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Expression of GalR1 and GalR2 Galanin Receptor Messenger Ribonucleic Acid in Proopiomelanocortin Neurons of the Rat Arcuate Nucleus: Effect of Testosterone¹

INSERM U422, Institut Fédératif de Recherches 22, Laboratoire de Neuroendocrinologie et Physiopathologie Neuronale (S.B., V.P., D.C., J.-C.B., V.M.), Place de Verdun, 59045 Lille Cedex, France; Merck Research Laboratories (A.H.), Rahway, New Jersey 07065; Rh\|[ocirc ]\|ne-Poulenc Rorer, Inc. (E.H.-O.), 94400 Vitry, France; and INSERM U413, IFRMP 23, Université de Rouen (S.J., H.V.), 76821 Mont-Saint-Aignan Cedex, France

Address all correspondence and requests for reprints to: Sebastien Bouret, INSERM U422, Laboratoire de Neuroendocrinologie et Physiopathologie Neuronale, 1 Place de Verdun, 59045 Lille Cedex, France. E-mail: bouret{at}biserte.lille.inserm.fr

	Abstract

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Previous studies have shown that galanin-containing fibers make synaptic contacts with POMC neurons in the arcuate nucleus. However, the ability of POMC neurons to express galanin receptors has never been assessed. The present study was designed to investigate whether POMC neurons express galanin receptor messenger RNA (mRNA) and whether testosterone could modulate galanin receptor gene expression. A dual-labeling in situ hybridization histochemistry, using ³⁵S-labeled (galanin receptors GalR1 or GalR2) and digoxigenin-labeled (POMC) riboprobes, was performed on brain sections from intact, castrated, and testosterone-replaced adult male rats. For analysis, the arcuate nucleus was divided into four rostro-caudal areas. The results revealed that both GalR1 and GalR2 mRNAs were expressed in POMC neurons. Most POMC neurons expressing galanin receptor mRNAs were found in the rostral parts of the nucleus. Castration reduced the labeling density of galanin receptor mRNAs in POMC neurons, and testosterone prevented the effects of castration in all rostro-caudal subdivisions of the arcuate nucleus. Taken together, these data indicate that galanin can directly modulate the activity of POMC neurons, via an action on GalR1 or GalR2 receptors, particularly in the rostral-arcuate nucleus. In addition, testosterone can modulate the expression of GalR1 and GalR2. Because POMC neurons located in the rostral part of the nucleus are known to project preferentially to the preoptic area, POMC neurons expressing the galanin receptor genes may play an important role in the regulation of the GnRH neuroendocrine axis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
POMC IS THE precursor molecule for the opioid peptide ß-endorphin, as well as for several other bioactive peptides (including -MSH and ACTH). The prominent group of POMC neurons, located in the arcuate nucleus of rat hypothalamus (1), send projections to multiple brain regions. In particular, numerous POMC-containing fibers innervate the preoptic area (2) where GnRH cell bodies are located. Several lines of evidence indicate that ß-endorphin and -MSH are implicated in the regulation of reproduction and feeding behavior (3, 4, 5) and that the opioid system, notably ß-endorphin, exerts a tonic inhibitory influence on gonadotropin release (4, 6). Previous studies have demonstrated that the activity of POMC neurons is under the influence of various factors, including gonadal steroids (2, 7, 8, 9), leptin (10, 11), and neuropeptides (4). The neuropeptide galanin, which is involved in the control of pituitary functions and feeding behavior (4, 12), may play a pivotal role in the regulation of POMC neurons, because synaptic connections between galanin-immunoreactive fibers and ß-endorphin-containing perikarya and dendrites have been observed in the arcuate nucleus (13). However, the existence of morphofunctional interactions between galanin and POMC neurons depends on the possibility that POMC neurons express galanin receptors. Two types of galanin receptors, the GalR1 (14, 15) and the GalR2 (16, 17, 18), are expressed in the brain. A third subtype of galanin receptor, named GalR3, has recently been cloned (19, 20, 21); but it is not clear whether this receptor is expressed in the brain. In contrast, the GalR1 and GalR2 galanin receptors are clearly expressed throughout the arcuate nucleus (22, 23, 24); and their distribution pattern exhibits similarities with that of the POMC neurons.

