, then in 0.05% DAB, 0.001% hydrogen peroxide in 0.05 M Tris buffer, pH 7.4, for 10 min and finally rinsed in PBS. Tissue labeled with DAB was counterstained with methyl green, dehydrated and cover-slipped with Permount. For Chromo-red labeling, tissue was rinsed in PBS, followed by a 5 min incubation in 0.01 M acetate buffer, pH 5.2, and a 20 min incubation in 0.02% 3-amino-9-ethylcarbazole, 5.55% N,N-dimethylformamide, 0.33% hydrogen peroxide in 0.01 M acetate buffer, pH 5.2. Following a rinse in PBS, tissue labeled with Chromo-red was coverslipped with glycerol gelatin (Sigma). For immunocytochemical control conditions, tissue was incubated in GnRH antisera preabsorbed overnight with 20 µM synthetic mammalian GnRH, or in preincubation solution containing no primary antisera. These incubations were followed by normal labeling procedures. Tissue was observed with an Olympus microscope (Melville, NY) and digital images were acquired with a SPOT camera (Diagnostic Instruments, Sterling Heights, MI).
Receptor binding autoradiography
Voles were anesthetized by halothane inhalation and killed by decapitation. Vomeronasal organs were quickly removed and the mucosa was dissected from the bone, frozen in liquid nitrogen and fresh-frozen sectioned (30 µm) coronally with a cryostat microtome. Tissue sections were placed directly onto three sets of Superfrost Plus slides (Fisher) and two sets were preincubated in 5 mM Tris-HCI, pH 7.4, containing 0.2% bovine serum albumin at 4°C for 30 min. Tissue was then incubated in the same buffer containing [125I]buserelin (200 000 c.p.m./ml; 1 mCi/g) with or without unlabeled GnRH (25 µM) for 2 h at 4°C. Following four 1 min buffer rinses and two 30 s water rinses, tissue was dried under a cold stream of air and exposed to P-Max Hyperfilm (Amersham Pharmacia, Piscataway, NJ). Experimental and control tissues were processed together and exposed to the same Hyperfilm to allow direct comparisons. Following a 3 day exposure, autoradiographs were developed and slides for which binding was apparent were vapor-fixed with paraformaldehyde and dipped in autoradiographic emulsion (Ilford NTB-2). Slides were developed after a 1 month exposure. The third set of tissue was fixed with paraformaldehyde and stained immediately with cresyl violet. Because buserelin binding requires the use of unfixed tissue, the morphology of tissue is not optimal for visualization of specific cells. However, since the sensory epithelium of the vomeronasal organ is very well localized to the medial wall of the organ and since the sensory cells form a thick epithelium, a regional analysis of the binding of buserelin can be readily accomplished.
For visualization of relative silver grain densities generated from buserelin binding, computer-generated color images were obtained from film autoradiographs. Autoradiographic film images were digitized with 241 density levels. Six colors were chosen for ranges of silver grain densities. For numerical silver grain density measurements, 10 selected areas from all experimental and adjacent competition control vomeronasal tissues sections were analysed using NIH image software. Density values were taken from randomly chosen vomeronasal sensory cell areas of varying size and location. The possible numerical value range extended from 1 (no silver grains, i.e. white) to 241 (completely filled with silver grains, i.e. black). Student's t-test (two-tail) was used to compare the densities between the various regions (unpaired values).
