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Galanin-Like Peptide Stimulates the Release of Gonadotropin-Releasing Hormone in Vitro and May Mediate the Effects of Leptin on the Hypothalamo-Pituitary-Gonadal Axis

Division of Metabolic Medicine, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Campus, London W12 ONN, United Kingdom

Address all correspondence and requests for reprints to: Professor S. Bloom, Division of Metabolic Medicine, Faculty of Medicine, Imperial College of Science Technology and Medicine, Hammersmith Campus, Du Cane Road, London W12 ONN, United Kingdom. E-mail: s.bloom{at}ic.ac.uk.

	Abstract

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Leptin regulates the hypothalamo-pituitary-gonadal axis in relation to nutritional status. The mechanism through which leptin mediates its effects on neuroendocrine reproductive circuits remains unclear. Galanin-like peptide (GALP) is a recently identified hypothalamic peptide, localized in the arcuate nucleus, which seems to be regulated by leptin and stimulates LH when administered centrally. Here, we demonstrate that leptin stimulates the release of GALP and GnRH in vitro from hypothalamic explants harvested from male rats. In addition, we show that GALP stimulates the release of GnRH from hypothalamic explants and GT1–7 cells. Furthermore, we demonstrate that GALP antiserum blocks the stimulatory action of leptin on GnRH release from hypothalamic explants. GALP is a ligand of the galanin receptors. We therefore investigated whether the effect of GALP on GnRH release may be mediated via a known galanin receptor. GALP-stimulated GnRH release from hypothalamic explants was attenuated (but not abolished) by the galanin receptor antagonist galantide. However, GALP-stimulated GnRH release from GT1–7 cells was not diminished by the coadministration of galantide. In addition, none of the cloned galanin receptors were expressed in GT1–7 cells by RT-PCR. These observations suggest that GALP may stimulate GnRH release through an indirect pathway involving a galanin receptor and via a direct action on GnRH neurons, possibly through a novel receptor. These findings suggest that GALP may mediate the actions of leptin on the reproductive axis and provide a link between nutrition and fertility.

	Introduction

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ENERGY STATUS AND fertility are highly regulated in mammals. The adipocyte hormone leptin, which is secreted in proportion to body adiposity, acts as a metabolic signal to the neuroendocrine system to integrate feeding behavior, metabolism, and pituitary function with the level of energy reserves (1). Several studies have indicated leptin may have a permissive role in the regulation of the reproductive axis, because low or absent leptin inhibits the hypothalamo-pituitary-gonadal (HPG) axis and impairs reproductive function. In both rodents and humans, the absence of leptin results in infertility attributable to hypogonadotropic hypogonadism (2, 3); reproductive function can be restored by peripheral injections of recombinant leptin (3, 4). Furthermore, in both rodents and humans, fasting, and therefore low leptin (5, 6), suppresses pulsatile LH secretion (7, 8). Again the exogenous administration of leptin in physiological replacement doses prevents these fasting-induced changes of the HPG axis (1, 9).

The mechanism of leptin’s effects on the HPG axis remains unclear. Leptin is known to act on hypothalamic neuronal targets primarily located in the arcuate nucleus (ARC). However, the leptin receptor has not been identified on GnRH neurons in vivo (10), and the intermediaries in the signal transduction pathway between leptin and GnRH have yet to be conclusively established. Galanin-like peptide (GALP) is a novel peptide recently isolated from the porcine hypothalamus (11), which is expressed solely in the ARC (12, 13, 14). The majority of GALP neurons in the ARC colocalize with the leptin receptor (12), and leptin treatment has been shown to increase GALP mRNA expression in ovariectomized female rats (13) and ob/ob mice (15). GALP immunoreactive (IR) fibers project from the ARC to the medial preoptic area (MPOA) and are often seen in close apposition with GnRH-IR perikarya (12). Intracerebroventricular (ICV) administration of GALP increases serum LH and testosterone in intact males (16, 17) and induces c-fos activation in GnRH neurons in the MPOA (16).

GALP shares sequence homology with galanin (11). To date, three G protein-coupled receptors, termed GAL R1, GAL R2, and GAL R3, with high affinity for galanin have been identified (18, 19, 20). GALP also shows high affinity for GAL R1 and GAL R2 (its affinity for GAL R3 is unknown) (11). Galanin has also been reported to act on the HPG axis. Galanin stimulates the release of GnRH from median eminence-ARC fragments in vitro (21), and ICV administration of galanin stimulates LH release in females in vivo (22). The receptor mediating the effects of galanin on the HPG axis remains unknown, given that selective galanin receptor antagonists are not available.

