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Endocrinology Vol. 144, No. 11 4967-4974
Copyright © 2003 by The Endocrine Society

Origin of Neuropeptide Y-Containing Afferents to Gonadotropin-Releasing Hormone Neurons in Male Mice

Gergely F. Turi, Zsolt Liposits, Suzanne M. Moenter, Csaba Fekete and Erik Hrabovszky

Department of Endocrine and Behavioral Neurobiology (G.F.T., Z.L., C.F., E.H.), Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083 Budapest, Hungary; and Departments of Medicine and Cell Biology (S.M.M.), University of Virginia, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Erik Hrabovszky, Department of Endocrine- and Behavioral Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony u. 43., 1083 Budapest, Hungary. E-mail: hrabovszky{at}koki.hu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The origin of neuropeptide Y (NPY) afferents to GnRH neurons was investigated in male mice. Neonatal lesioning of the hypothalamic arcuate nuclei (ARC) with monosodium glutamate markedly reduced the number of NPY fibers in the preoptic area as well as the frequency of their contacts with perikarya and proximal dendrites of GnRH neurons. Dual-label immunofluorescence studies to determine the precise contribution of the ARC to the innervation of GnRH neurons by NPY axons were carried out on transgenic mice in which enhanced green fluorescent protein was expressed under the control of the GnRH promoter (GnRH-enhanced green fluorescent protein mice). The combined application of red Cy3 and blue AMCA fluorochromogenes established that 49.1 ± 7.3% of NPY axons apposed to green GnRH neurons also contained agouti-related protein (AGRP), a selective marker for NPY axons arising from the ARC. Immunoelectronmicroscopic analysis detected symmetric synapses between AGRP fibers and GnRH-immunoreactive perikarya. Additional triple-fluorescence experiments revealed the presence of dopamine-ß-hydroxylase immunoreactivity within 25.4 ± 3.3% of NPY afferents to GnRH neurons. This enzyme marker enabled the selective labeling of NPY pathways ascending from noradrenergic/adrenergic cell populations of the brain stem, thus defining a second important source for NPY-containing fibers regulating GnRH cells. The absence of both topographic markers (AGRP and dopamine-ß-hydroxylase) within 26% of NPY contacts suggests that additional sources of NPY fibers to GnRH neurons exist. Future studies will address distinct functions of the two identified NPY systems in the afferent neuronal regulation of the GnRH system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROPEPTIDE Y (NPY), A 36-AMINO-ACID peptide of the pancreatic polypeptide family plays important roles in the regulation of reproduction via acting at both the hypothalamic and pituitary levels (1). Centrally, a chronic increase in NPY tone has been implicated in hypogonadism that accompanies malnutrition (2), obesity (3), or diabetes (4). Inhibition of the gonadotropic axis can also be induced experimentally in male rats and mice by the central infusion of either NPY or Y5 receptor agonist (5). Furthermore, high central NPY levels coincide with the lack of fertility in the leptin-deficient ob/ob mice (6). The inhibition of reproduction is likely caused by the hyperfunction of leptin receptor-expressing NPY neurons in the hypothalamic arcuate nucleus (ARC) (7) because leptin replacement to these animals suppresses hypothalamic NPY gene expression with a concomitant occurrence of normal LH secretion and fertility (6). Moreover, cross-breeding of ob/ob mice with NPY-knockout animals also results in significant improvement of the reproductive phenotype (8).

Although chronic increase in NPY tone inhibits gonadotropin release (5), delays sexual maturation (9), and suppresses estrous cyclicity (10), 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 (11, 12, 13, 14, 15, 16), whereas, in intact or gonadectomized and steroid-primed rodents, NPY increases serum gonadotropin levels (5, 12, 15, 16, 17, 18).

NPY influences gonadotropin secretion via acting at multiple levels of the reproductive axis. It potentiates gonadotropin release in response to a GnRH challenge (19) by increasing the number of GnRH binding sites on pituitary gonadotrophs (20). In addition, it also stimulates GnRH secretion via acting centrally on GnRH axon terminals in the mediobasal hypothalamus (21, 22, 23). The major site of central NPY actions appears to be the preoptic area (POA). The majority of GnRH neurons are located in this region, gain abundant innervation from NPY-containing axon terminals (24, 25, 26), and express the Y5 receptor isoform (27).