The primary objective of the present study was to determine whether POMC neurons of the arcuate nucleus express GalR1 and GalR2 messenger RNAs (mRNAs). Because the expression of POMC mRNA is controlled by gonadal steroids (2, 7, 8, 9) and because gonadal steroids enhance galanin binding in several hypothalamic nuclei (25), we have also investigated the possible effects of testosterone replacement on the expression of GalR1 or GalR2 mRNA in POMC neurons. Using a dual-labeling in situ hybridization technique, we report that some POMC neurons can express GalR1 or GalR2 mRNA. We also show that testosterone modulates the expression of GalR1 and GalR2 galanin receptor mRNAs in POMC neurons.

	Materials and Methods

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Animals and tissue preparation
Adult male Wistar rats (300–350 g; CERJ, Saint-Berthevin, France) were maintained on a 14-h light, 10-h dark cycle (lights on at 0500 h), with food and water available ad libitum. The animals were castrated (n = 10) or sham-operated (n = 5) under ether anesthesia. Immediately after castration, animals received sc a 30-mm SILASTIC brand (Dow Corning, Midland, MI) capsule (id, 1.57 mm; od, 3.18 mm; Harvard Apparatus, Les Ulis, France) that was either empty (n = 5) or filled with crystalline testosterone (n = 5). These capsules were prepared as previously described (26) and were soaked for 3 days in phosphate buffer (solutions were changed twice daily). Four days after surgery, animals were anesthetized with ketamine (20 mg/kg) and xylazine (0.2 ml/kg). Blood was collected and plasma was stored at -20 C for testosterone RIA. Animals were perfused intracardially with 5–10 ml saline, followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed and immersed in the same fixative for 2 h. The tissues were washed overnight in 0.05 M Coons veronal buffer (pH 7.4) containing 20% sucrose, embedded in Tissue-Tek (Miles Laboratories, Naperville, CA), and frozen in liquid nitrogen. Frozen 14-µm coronal sections were collected from the level of -1.8 to -4.8 mm, relative to Bregma, according to the atlas of Paxinos and Watson (27). The sections were mounted onto gelatin-coated slides and stored at -80 C until used for in situ hybridization. All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/European Economic Community), regarding mammalian research.

Serum testosterone assay
Testosterone was measured in plasma using an RIA kit (SORIN Biomedica, Antony, France). The sensitivity of the assay was 2 pg/tube, and the intraassay coefficient of variation was 6.5%. All samples were analyzed within the same assay.

³⁵S-Labeled GalR1 and GalR2 receptor complementary RNA probes
The plasmid vector pBluescript IIKS containing a HindIII/BamHI fragment of 1600 bp of the full-length rat GalR1 receptor (15) and a pcDNA3 vector containing a HindIII/NotI fragment of 1900 bp isolated from a rat hypothalamic cDNA (complementary DNA) library and containing the full-length GalR2 receptor region (16) were used. BamHI and T7 made the antisense probe, and HindIII with T3 RNA polymerase produced the sense probe for GalR1. NotI plus T7 made the GalR2 antisense probe, and HindIII with Sp6 RNA polymerase produced the sense probe. The radioactive probes were generated by labeling with 200 µCi of [³⁵S]cytidine 5'-triphosphate (Amersham Pharmacia Biotech, Les Ulis, France) using 1 µl of the appropriate RNA polymerase in a 40-µl transcription reaction volume containing 8 µl of 5x transcription buffer, 2 µl of 0.1 M dithiothreitol (DTT), 1 µg linearized plasmid (50 ng/ml), 1 µl Escherichia coli transfer RNA (5 mg/ml), 20 U of RNasin, and 1 µl of a 10-mM stock solution of ATP, GTP, and uridine 5'-triphosphate. The transcription reagents were incubated for 4 h at 39 C. Labeled probes were extracted with phenol-chloroform and separated from nonincorporated nucleotides on a Sephadex-G50 column. The ³⁵S-labeled riboprobes were diluted with hybridization buffer to a final concentration of 30,000 dpm/µl.