In our current study, we have examined the hypothesis that GALP mediates the effects of leptin on the reproductive axis, and we have also compared the effects of GALP with those of galanin. We have investigated, first, the effect of leptin on the release of GALP, galanin, and GnRH from hypothalamic explants in vitro; and second, the effects of GALP and galanin on the release of GnRH from hypothalamic explants. Furthermore, we have investigated the effects of GALP antiserum on leptin-stimulated GnRH release. To determine whether GALP or galanin has a direct effect on the GnRH neuron, we have examined the effect of both peptides on GnRH release from the immortalized GnRH cell line GT1–7 cells. Finally, because GALP is a ligand of the galanin receptors, we aimed to identify the galanin receptor mediating these effects on GnRH release.

	Materials and Methods

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Materials
Rat GALP and galantide were purchased from Bachem (UK) Ltd. (Merseyside, UK). Rat galanin was synthesized using an automated peptide synthesizer (model 6 MPS, Advanced Chemotech, Louisville, KY). Peptides were purified to homogeneity by reversed-phase HPLC on a C8 column, and molecular weight was checked by mass spectroscopy (23). Recombinant murine leptin was a gift from M. Chiesi and N. Levens from Novartis (Basel, Switzerland). Reagents for the hypothalamic explant experiments were supplied by BDH (Poole, Dorset, UK). GALP antibody was purchased from Phoenix Pharmaceuticals (Belmont, CA).

Animals
Adult male Wistar rats, weighing 200–250 g, were maintained in cages of five, under controlled temperature (21–23 C) and light (12-h light, 12-h dark cycle; lights on at 0700 h). Animals had ad libitum access to food (RM1 diet SDS, Witham, UK.) and water unless otherwise stated. Adult male C57BCK6/CBA mice (20–25 g) were maintained as above. All animal procedures undertaken were approved by the British Home Office Animals (Scientific Procedures) Act, 1986.

Static incubation of hypothalamic explants
The static incubation system was used as previously described (24). Rats were killed by decapitation. The upper skull was removed and the brain dissected free of dura; the frontal lobes were then lifted, and the exposed optic nerves were cut. The whole brain was then reflected. The brain was mounted with ventral surface uppermost and placed in a vibrating microtome (EnergyBeam Sciences Inc., Agawam, MA). A 1.7-mm explant was taken from the basal hypothalamus. Extrahypothalamic tissue was removed by dissection through the mamillary bodies posteriorly and laterally through the Circle of Willis. The explant includes the MPOA. The explant was incubated in artificial cerebrospinal fluid (aCSF) (20 nM NaHCO₃, 126 mM Nacl, 0.09 mM Na₂HPO₄, 6 mM KCl, 1.4 mM CaCl₂, 0.09 mM MgSO₄, 5 mM glucose, 0.18 mg/ml ascorbic acid, and 100 µg/ml aprotinin), equilibrated with 95% O₂ and 5% CO₂ at 37 C. After an initial 2-h equilibration period, the hypothalami were incubated for 45 min in aCSF (basal period) before being challenged with peptide for 45 min. At the end of the study, the viability of the tissue was verified by a 45-min exposure to 56 mM KCl; isotonicity was maintained by substituting K⁺ for Na⁺. Explants were excluded from analysis if release during the K⁺ exposure was less than basal release (<10% of explants excluded). The aCSF collected at the end of each period was frozen at -20 C until measurement of peptide-IR by RIA.

GT1–7 cell culture and secretion experiments
The immortalized hypothalamic GnRH-producing neurone subclone GT1–7 cells (25) were maintained at 37 C in 5% CO₂ in DMEM with 4.5 g/liter glucose, 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml). GT1–7 cells were plated on poly-L-lysine-coated 48-well plates. After growing for 2–3 d to confluence, cells were washed twice in DMEM with 4.5 g/liter glucose, penicillin (100 IU/ml), streptomycin (100 µg/ml), and 0.1% BSA. The cells were then preincubated for 2 h in serum-free medium. Thereafter, the medium was discarded, and the cells were incubated in 500 µl of serum-free medium plus the appropriate test substance [GALP/galanin/galantide (0.01–100 nM) or 56 mM KCl]. The cells were incubated with the test substances for 60 min, after which the medium was removed and frozen at -20 C until measurement of GnRH-IR by RIA. Protein content per well was assayed using the Pierce bicinchoninic acid (BCA) protein assay (Sigma, Poole, Dorset, UK). Protein content was consistent between wells (<10% variation).