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RT-PCR |
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Two male voles were killed by carbon dioxide inhalation and decapitation. Vomeronasal mucosa, olfactory epithelium and pterygopalatine ganglia were removed bilaterally and each tissue type was pooled and frozen separately in liquid nitrogen. Tissue was stored at -80°C until use. For RNA isolation, tissue was homogenized and RNA extracted using an RNeasy Total RNA Isolation Kit (Qiagen, Santa Clarita, CA), according to the manufacturer's instructions. Extracted RNA was further treated with DNase to remove all genomic DNA, using a Message Clean Kit (GenHunter Corp., Nashville, TN). Total RNA (1 µg/sample) was reverse transcribed (RT) to cDNA with the use of a RNA PCR kit (Perkin-Elmer, Norwalk, CT) in a total volume of 10 µl. The cDNA was then amplified by PCR using primers complementary to the cloned rat GnRH receptor sequence to yield a 257 bp fragment. The 5' primer sequence was [5'-TGA TTA GCC TGG ATC GCT CC-3'] and the 3' primer sequence was [5'-GAA CAG GCA GCT GAA GGT-3'], which correspond to positions 401-420 and 657-638, respectively, and were synthesized by Integrated DNA Technologies Inc. (Coralville, IA). These primers were complementary to sequences on exons 1 and 2 of the rat GnRH receptor gene. In addition, both primers encoded portions of the third and fifth transmembrane regions of the receptor and therefore are highly conserved across species. The 5' primer sequence (positions 401-420) and the 3' primer sequence (positions 657-638) are both 90% homologous to the mouse sequence and the amplified region (positions 401-657) is 89% homologous to the mouse sequence (GenBank Accession No. L001119). In addition, since one intron is located between the two exons from which primers were designed, amplification of any possible contaminating genomic DNA would not result in a PCR product of the predicted size of 257 bp. PCR amplification was conducted with one cycle of 94°C for 4 min and then 35 cycles of 94°C for 30 s, 50°C for 1 min and 72°C for 1 min, followed by one cycle of 72°C for 7 min. The amplified cDNA (15 µl) was then electrophoresed on an agarose gel and stained with ethidium bromide.
Southern blot analysis
Full-length 32P-labeled GnRH receptor cDNA was synthesized from
a Bluescript II plasmid containing the rat GnRH receptor insert, obtained
courtesy of Dr William Chin (Kaiser et
al., 1992). The plasmid was excised with BamHI (Promega,
Madison, WI) and isolated by agarose gel electrophoresis and extracted using a
Qiaex II Gel Extraction Kit (Qiagen, Santa Clarita, CA). The cDNA was
end-labeled with [g-32P]dCTP (3000 Ci/mmol; ICN Pharmaceuticals,
Inc., Costa Mesa, CA), using a Multiprime DNA Labeling Kit (Amersham
Pharmacia, Piscataway, NJ) according to the manufacturer's instructions. The
labeled probe was purified on a Sephadex G-50 Quick Spin column (Boehringer
Mannheim, Indianapolis, IN).
PCR products electrophoresed on an agarose gel were transferred to a Zetabind nylon membrane (Cuno Laboratory Products, Meriden, CT). The cDNA immobilized on the membrane was denatured and the membrane was incubated overnight with the 32P-labeled GnRH receptor cDNA probe at 50°C. The membranes were washed under high-stringency conditions and the hybridized probe was visualized by film autoradiography.
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Results |
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Immunocytochemistry
Immunohistochemical control conditions did not produce labeling of any neurons. Following incubations in GnRH antisera, neural cell bodies and fibers containing GnRH-ir were observed along the entire vomeronasal system projection to the accessory olfactory bulb in both neonatal and adult voles (Figure 1). The GnRH-ir was confined to bipolar and unipolar neurons of the nervus terminalis system.
In neonatal vole tissue, GnRH-ir cell bodies and fibers were observed within: (1) vomeronasal organ sensory epithelium (Figure 2A,B,D); (2) vomeronasal nerve fascicles within the vomeronasal organ (Figure 2C); and (3) vomeronasal nerve fascicles projecting through the lamina propria of the nasal septum (Figure 2E) and vomeronasal nerve fascicles traveling along the medial surface of the olfactory bulb (Figure 4A) and projecting into the accessory olfactory bulb. A plexus of GnRH-ir cell bodies and fibers also existed along the medial surface of the olfactory bulb ventral to the vomeronasal nerve projection to the accessory olfactory bulb (Figure 4A). Fibers from this plexus wrapped around the olfactory peduncle. The accessory olfactory bulb received a dense innervation of GnRH-ir fibers (Figure 2F).