Digoxigenin-labeled complementary RNA POMC probe
A 409-bp DNA fragment (position 221–629 bp of the POMC gene exon III) was amplified by PCR from the rat POMC gene subcloned in pBR322 (provided by Dr. J. Drouin, Montréal, Canada). The PCR fragment was subcloned into the vector pCRôII. HindIII and T7 made the antisense probe, and XhoI with Sp6 polymerase produced the sense probe for POMC. The riboprobes were synthesized in vitro with 1 µg linearized POMC cDNA, 1 x digoxigenin RNA labeling mixture (Roche Diagnostics, Meylan, France), RNA polymerase, and 1x transcription buffer. The mixture was incubated at 37 C for 2 h. Residual DNA was digested with deoxyribonuclease. The probes were diluted 1:1000 with hybridization buffer for dual-labeling in situ hybridization.

Dual-labeling in situ hybridization
Sections were removed from storage at -80 C and placed into 0.1 M glycine-0.2 M Tris-HCl (pH 7.4) for 10 min before treatment with proteinase K (1 mg/ml in 100 mM Tris [pH 8.0] and 50 mM EDTA) for 15 min at 37 C. Slides were then immersed in 4% paraformaldehyde/0.1 M phosphate buffer for 15 min and treated with 0.1 M triethanolamine (pH 8.0) for 10 min, followed with 0.25% acetic anhydride for 10 min. The sections were dehydrated in graded concentrations of ethanol and hybridized in a 55-C oven overnight in diluted probes/hybridization buffer containing 50% formamide, 10% dextran sulfate, 0.3 M NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1 x Denhardt’s solution, 0.5 mg/ml Escherichia coli transfer RNA, 100 mM DTT, and 1% salmon sperm DNA. Four sets of two adjacent sections from each animal were simultaneously hybridized with the digoxigenin-labeled cRNA probes for POMC and with the radiolabeled cRNA probes for GalR1 or GalR2. The slides were washed twice with 4 x SSC for 30 min and 10 mM DTT for 1 h, in 0.3 M NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, and 50% formamide for 30 min. After treatment with ribonuclease A (20 µg/ml in 0.1 M Tris [pH 8.0], 0.5 M NaCl, and 0.5 M EDTA) for 30 min at 37 C, the sections were rinsed in 2 x SSC for 15 min at 60 C and 0.1x SSC for 15 min at 60 C. The sections were then washed in buffer 1 (100 mM Tris-HCl and 150 mM NaCl [pH 7.4]) and incubated for 30 min in blocking buffer (1% Boehringer blocking agent in buffer 1). The sections were incubated for 4 h in buffer 1 containing antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Roche Diagnostics) diluted 1:250, 1% normal sheep serum, and 2.4 mg levamisole in 10 ml buffer 1. After rinsing for 10 min in buffer 1 and 10 min in buffer 2 (100 mM Tris-HCl, 50 mM MgCl_2, and 100 mM NaCl [pH 9.5]), the sections were incubated in the chromogen solution (buffer 2 containing tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate). The reaction was stopped after 3 h by rinsing twice for 15 min in TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA). The slides were dehydrated in 70% ethanol in ammonium acetate and 100% ethanol, and dipped in K5 emulsion (Ilford, Saint-Priest, France). All sections were developed after a 20-day exposure.

Controls
The controls for specificity included incubation of the sections with [³⁵S]- and digoxigenin-labeled sense probes, pretreatment with ribonuclease, and coincubation with a 100-fold excess of unlabeled antisense probe. No significant labeling was observed on control sections.

Rostro-caudal subdivision of the arcuate nucleus
Using the rat brain atlas of Paxinos and Watson (27) as an anatomical guide, the arcuate nucleus was divided into four areas of approximately equal length in the rostral-caudal plane, as commonly used by some authors (2, 8, 28, 29, 30). Area A began rostrally at the retrochiasmatic area, where the first POMC-labeled cells are found, and extended caudally to the elongation of the third ventricle. Area B extended caudally to the onset of the dorsomedial nucleus of the hypothalamus (DM). Area C extended caudally through the DM to the beginning of the infundibular stalk. Area D began with the most caudal portion of the DM and extended caudally to the end of the arcuate nucleus and/or the disappearance of POMC-labeled neurons.