RIAs
GnRH-IR levels were measured using reagents and methods kindly provided by H. M. Fraser, Medical Research Centre Reproductive Biology Unit, Edinburgh (26). The intraassay and interassay variations were 8 and 12%, respectively, and the sensitivity was 0.25 fmol/tube. Rat GALP-IR was measured using a commercially available RIA from Phoenix Pharmaceuticals, CA. The intraassay and interassay variations were 4 and 15%, respectively, and the sensitivity was 1 pg/tube (0.16 fmol/tube). An in-house rat galanin RIA was used to measure galanin immunoreactivity, as previously described (27). Briefly, galanin antiserum raised in rabbits to unconjugated synthetic rat galanin was used at a final dilution of 1:320,000. The tracer prepared by the iodogen method had a specific activity of 48 Bq/fmol. The intraassay and interassay variations were less that 15%, and the sensitivity was 2 fmol/tube.

RT-PCR analysis
Generation of total RNA.
Total RNA was extracted from GT1–7 cells, mouse hypothalami, and rat hypothalami using Tri-reagent (Helena Biosciences, Sunderland, Tyne and Wear, UK) according to the manufacturer’s protocol. Hypothalami were harvested from male C57BL6/CBA mice (20–25 g) and male Wistar rats (200–250 g). The tissue removed was bordered rostrally by the anterior edge of the optic chiasma, laterally by the hypothalamic fissures, and caudally by the mamillary bodies.

RT-PCR.
RT-PCR was performed as previously described (28). Briefly, total RNA was reverse transcribed using avian myoblastoma virus reverse transcriptase (RT) (Promega, Southampton, UK) in a reaction primed using oligo(dT) (12, 13, 14, 15, 16, 17, 18). The RT reaction was subjected to PCR using primers obtained from the published sequence of the rat GAL R1, GAL R2, and GAL R3 (accession nos. U33193, AF010318, and AF079844, respectively). The primers were synthesized by Oswel DNA services (Southampton, UK). The primers used for GAL R1 were agtcggatccccaaggttctcaatcatctgc and agtcgaattccggggtatctattcggttct corresponding to positions 659–679 and 997-1016 of the GenBank nucleotide sequence; for GAL R2, the primers were agtcggatcccacacctcgaaacgcgctggc and agtcgaattcgtgcagttgggaagtgcggg corresponding to sequence positions 432–451 and 1086–1105; and for GAL R3, the primers were agtcggatccctcatcttcctgttgggcatg and agtcgaattcgtagatgagcagatgtaccg, corresponding to sequence positions 76–96 and 289–309. Using these primers, amplified fragments of 357 bp for GAL R1, 673 bp for GALR2, and 233 bp for GAL R3 would be expected. PCR cycles were as follows: 95 C for 1 min, 55 C for 1 min (decreasing by 2 C each cycle for 5 cycles), then 95 C for 1 min, 50 C for 1 min (decreasing by 1 C each cycle for 8 cycles) then 95 C for 1 min, 45 C for 45 sec, 72 C for 1 min for 12 cycles. To check for possible artifacts generated by amplification of remnants of genomic DNA, control RT-PCRs were performed and treated in an identical way but without RT added to the RT-reaction mixture. Amplified fragments were resolved by 1% agarose gel electrophoresis and visualized by ethidium bromide staining.

Statistical analysis
Results are shown as mean values ± SEM. Data from the hypothalamic explant experiments was analyzed by paired t test between basal and treatment groups. Pairwise comparison between groups, where appropriate, was performed using a one-way ANOVA with a Student-Newman-Keuls post hoc test. For the secretion experiments, the data were analyzed using a one-way ANOVA with a Dunnet’s post hoc analysis, to compare the different groups with the basal control. In all cases, the level of statistical significance was set at P < 0.05.