One putative source of NPY-containing afferents to GnRH neurons is the ARC because neonatal lesioning of this region by monosodium glutamate (MSG) treatment markedly reduced the density of NPY fibers in the POA (28). Indeed, injection of retrograde tracer around GnRH neurons labeled a population of NPY cells in the ARC (26), and anterograde tracing of NPY axons from the ARC revealed their direct appositions to GnRH neurons of the POA (25). Nearly all NPY neurons in the ARC express another orexigenic peptide, agouti-related protein (AGRP). Given that these cells represent a unique source for AGRP in the central nervous system (28), the presence of this neuropeptide can serve as a distinctive marker for NPY axons arising from the ARC. An additional likely source for the origin of NPY afferents to GnRH neurons is the brain stem in which NPY colocalizes with epinephrine and norepinephrine (26, 29, 30). Retrograde tracing studies found that norepinephrine neurons projecting to the vicinity of GnRH cells coexpress NPY in the ventrolateral medulla (A1 catecholamine cell group) but not in the nucleus tractus solitarii (A2 cells) (26). The putative functional heterogeneity of central NPY pathways motivated the present studies to determine the relative contribution of neurochemically distinct NPY systems to the innervation of GnRH neurons.

We performed a series of immunocytochemical experiments to quantitatively address the involvement of the ARC as well as noradrenergic/adrenergic cell groups of the brain stem in the afferent regulation of GnRH neurons by NPY. To estimate the contribution of NPY cells in the ARC to this innervation, first we compared the frequency of AGRP-containing vs. NPY-containing axonal contacts on GnRH neurons, using dual-label immunocytochemistry for bright-field light microscopy. As a second approach, we determined the lost fraction of NPY-immunoreactive juxtapositions to GnRH neurons in neonatally MSG-treated mice with lesioned ARC. The ultrastructural relationship between AGRP-immunoreactive NPY afferents form the ARC and GnRH neurons was also addressed by immunoelectronmicroscopy. Finally, studies using three-color fluorescent microscopy were conducted in preoptic sections of GnRH-enhanced green fluorescent protein (GFP) transgenic mice (31). The immunofluorescent identification of AGRP in NPY afferents to GnRH-GFP neurons was used to label innervation from the ARC, whereas a second set of experiments used the detection of dopamine-ß-hydroxylase (DBH) in NPY-containing contacts to identify afferents ascending from noradrenergic/adrenergic cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Adult (8 wk old) male GnRH-GFP transgenic mice (n = 16) in which the GnRH promoter drives selective GFP expression in the majority of GnRH neurons (31) were bred and housed at the Institute of Experimental Medicine under conditions of 12 h of light (lights on at 0700 h) with constant access to food and water. The animals were treated in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. All experimental protocols were reviewed and approved by the Animal Welfare Committee at the Institute of Experimental Medicine.

Neonatal MSG treatment
To eliminate NPY fibers arising from the ARC, the chemical lesion of this region was performed by MSG treatment of four neonatal mice. Briefly, the neonatal animals were injected sc with increasing volumes of an 8% MSG solution (dissolved in water), using a treatment paradigm adapted from Légrádi and Lechan (32): postnatal d 1 and 3, 4 mg/g body weight (bw); postnatal d 5, 7, and 9, 8 mg/g bw. 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.

Transcardiac perfusion
All animals used in these studies were deeply anesthetized with sodium pentobarbital (45 mg/kg bw, ip) and perfused via the ascending aorta first, with 20 ml of 0.01 M PBS (pH 7.4) and then with 100 ml fixative solution. Tissues were fixed with 4% paraformaldehyde (Sigma Chemical Co., St. Louis, MO) in 0.01 M phosphate buffer (PB) for light and fluorescent microscopy and with a mixture of 2% paraformaldehyde and 4% acrolein (Aldrich Chemical Co., Milwaukee, WI) in PB for electron microscopy.

Section preparation
Brains used for light and fluorescent microscopy were immersed into 30% sucrose in PBS overnight, snap frozen on powdered dry ice, and sectioned at 25 µm with a Leica CM 3050 S cryostat (Meyer Instruments, Houston, TX). For preembedding immunoelectronmicroscopy, preoptic sections (35 µm) were prepared on a Vibratome (Technical Products International, St. Louis, MO).