Quantitative analysis
Four tissue sections per rostro-caudal area from each animal were analyzed (16 slices per animal) for each receptor subtype. For quantitative analysis of autoradiographic grain density, a computer-assisted quantitative system (Densirag computerized program; Biocom, Les Ulis, France) interfaced to an Axiophot microscope (60x epiillumination dark-field objective; Carl Zeiss, Göttingen, Germany) was used. During a first analysis, the grain density corresponding to GalR1 or GalR2 mRNA levels was quantified in non-POMC cells. Cells were identified as labeled with the GalR1 or GalR2 probe if the silver grain optical density over the perikaryon was at least three times higher than the background. During a second analysis, POMC neurons were analyzed for the expression of GalR1 or GalR2 mRNA. POMC mRNA-expressing cells were first isolated under bright-field illumination, and the density of grains atop all POMC neurons was determined. Two types of analysis were applied, giving complementary information. The first approach consisted of counting the grain density over all POMC neurons of each area and calculating the labeling ratio in each neuron. The labeling ratio was defined as the ratio of the total grain density over the POMC neuron to the background measured in a cell-sized region nearby (density of silver grains caused by hybridization/mean background density of silver grains). The data were expressed as frequency distribution of GalR1 or GalR2 mRNA labeling in POMC neurons. This approach which avoided determining an arbitrary threshold might be more representative of the total number of double-labeled POMC neurons. This type of approach has been previously reported for analysis of double-labeled neurons (2). The second approach consisted of analyzing the POMC neurons having a number of silver grains over the perikaryon at least three times higher than the background. The mean cellular GalR1 or GalR2 mRNA content in POMC neurons was thus determined. In this approach, statistical analysis could be performed.

Statistical analysis
The mean density of grains per cell (±SEM) was calculated for each group of rats. Differences among areas of the arcuate nucleus were assessed with a one-way ANOVA, followed by a post hoc Bonferroni’s t test, to compare the levels of GalR1 or GalR2 receptor mRNA expression in non-POMC and in POMC neurons in the four areas of the arcuate nucleus. Differences between the areas were regarded as significant when P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The distribution of POMC, GalR1, GalR2, and POMC/GalR1 or POMC/GalR2 mRNA-expressing cells is presented on schematic drawings of frontal sections that are representative of the four rostro-caudal areas of the arcuate nucleus examined in the present study (Fig. 1).

View larger version (14K): [in this window] [in a new window]   Figure 1. Schematic drawings depicting the distribution of cells expressing GalR1 mRNA (left panels, open triangles) and GalR2 mRNA (right panels, open stars), POMC mRNA (circles), POMC and GalR1 (solid triangles), or POMC and GalR2 (solid stars) mRNAs, in the four rostro-caudal subdivisions (A–D) of the arcuate nucleus. A is the most rostral, and D is the most caudal subdivision of the nucleus. Each symbol represents two galanin receptor mRNA-expressing cells or two POMC mRNA-expressing cells. f, Fornix; ot, optic tract; s, supraoptic decussation; mt, mammillothalamic tractus. V, third ventricle; VM, ventromedial nucleus.

GalR1 mRNA-expressing cells
GalR1 mRNA-expressing cells were observed throughout the rostro-caudal extent of the arcuate nucleus. GalR1 mRNA-expressing cells were distributed in all subdivisions of the nucleus, with a preferential distribution in the ventrolateral subdivision, as previously described (22). In intact animals, the mean number of GalR1 mRNA-expressing cells did not exhibit significant variations (P > 0.05) across the four rostro-caudal areas of the nucleus (Table 1). The levels of GalR1 mRNA in arcuate cells varied across the four rostro-caudal areas, the highest levels being observed in the caudal parts of the nucleus (P < 0.05) (Table 1).

View larger version (14K): [in this window] [in a new window]   Figure 2. Mean number of arcuate cells expressing GalR1 mRNA (black columns) or GalR2 mRNA (white columns) per hemisection (A) and relative amount of GalR1 mRNA (black columns) or GalR2 mRNA (white columns) (B), as reflected by density of grains per cell, in intact, castrated, and testosterone-replaced male rats. The values are given as the mean ± SEM. Significant differences (P < 0.05) among the average values are noted as a vs. b and c vs. d (statistical analysis with the Bonferroni’s test).