	Results

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Study 1: a dose-response curve for the effect of leptin (1, 10, and 100 nM) on GALP release from hypothalamic explants harvested from fed animals
Hypothalamic explants, harvested from ad libitum-fed animals, were exposed to leptin (1, 10, and 100 nM) for 45 min. All doses of leptin significantly increased GALP release from hypothalamic explants, when compared with basal release [GALP release: basal 124.3 ± 10 fmol/explant, GALP (1 nM) 139.4 ± 23 fmol/explant, P < 0.05; GALP (10 nM) 149.6 ± 14 fmol/explant, P < 0.05; GALP (100 nM) 161.0 ± 26 fmol/explant, P < 0.05, n = 9–11] (Fig. 1).

View larger version (15K): [in this window] [in a new window]   FIG. 1. A dose-response curve for the effect of leptin (1, 10, and 100 nM) on GALP release from hypothalamic explants. *, P < 0.05, basal vs. GALP (dose).

Study 3: the effect of GALP and galanin on the release of GnRH from hypothalamic explants harvested from fed animals
Hypothalamic explants, harvested from ad libitum-fed animals, were exposed to GALP (100 nM) for 45 min. GALP treatment significantly increased the release of GnRH, when compared with basal release (GnRH release: basal 8.1 ± 2.3 fmol/explant, GALP 18.2 ± 2.4, P < 0.01, n = 14) (Fig. 2A). Exposure to galanin (100 nM) also significantly increased GnRH release (GnRH release: basal 9.1 ± 1.4 fmol/explant, galanin 14.0 ± 1.4 fmol/explant, P < 0.01, n = 10) (Fig. 2B).

View larger version (11K): [in this window] [in a new window]   FIG. 2. A, The effect of GALP (100 nM) on GnRH release from hypothalamic explants (**, P < 0.01, GALP vs. basal). B, The effect of galanin (100 nM) on GnRH release from hypothalamic explants (**, P < 0.01, galanin vs. basal).

View larger version (14K): [in this window] [in a new window]   FIG. 3. The effect of leptin (100 nM), leptin (100 nM) and GALP antiserum (1:300 dilution), and GALP antiserum (1:300 dilution) alone on GnRH release from hypothalami harvested from 24-h-fasted rats (**, P < 0.001, leptin vs. basal; #, P < 0.05, leptin vs. leptin + GALP antiserum). ab, Antibody.

View larger version (15K): [in this window] [in a new window]   FIG. 4. The effect of GALP (100 nM), both GALP (100 nM) and galantide (100 nM), and galantide (100 nM) alone on GnRH release from hypothalamic explants (**, P < 0.001, GALP vs. basal).

View larger version (24K): [in this window] [in a new window]   FIG. 5. The effect of GALP (0.01–100 nM) on the release of GnRH from GT1–7 cells. (*, P < 0.001 vs. basal).

View larger version (19K): [in this window] [in a new window]   FIG. 6. The effect of galanin (0.01–100 nM) on the release of GnRH from GT1–7 cells. (*, P < 0.0001 vs. basal).

View larger version (26K): [in this window] [in a new window]   FIG. 7. The effect of galanin antagonist galantide (0.1–100 nM) on GnRH release from GT1–7 cells. (*, P < 0.0001 vs. basal).

View larger version (16K): [in this window] [in a new window]   FIG. 8. The effect of galanin antagonist galantide (100 nM) on GALP (10 nM)-induced GnRH release from GT1–7 cells. (*, P < 0.001 vs. basal).

View larger version (24K): [in this window] [in a new window]   FIG. 9. Galanin receptor (Gal R) mRNA expression in the GT1–7 cell line as determined by RT-PCR. M, 1-kb ladder; H, mouse hypothalamic RNA; G+, GT1–7 RNA + RT; G-, GT1–7 RNA - RT; W, water. The size of the expected PCR product for GAL R1 was 357 bp; for GAL R2, it was 673 bp; and for GAL R3, 233 bp.

	Discussion

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In these studies, we demonstrate that in vitro leptin significantly stimulates the release of GALP peptide from hypothalamic explants harvested from male rats in a dose-dependent manner. The observation that GALP peptide is positively regulated by leptin is supported by previous studies demonstrating that GALP mRNA expression is up-regulated by leptin. ICV injection of leptin for 7 d in ob/ob mice significantly increased GALP expression, when compared with vehicle (15); twice daily peripheral injections of leptin administered to 48-h fasted ovariectomized female rats for 2 d significantly increased GALP mRNA (13); and GALP mRNA is reduced in male Zucker obese (fa/fa) rats (rats with defective leptin signaling), compared with age-matched Zucker lean (Fa/fa or Fa/Fa) rats (30). The literature therefore suggests that chronic manipulation of leptin alters GALP expression in the ARC; however, this is the first demonstration that GALP peptide release can be acutely regulated by leptin.