Immunocytochemical studies
A series of immunocytochemical studies were performed using different chromogen combinations for bright-field light microscopy, electron microscopy, and fluorescent microscopy. Specificity of labeling was tested by the omission of primary or secondary antibodies, which resulted in the absence of any staining with the AGRP-, DBH-, NPY-, and GnRH antisera. The distribution of immunoreactive neuronal structures was in agreement with data of previous mapping studies for these antigens.

Experiment 1: light microscopic analysis of NPY- and AGRP-containing fibers in contact with GnRH neurons of neonatally MSG-treated mice vs. untreated controls
Single-label immunocytochemistry.
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 argyrophilia (33) and with 0.5% H2O2 in Tris-buffered saline (TBS) (0.1 M Tris-HCl with 0.9% NaCl; pH 7.8) for 15 min to eliminate endogenous peroxidase activity. Nonspecific antibody binding sites were blocked and tissues permeabilized with 2% normal horse serum in TBS/0.4% Triton X-100 (Sigma) 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) (34), which was generously provided by Dr. István Merchenthaler (Wyeth Research, Collegeville, PA), and applied to the sections for 48 h at 4 C. 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) (35). 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 diaminobenzidine (DAB), 30 mg nickel-ammonium-sulfate, and 0.002% H2O2 in 24 ml TBS. Then silver intensification of the peroxidase reaction product was carried out as described by Liposits et al. (33).

Dual-label immunocytochemistry.
The majority of sections immunostained for AGRP and 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, Montréal, 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 single- and dual-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 analysis of dual-labeled sections
Sections dual immunostained 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 according to a pattern that allowed later identification of section sources. The slides were coverslipped, sections individually labeled with a code, and in each the number of axonal contacts along the outlines of GnRH neurons analyzed using a x100 objective lens with immersion oil by an investigator who was blind to the experimental procedures and the pattern of section mounting. Studies of sections in random sequence ensured the homogenous analysis of groups to compare. A case was considered contact based only on highly stringent criteria that were applied consistently. The axon and GnRH neuron had to occur in the same focus 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 AGRP immunoreactive 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 final results of the analysis were expressed as mean ± SEM.

Experiment 2: electron microscopic analysis of AGRP afferents to GnRH neurons
Preembedding dual-label immunoelectronmicroscopy was used for the ultrastructural analysis of neuronal contacts between AGRP-immunoreactive axons and GnRH cells of the POA. The methodology for dual labeling was adapted from a procedure published recently (36). Briefly, 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 d, biotinylated antirabbit antibodies (Jackson ImmunoResearch Laboratories; 1: 1000) for 2 h, and ABC working solution (Vector Laboratories; 1: 1000) 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-d 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 antirabbit IgG conjugated with 0.8 nm colloidal gold (Electron Microscopy Sciences; diluted at 1:100 with the blocking reagent). The sections were rinsed briefly with the same blocking reagent and then with PBS, 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 2% 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 C. Ultrathin section (50–60 nm) were cut from the resin blocks with an ultracut UCT ultramicrotome (Leica Microsystems AG, Wetzlar, Germany), collected onto Formvar-coated single-slot grids, and examined with a Jeol-100C transmission electron microscope (JEOL, Tokyo, Japan).

Experiment 3: triple-fluorescent analysis of AGRP/NPY and DBH/NPY contacts with GnRH-GFP neurons
To determine the relative contributions of the ARC and brain stem to the innervation of GnRH neurons, a triple-fluorescence strategy was developed, 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 brain stem. Although DBH is present exclusively in adrenergic/noradrenergic cells of the brain stem, the fine distinction among various catecholamine cell groups that express NPY differentially (26, 29, 30) 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) (37) was applied to the sections for 48 h at 4 C. This was followed by sequential incubations in a mixture of secondary antibodies, donkey biotin-conjugated antirabbit 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) for 1 h each. The detection of AGRP and DBH fibers was completed by treating sections with biotin tyramid working solution (described below) for 30 min and finally with avidin-conjugated AMCA fluorochrom (1:200; Vector Laboratories). To prepare the biotin tyramid solution, a stock solution was first synthesized according to instruction by Adams (38). This stock was stored in frozen aliquots and diluted at 1:1000 in TBS/0.005% H2O2 immediately before use. Following steps of this 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: for Cy3, excitation of 540–590 nM, bandpass of 595 nm, and emission of 600–660 nm; for AMCA, excitation of 320–400 nm, bandpass of 400 nm, and emission of 430–490 nm; for GFP, excitation of 460–500 nm, bandpass of 505 nm, and emission of 510–560 nm.