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In adult vole tissue, GnRH-ir cell bodies and fibers were observed within vomeronasal nerve fascicles projecting along the nasal septum (Figure 3A) and along the medial surface of the olfactory bulb (Figure 4B). As in the neonatal vole, a plexus of cell bodies and fibers also existed along the medial surface of the olfactory bulb ventral to the vomeronasal nerve projection to the accessory olfactory bulb (Figure 4B). Some GnRH-ir fibers followed the vomeronasal nerve into the accessory olfactory bulb to provide a dense innervation of mainly the superficial layers of the accessory olfactory bulb (Figure 3B).
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In both neonatal animals and adults, the GnRH-ir nervus terminalis courses over the medial surface of the olfactory bulbs (Figure 4), before entering the ventral forebrain on the medial surface of the anterior olfactory nucleus, just rostral to the anterior cerebral artery. Upon entering the forebrain, the peripheral fibers of the nervus terminalis project into a cluster of GnRH-ir neurons, the terminal ganglion (Figure 1). Caudal to the terminal ganglion, isolated GnRH-ir cells and their fibers project toward the septum and hypothalamus. On the medial surface of the olfactory bulbs, some fibers diverge from the main branch of the nervus terminalis and project to the accessory olfactory bulb within the vomeronasal nerve. In addition, other fibers project to the neck of the olfactory bulbs and completely encircle the neck of the olfactory bulb on its medial and lateral surfaces (Figure 1).
Receptor binding autoradiography
Visual examination of film autoradiographs, computer-generated color images and tissue autoradiographs demonstrated robust binding of [125I]buserelin greater than that of control levels—i.e. incubation in [125I]buserelin with excess non-radioactive GnRH—in the vomeronasal sensory epithelium (Figure 5). Microscopic examination of tissue autoradiographs showed specific binding in sensory cell bodies of the vomeronasal epithelium (Figure 5D).
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Densitometric analysis of tissue autoradiographs revealed a higher density of silver grains (i.e. 2.5 times higher than non-specific density) in the vomeronasal sensory epithelium following incubation in [125I]buserelin than in control incubations. The average density of silver grains in tissue incubated with [125I]buserelin (75.7 densitometry units; 31.4% of maximum density) was significantly greater than for control incubations (32.6 densitometry units; 13.5% of maximum density). Statistical analysis showed that this difference was significant (t = 13.43, d.f. = 18, P < 0.0000000001).
RT-PCR and Southern blotting
All PCR bands demonstrated positive labeling by Southern blot analysis (Figure 6). As expected, the vomeronasal mucosa revealed the highest degree of labeling by Southern blotting. The pterygopalatine ganglion demonstrated the least PCR product as indicated by Southern blotting. This may have been due to the small amount of GnRH-receptor-expressing cells in the tissue sample.
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We have previously PCR-amplified GnRH receptor mRNA from rat vomeronasal
mucosa, olfactory epithelium and several other tissues with these primers and
have found positive labeling by Southern blot analysis using the same
full-length 32P-labeled GnRH receptor cDNA
(Wirsig-Wiechmann et al.,
2000). In addition, we cloned PCR bands from rat vomeronasal
mucosa and olfactory epithelium into pCRII vectors and sequenced these to
confirm the identity of the amplified cDNAs. The nucleotide sequences from the
cloned cDNAs demonstrated 100% bp identity with the published sequence for the
mammalian GnRH receptor (GenBank Accession No. L07646).