We have also found that exposure of hypothalamic explants to GALP dramatically stimulates the release of GnRH. This is the first direct demonstration that GALP increases hypothalamic GnRH release in vitro, and it suggests that the increase in LH seen after ICV GALP administration is attributable to the activation of hypothalamic GnRH neurons (16). In addition to this, we have demonstrated that GALP antiserum blocked leptin-induced GnRH release from hypothalamic explants. This finding provides support for the hypothesis that GALP may mediate the actions of leptin on the HPG axis. The following model can therefore be proposed. In freely fed rats, the level of circulating leptin is high. Leptin interacts with leptin receptors found on GALP neurons in the ARC, resulting in the release of GALP. Consequently, GALP stimulates the release of GnRH and, thus, further-downstream components of the HPG axis. Under conditions of prolonged food restriction, however, the low levels of circulating leptin may be insufficient to stimulate the release of GALP. This would remove a stimulatory input on the HPG axis and may be one mechanism through which the HPG axis is suppressed during fasting.

Recent studies have indicated that the main physiological role of leptin is to mediate the metabolic and endocrine responses necessary to adapt to changes in nutritional status (31). Thus, leptin is involved in the regulation of the thyroid, reproductive, growth, and adrenal axes in addition to regulating feeding behavior. The mechanism through which leptin regulates food intake and energy expenditure is well documented (32, 33, 34). Two populations of neurons within the ARC, one expressing the orexigenic peptides neuropeptide Y and agouti-related protein and another expressing the anorexic peptides -melanocyte stimulating hormone and cocaine and amphetamine-regulated transcript, are believed to be the principal mediators of leptin’s effects on energy homeostasis. However, the mechanisms through which leptin controls the reproductive, growth, and stress axes have not been investigated as thoroughly. The data presented here identifies a new neuronal population in the ARC, which may mediate the effects of leptin on the reproductive axis. These findings develop our understanding of how the integration of nutritional status and fertility is achieved within the hypothalamus and may help to distinguish between the complex effects of leptin on the neuroendocrine axes and metabolism.

In contrast to GALP, leptin had no significant effect on the release of galanin from hypothalamic explants. The effect of leptin on galanin expression seems to be dependent on the region of the hypothalamus examined. Galanin is found in several hypothalamic nuclei, including the ARC and dorsomedial nucleus (DMN) (35), areas with high expression of the leptin receptor. However, no colocalization between galanin IR neurons and the leptin receptor has been observed (10). In the ob/ob mouse, treatment with leptin significantly decreased galanin expression in the periventricular nucleus but had no effect in the other nuclei examined, including the ARC and DMN (36). In the obese Zucker rat, galanin expression in the PVN is double that of age-matched lean controls. However, expression in the median eminence is significantly reduced, and there are no changes in the ARC or DMN (37). These studies indicate that the regulation of galanin expression by leptin is complex. This may be attributable, in part, to the widespread distribution of galanin in the hypothalamus and the diverse functions attributed to it (35).

We also found that galanin stimulated the release of GnRH from hypothalamic explants. This observation is consistent with previous studies in which galanin was found to stimulate GnRH release from arcuate-median eminence fragments from both male rats and ovarian steroid-primed ovariectomized female rats (21, 38). The median eminence contains the highest concentration of galanin peptide in the hypothalamus, and it has been hypothesized that galanin may act presynaptically to potentiate the release of GnRH from nerve terminals in the median eminence (35). The finding that both GALP and galanin stimulated GnRH release suggested that this effect may be mediated by a galanin receptor. Therefore, we examined the ability of the galanin receptor antagonist, galantide, to block GALP-induced GnRH release. Galantide is a chimeric peptide that binds with high affinity to all three galanin receptors and has been shown to block the actions of galanin on the HPG axis (21, 39, 40). An equimolar concentration of galantide completely blocks galanin-stimulated GnRH release from arcuate-median eminence fragments from steroid-primed rats (21). In these studies, galantide had no effect on basal release, but an equimolar concentration was able to reduce GALP-stimulated GnRH release from hypothalamic explants. Taken together with the finding that galanin stimulates GnRH from hypothalamic explants, this data suggests that GALP-stimulated GnRH release may be mediated, at least in part, via the galanin receptors. However, GALP-induced GnRH release was not entirely abolished by galantide. This observation suggests that galanin receptor-independent pathways may also contribute to GALP’s effects on GnRH.