To determine the percent ratios of dual-labeled NPY fibers in each set of experiment, individual NPY juxtapositions were analyzed at high magnification 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 a x100 objective lens with 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: light microscopic analysis 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. 1BGo) vs. untreated controls (Fig. 1AGo) showed a marked reduction in the size of the ARC, whereas neurons of the ventromedial nuclei remained intact.



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  		FIG. 1. Effects of neonatal 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 reflect death of neurons that synthesize both AGRP and NPY in the ARC. Scale bar, 100 µm (A–F).

 
Results of immunocytochemical experiments showed that the density of NPY-immunoreactive fibers was largely reduced in the POA of ARC-lesioned animals (Fig. 1DGo), in comparison with untreated controls (Fig. 1CGo). 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. 1EGo) formed a somewhat less dense plexus in the POA than NPY fibers (Fig. 1CGo) in control animals. As opposed to NPY immunostaining (Fig. 1DGo), AGRP immunoreactivity was almost completely absent from the POA (Fig. 1DGo) in MSG-treated animals, corroborating previous data that the ARC is the only brain region in which this neuropeptide is synthesized (28).

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. 2AGo). 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. 2BGo). The number of AGRP immunopositive contacts was 56.5 ± 9.8% of that of NPY immunoreactive contacts. Furthermore, significantly less (P < 0.05) NPY-containing neuronal contacts were visible in neonatally MSG-treated animals (Fig. 2CGo) vs. controls. The lesion of the ARC caused a 63.7 ± 5.0% loss of NPY-immunopositive 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.



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  		FIG. 2. Innervation of GnRH neurons by NPY and AGRP fibers. Effects of neonatal 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).

 
Experiment 2: electron microscopic analysis of AGRP afferents to GnRH neurons
At the ultrastructural level, AGRP-immunoreactive fibers contained electron-dense DAB deposits, and they were frequently apposed to GnRH neurons accumulating highly electron-dense silver-gold particles (Fig. 3Go, A and B). Synapses of symmetric morphology were observed between AGRP axons and GnRH neurons as well as non-GnRH structures (Fig. 3BGo). 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.



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  		FIG. 3. Electron microscopic evidence for synaptic communication between AGRP-containing 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 silver-intensified 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.

 
Experiment 3: triple-fluorescent analysis of AGRP/NPY and DBH/NPY contacts with GnRH-GFP neurons
The distributions of NPY- (Fig. 4AGo) and AGRP-containing (Fig. 4BGo) fibers largely overlapped (Fig. 4CGo) 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. 4Go, D–F) of GnRH-GFP neurons, which exhibited bright green fluorescence. High-power analysis of the fluorescent specimen established that 49.1 ± 7.3% of NPY-containing neuronal contacts also contained AGRP (Figs. 4Go, D–F). In addition, nearly all AGRP fibers were immunoreactive to NPY.



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  		FIG. 4. Immunofluorescent identification of AGRP and DBH in NPY afferents to GnRH neurons. A–C, Both NPY (A; red Cy-3 fluorochrom) and AGRP (B; blue AMCA fluorochrom) immunoreactive 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 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 axons (G; red Cy-3 fluorochrom) also overlaps with that of DBH containing fibers (H; blue AMCA fluorochrom) in the MPOA. Note that this match (purple-to-white color in the merged panel; I) is of lower degree than in case of NPY and AGRP immunoreactive 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, DBH-immunoreactive axons (blue color) often remain immunonegative for NPY. Scale bars, 100 µm in A–C and G–I and 10 µm in D–F and J–L.