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Discussion |
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TopAbstractIntroductionMaterials and methodsRT-PCRResultsDiscussionReferences |
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This study has demonstrated that GnRH-ir fibers project directly to the vomeronasal organ in prairie voles. It also supports our hypothesis that GnRH binds specifically to vomeronasal sensory epithelium and that vomeronasal mucosa expresses the GnRH receptor. GnRH-ir neural cell bodies and fibers were mainly associated with the vomeronasal systems in both neonatal and adult voles. Very little GnRH-ir was observed in association with olfactory structures at both peripheral and central levels. These anatomical observations suggest that GnRH acts mainly on the vomeronasal system in this species. A similar tight anatomical association of GnRH-ir neurons with the vomeronasal system has been reported in other rodents such as hamster (Wirsig and Leonard, 1986
) as
well as in developing rat vomeronasal nerves (Yoshida et al., 1995,
1999). However, it should be noted that GnRH could be secreted locally into
the blood stream to travel to other chemosensory regions in the nasal cavity
(Jennes, 1986). This is
supported by the observation that GnRH modulates olfactory neuron responses to
odors (Wirsig-Wiechmann et al.,
2000).
Binding of the GnRH agonist, buserelin, was the highest over the
chemosensory vomeronasal neurons. This finding represents the first
description of GnRH binding in the chemosensory epithelium of mammals and the
second report of GnRH binding in the chemosensory epithelium of vertebrates.
Our previous work demonstrated GnRH agonist binding in the olfactory mucosa of
tiger salamanders (Wirsig-Wiechmann and
Jennes, 1993). Together, these studies suggest that GnRH can
influence the processing of chemosensory information, first at the receptor
cell level, and that this modulatory role of GnRH in the nasal cavity is
common among vertebrates. This hypothesis has been supported by our
patch-clamp studies of mudpuppy olfactory mucosa, in which we showed that GnRH
modulated voltage-dependent currents
(Eisthen et al.,
2000), and isolated rodent olfactory and vomeronasal receptor
cells, in which we showed that GnRH modulated odor-induced currents
(Wirsig-Wiechmann et al.,
2000). In fact, in this latter study in rats, the odor response of
one vomeronasal sensory neuron was completely eliminated by GnRH.
A likely natural source of GnRH in the chemosensory mucosa is the nervus terminalis, a GnRH-ir neural plexus in the nasal cavity and rostral forebrain. Our observation of GnRH neural cell bodies and fibers within the vomeronasal epithelium of the neonatal animal may represent a late developmental stage of this system, when neurons are still migrating centrally from the vomeronasal organ. Cell bodies and fibers were not observed in the adult sensory vomeronasal epithelium. The most peripheral immunoreactive fibers in the adult were found within vomeronasal nerves on the nasal septum, just dorsal to the vomeronasal organ. Therefore, in the adult, GnRH would have to diffuse along vomeronasal nerves and through the lamina propria of the vomeronasal organ to reach the sensory epithelium. Another route to the vicinity of the sensory epithelium could be through the microvasculature supplying the lamina propria of the vomeronasal organ. There is an extensive vascular network surrounding the vomeronasal mucosa. We have observed GnRH-ir tangles of fibers surrounding blood vessels just dorsal to the vomeronasal organ, especially in neonatal animals.
Receptors for GnRH are found in many tissues, including the central and
peripheral nervous systems. In the brain, GnRH binding sites are abundant in
olfactory and limbic areas (Jennes et
al., 1988), suggesting that there may be intracerebral
modulation of olfactory function by GnRH. In support of this, we have observed
a dense innervation of the accessory olfactory bulb and entire posterior bulb
neck region by GnRH-ir fibers in voles as well as in hamster
(Wirsig and Leonard, 1986).
This latter region contains `necklace glomeruli' (Shinoda et al.,
1989,
1993;
Zufall and Munger, 2001) that
receive projections from several specialized groups of olfactory receptors
(Juilfs et al., 1997;
Ring et al., 1997).