To further investigate the mechanism through which GALP and galanin stimulate the release of GnRH, we used the GnRH immortalized cell line GT1–7 cells. GT1–7 cells exhibit many of the known physiological characteristics of GnRH neurons in situ; they respond appropriately to depolarization, release GnRH in a pulsatile manner (41), and have been shown to respond to known GnRH secretogogues such as neuropeptide Y and glucagon-like peptide 1 (26, 42). In whole hypothalamic tissue, both GALP and galanin stimulated the release of GnRH. However, only GALP stimulated GnRH release from GT1–7 cells. Moreover, the addition of galantide did not attenuate GALP-induced GnRH release from GT1–7 cells. This suggests that GALP-induced GnRH release from GT1–7 cells may not be mediated by a cloned galanin receptor. In keeping with this, RT-PCR analysis failed to detect the presence of transcript for any of the cloned galanin receptors in GT1–7 cells. To date, no significant colocalization between the galanin receptors and GnRH neurons has been reported, despite all three receptors being expressed in the MPOA (19, 20, 43). However, further studies are required to establish definitively whether GnRH and galanin receptors colocalize in vivo.

Taken together, these studies indicate that in vitro GALP may stimulate GnRH through two distinct mechanisms: first, through an indirect action mediated by a galanin receptor; and second, through direct activation of GnRH neurons, possibly via a novel receptor. It should be noted, however, that the different mechanisms observed in hypothalamic explants and GT1–7 cells could be explained by differences between GnRH neurons from animal tissue and cultured GnRH neurons. It is possible that the lack of input from afferent neurons and supporting glia may alter the pattern of receptor expression in GT1–7 cells. In vivo colocalization studies would be of great interest. From the experiments carried out in this investigation, it is difficult to identify whether the action of GALP on GnRH release occurs at the level of the cell body or at the terminals in the median eminence. It would be interesting to examine the effect of GALP on the release of GnRH from median eminence explants.

The absence of transcript for the known galanin receptors, in addition to the observation that galanin has no effect on GnRH release from GT1–7 cells, suggests that GALP’s direct effects on GnRH release in GT1–7 cells may be mediated through a novel receptor. The presence of a specific GALP receptor has been suggested previously because of the differential effects of GALP and galanin on food intake (44), LH release (16), and the differential effects of galanin and GALP on c-fos induction (45). Within the GALP sequence, there are two highly conserved and unique amino acid sequences not shared with galanin (GALP 1–8 and 38–54). It is possible that these sequences may dictate its interaction with a unique GALP receptor and mediate its physiological effects. Further work is required to identify the receptor mediating the effects of GALP in GT1–7 cells. A summary of the proposed interactions among leptin, GALP, and GnRH in the regulation of the reproductive axis is illustrated in Fig. 10.

View larger version (22K): [in this window] [in a new window]   FIG. 10. A proposed model for the interaction among leptin, GALP, and GnRH in the regulation of the neuroendocrine reproductive axis. Circulating leptin passes across the blood-brain barrier into the ARC of the hypothalamus and binds to the leptin receptor (Ob-R) expressed on GALP neurons. This stimulates the release of GALP, which acts on GnRH neurons through two mechanisms: first, through a direct activation of GnRH neurons via a novel receptor; and second, through an indirect pathway that involves a galanin receptor (Gal-R). The net result is an increase in GnRH release.

	Footnotes

 
This work was supported by an Medical Research Council (MRC) program grant, an MRC studentship for A.S., and a Biotechnology and Biological Sciences Research Council-GlaxoSmithKline case studentship for P.J.

Abbreviations: aCSF, Artificial cerebrospinal fluid; ARC, arcuate nucleus; DMN, dorsomedial nucleus; GALP, galanin-like peptide; HPG, hypothalamo-pituitary-gonadal; ICV, intracerebroventricular; IR, immunoreactive; MPOA, medial preoptic area; n.s, not significant; RT, reverse transcriptase.

Received July 14, 2003.

Accepted for publication October 16, 2003.

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