 
Similar to AGRP immunoreactive axons, fibers containing DBH (Fig. 4GGo) showed overlapping distribution with that of NPY axons in the POA (Fig. 4HGo). High-power analysis of immunostained axons established the presence of DBH in 25.4 ± 3.3% of NPY-containing fibers in contact with GnRH neurons (Fig. 4Go, J–L). Other types of fibers observed in the POA contained NPY without DBH or DBH without NPY (Figs. 4Go, J–L), and they were apposed to GnRH neurons.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NPY plays a rather complex role in the regulation of reproduction. Although evidence exists for direct stimulation of pituitary gonadotrophs by NPY (19, 20, 21, 39), the majority of work indicates that NPY mainly acts centrally to modulate gonadotropin secretion (3, 5, 9, 10, 11, 12, 13, 14, 16, 17, 18, 21, 22, 40, 41) and that GnRH neurons of the POA are direct target cells to central NPY actions (23, 24, 25, 26, 27). In the present study, the origins of NPY-containing neurons 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 brain stem. These quantitative data supplement results of a previous report by Simonian et al. (26), who described retrograde labeling of NPY neurons in both the ARC and ventrolateral medulla of the rat after tracer injection around GnRH neurons.

Receptors mediating NPY effects belong to the family of the seven-transmembrane domain, G protein-coupled receptors, which act primarily via inhibiting adenylate cyclase (42). Pharmacological studies using selective ligands to Y receptor subtypes for intracerebroventricular acute injections to castrated rats (13) and chronic infusions to intact male rats and mice (5) provided evidence that the Y5 receptor isoform plays a crucial role in the inhibitory control of gonadotropin release by NPY. Given that 55% of GnRH neuronal perikarya bear this receptor isoform (27), the Y5 receptor-mediated reproductive actions of NPY may be exerted, at least partly, on GnRH cells. In pentobarbital-blocked, proestrous rats, iv pulses of Y1 receptor antagonist blocked the endogenous LH surges and prevented the amplification of the LHRH-induced LH surges by NPY, implicating Y1 receptor activation in the stimulatory control of LH secretion (43). Morphological evidence supports the presence of the Y1 receptor isoform in axon terminals of GnRH neurons in the median eminence as well as in NPY fibers that innervate GnRH neurons from the ARC (25). It is reasonable to speculate that Y1 receptors presynaptic to GnRH neurons may modulate the synaptic release of NPY, AGRP, or a putative major neurotransmitter of these neurons, {gamma}- aminobutyric acid (GABA) (44). Accordingly, NPY agonism on presynaptic Y1 and Y2 receptors has been shown to inhibit glutamate (45) GABA (46) and norepinephrine (47) release.

The use of AGRP immunoreactivity as a marker for NPY axons of ARC origin was based on previous evidence that AGRP and NPY neurons of the ARC are essentially identical (28). 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 neurons. A similar ratio for NPY axons of ARC origin (56%) was calculated in experiment 1 from the comparison of AGRP-immunoreactive vs. NPY-immunoreactive contacts on GnRH neurons. A somewhat heavier innervation appear to originate from the ARC (64%) if the lost fraction of NPY contacts in MSG-treated animals is considered. 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 axons establish synapses with GnRH neurons. From a functional viewpoint, it is important to note that AGRP 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 (24). Furthermore, this synaptic morphology also characterizes GABAergic synapses (48). Therefore, the identification of the biosynthetic enzyme of GABA, GAD-65, in 30% of NPY neurons in the ARC (49) along with the light and electron microscopic demonstration of GABA immunoreactivity in subsets of NPY axons in the POA (44) highly indicate that at least some AGRP/NPY terminals that innervate GnRH neurons may cocontain GABA. Although this hypothesis awaits confirmation, recent evidence supports the physiological importance of a GABA/NPY interplay in the regulation of gonadotropin secretion (44).

Little is currently known about the role of AGRP, an endogenous antagonist of melanocortin 3 and 4 receptors (MC3-R and MC4-R), 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 (50). Furthermore, AGRP also stimulated GnRH release from mediobasal hypothalamic explants in vitro, and this effect was prevented by the presence of {alpha}-MSH in the medium (50), indicating that AGRP partially acts via antagonizing melanocortin receptors in the ARC-median eminence region. The synaptic communication revealed by our studies between AGRP axons and GnRH neurons of the POA represents an additional anatomical route for AGRP to influence the reproductive axis. The type and location of receptors involved in this synaptic interaction requires clarification. 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.