The function of the necklace glomeruli throughout an animal's life is not yet
known, but there is evidence that it may play a role in pheromone detection
during suckling in neonatal rats (Greer
et al., 1982). The density of GnRH fibers in proximity to
these glomeruli suggests to us that they may have a general function related
to different aspects of reproduction, perhaps the initial detection of
pheromones by the olfactory system and the setting up of odor attractions.
In contrast, in the tiger salamander, GnRH-ir projections are concentrated
in medial olfactory nerve projections to rostral regions of the olfactory
mucosa and within the olfactory bulb
(Wirsig and Getchell, 1986),
but are less prevalent in vomeronasal nerves and are rarely seen within the
accessory bulb. This species difference in the location of GnRH-ir neurons and
fibers may not have significant functional relevance if GnRH accesses the
various chemosensory regions via the vasculature. In mammals and most other
vertebrates, GnRH accesses the pituitary via the vascular portal system.
However, in fish the GnRH fibers project directly into the anterior pituitary
(Yamamoto et al.,
1998). In both cases, GnRH acts to release hormones from the
pituitary. Perhaps, the proximity of GnRH-ir fibers to specific areas
indicates the regions of acute effects of GnRH, while those areas at greater
distance experience the longer-term chronic effects of GnRH. In support of
this, the hippocampus has the highest number of GnRH receptors in the brain
(Ruebi and Maurer, 1985;
Jennes et al., 1988,
1990,
1997) and yet contains very
few, if any, GnRH fibers (Barry et
al., 1985).
GnRH is continually released from GnRH neurons along the nervus terminalis
by their autonomous activity (Oka,
1992; Abe and Oka,
2000), but this release may increase or decrease depending on
hormonal—for example melatonin
(Wirsig-Wiechmann,
1993)—and neural inputs
(Yamamoto and Ito, 2000). In
fact, GnRH itself can be self-regulating; GnRH released from one nervus
terminalis neuron can increase the activity and therefore secretion of other
GnRH neurons (Abe and Oka,
2000).
The effect of GnRH on peripheral chemosensory cells may involve changes in
the concentration of GnRH rather than its presence or absence, as well as
changes in the number of GnRH receptors in the target tissue.
Electro-physiological recording of mudpuppy olfactory neurons has demonstrated
that twice as many olfactory receptor neurons respond to GnRH during the
breeding season as at other times. The amount of GnRH released may depend on
factors controlling its synthesis. Estrogen has been shown to increase the
synthesis of GnRH in brain neurons (Rosie
et al., 1990) and also appears to increase GnRH synthesis
in the nervus terminalis in Xenopus laevis as demonstrated by
increased immunoreactivity
(Wirsig-Wiechmann and Lee,
1999). Conversely, removal of estrogen via ovariectomy leads to a
depletion of GnRH in nervus terminalis neurons
(Wirsig-Wiechmann and Lee,
1999) due to increases in release
(Jennes and Stumpf, 1986).
The GnRH-containing component of the nervus terminalis thus has the potential to modulate chemosensory information, particularly in the vomeronasal system of rodents at multiple peripheral and central sites. This modulation likely occurs in a controlled fashion based on hormonal and neural inputs to the nervus terminalis GnRH system. In future studies we will attempt to determine the molecular mechanisms of GnRH action on chemosensory neurons and the mechanisms that ultimately control the frequency of GnRH release from the nervus terminalis.
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Acknowledgments |
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This work was supported by OCAST award HR00-078 from the Oklahoma Center for Science and Technology (C.R.W.), a Presbyterian Health Foundation Grant (C.R.W.) and National Science Foundation grant No. IBN 9896098 (C.R.W.). The authors thank Ms Angela Smith for excellent technical assistance, Dr Lothar Jennes for his kind contribution of [125I]buserelin as well as guinea pig anti-GnRH antisera, Dr William Chin for his kind contribution of plasmid containing the GnRH receptor cDNA and Drs John Lepri and Thomas Insel for their kind donation of voles.
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Accepted August 14, 2001
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