In addition to AGRP/NPY contacts of arcuate origin, our analysis found DBH immunofluorescence within 25% of NPY immunoreactive axonal contacts on GnRH neurons. This observation indicates that noradrenergic/adrenergic pathways directly regulate GnRH neurons and corroborates previous data by others (26, 51, 52) that noradrenergic cell groups project to the immediate vicinity of GnRH neurons in the rat. The adrenergic/noradrenergic input to GnRH cells is consistent with a large body of evidence in the literature indicating the important role of adrenergic stimuli in the regulation of the ovarian cycle and the steroid-induced gonadotropin surge (53). The present work did not address the ultrastructural characteristics of DBH-immunoreactive juxtapositions to GnRH neurons. One previous study (54) suggested that noradrenergic neurons can communicate with GnRH cells through classical synaptic mechanisms, whereas other investigators (55) 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 immunoelectronmicroscopic analysis of DBH contacts on GnRH cells, there is little doubt that the proposed nonsynaptic mechanisms often play a role in noradrenergic neurotransmission. For example, only a small fraction of DBH-immunoreactive juxtapositions establish classical synapses with neurons of the cerebral cortex and the subcellular distribution of {alpha}2A adrenoceptors is not restricted to the postsynaptic membranes (56). 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 (26, 29, 30). 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 (26, 29). Finally, while it is likely that at least some of the AGRP/NPY input that reaches GnRH neurons from the ARC is GABAergic (44, 49), the recent demonstration of vesicular glutamate transporter-2 in C1 adrenergic and other catecholamine neurons of the brain stem (57), which also synthesize NPY (29), raises the possibility that the excitatory neurotransmitter glutamate is coreleased with NPY and epinephrine/norepinephrine from afferents to GnRH neurons.

Although the ARC and brain stem are generally viewed as the most important sources of central NPY, neuronal perikarya-synthesizing NPY occur in many further brain regions, including the dorsomedial hypothalamic nucleus, bed nucleus of the stria terminalis, anterior horn of the anterior commissure, lateral preoptic area, dorsal hypothalamic area, mesencephalic central gray, and ventrolateral geniculate nucleus (58). It is likely that some of the above or other NPY cell groups contribute to the innervation of GnRH neurons. The calculated percentage of these alternative afferents can be up to 20–26% of NPY fibers form the ARC are 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 on the basis of their AGRP content. A subset of NPY neurons in the ARC (5%) seem to be devoid of AGRP (28), 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 low levels of DBH or AGRP in subsets of NPY axons, despite the high detection sensitivity provided by the tyramid signal amplification technique (38).

The AGRP and DBH contents of NPY afferents to GnRH neurons were analyzed in GnRH-GFP transgenic mice (31) in which green fluorescence is detectable in the vast majority of GnRH neurons, without a significant ectopic expression of the transgene (31). 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.

In summary, the quantitative data we present here reveal the relative contributions of two major NPY systems to the afferent regulation of GnRH neurons. Using different detection approaches, 49–64% of NPY contacts on GnRH neurons were found to originate from the ARC, cocontain AGRP, and establish symmetric synapses with GnRH neurons. An additional 25% of NPY afferents contain DBH, indicating their origin in noradrenergic/adrenergic cell groups of the brain stem. This information will stimulate future research for the functional characterization of NPY afferents to GnRH neurons and the input-specific use of pre-, post-, and/or extrasynaptic receptors by distinct afferent systems.


   Acknowledgments
 
The authors thank Dr. R. Benoit for the kind donation of the LR1 antibody to GnRH and Dr. I. Merchenthaler for the FJL no. 14/3A antiserum to NPY.


   Footnotes
 
This work was supported by grants from the National Science Foundation of Hungary (OTKA T031770 and T043407), the Hungarian Medical Research Council (ETT 280/2000), and the Fifth EC Framework Program (QLG3 2000-00844).

Abbreviations: AGRP, Agouti-related protein; ARC, arcuate nucleus; bw, body weight; DAB, diaminobenzidine; DBH, dopamine-ß-hydroxylase; GABA, {gamma}-aminobutyric acid; GFP, green fluorescent protein; MSG, monosodium glutamate; NPY, neuropeptide Y; PB, phosphate buffer; POA, preoptic area; TBS, Tris-buffered saline.

Received April 15, 2003.

Accepted for publication July 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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