View larger version (23K): [in this window] [in a new window]   Figure 3. Frequency distribution of GalR1 mRNA labeling in POMC neurons throughout the four rostro-caudal subdivisions of the arcuate nucleus, in intact and castrated animals (A) and castrated and testosterone-treated (T-replaced) animals (B). Area A is the most rostral, and area D is the most caudal subdivision of the nucleus. In all areas of the nucleus, castration shifted the distribution of POMC neurons containing GalR1 mRNA toward lower labeling ratio. Only the POMC neurons presenting a labeling ratio 2 (i.e. a specific labeling) were shown in this figure.

View larger version (20K): [in this window] [in a new window]   Figure 4. Relative amounts of GalR1 mRNA in POMC neurons, as reflected by density of grains per cell, throughout the four rostro-caudal subdivisions (A–D) of the arcuate nucleus in intact, castrated, and testosterone-replaced male rats. The values are the mean ± SEM. Significant differences (P < 0.05) among the average values for each area are noted as a vs. b (statistical analysis with the Bonferroni’s test).

View this table: [in this window] [in a new window]   Table 3. Total number of POMC neurons examined for the presence of GalR1 mRNA labeling in Fig. 3

View larger version (91K): [in this window] [in a new window]   Figure 5. Bright-field (A) and dark-field (B) photomicrographs of a representative section of the arcuate nucleus showing expression of GalR1 mRNA (silver grains) in POMC neurons (dark precipitate) in the central subdivision of the arcuate nucleus. Arrows point to neurons that are double-labeled for POMC and GalR1 mRNAs. Scale bars, 55 µm.

View larger version (85K): [in this window] [in a new window]   Figure 6. Bright-field photomicrographs of representative sections of the arcuate nucleus from intact (A and C) or castrated rats (B and D), showing POMC neurons (precipitate) expressing GalR1 (A and B) or GalR2 (C and D) galanin receptor mRNA (silvers grains). Note the lower level of expression of galanin receptor mRNAs in POMC neurons in castrated animals (B, D), compared with intact animals (A, C). Scale bar, 10 µm.

View larger version (20K): [in this window] [in a new window]   Figure 7. Frequency distribution of GalR2 mRNA labeling in POMC neurons throughout the four rostro-caudal subdivisions (areas A–D) of the arcuate nucleus, in intact and castrated animals (A) and castrated and testosterone-treated animals (B). In the areas A, B, and D of the nucleus, castration shifted the distribution of POMC neurons containing GalR2 mRNA toward lower labeling ratio. Only the POMC neurons presenting a labeling ratio 2 (i.e. a specific labeling) were shown in this figure.

View larger version (18K): [in this window] [in a new window]   Figure 8. Relative amounts of GalR2 mRNA in POMC neurons, as reflected by density of grains per cell, throughout the four rostro-caudal subdivisions (A–D) of the arcuate nucleus in intact, castrated, and testosterone-replaced male rats. The values are the mean ± SEM. Significant differences (P < 0.05) among the average values for each area are noted as a vs. b (statistical analysis with the Bonferroni’s test).

View this table: [in this window] [in a new window]   Table 4. Total number of POMC neurons examined for the presence of GalR2 mRNA labeling in Fig. 7

View larger version (87K): [in this window] [in a new window]   Figure 9. Bright-field (A) and dark-field (B) photomicrographs of a representative section of the rostral subdivision (area A) of the arcuate nucleus where a double-labeling in situ hybridization technique with riboprobes complementary to POMC (dark precipitate) and GalR2 mRNA (silver grains) was performed. Many POMC neurons expressed GalR2 mRNA (arrows) in this area. Scale bars, 55 µm.

View larger version (84K): [in this window] [in a new window]   Figure 10. Bright-field (A) and dark-field (B) photomicrographs of a representative section of the caudal subdivision (area D) of the arcuate nucleus where a double-labeling in situ hybridization technique with riboprobes complementary to POMC (dark precipitate) and GalR2 mRNA (silver grains) was performed. In this area, few POMC neurons expressed GalR2 mRNA (arrow), whereas numerous non-POMC cells expressed GalR2 mRNA. Scale bars, 55 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present report describes the occurrence of GalR1 and GalR2 galanin receptor mRNAs in POMC neurons of the arcuate nucleus of male rats and demonstrates that the expression of the GalR1 and GalR2 genes is regulated by androgens. The arcuate nucleus has been divided into four rostro-caudal subdivisions, and each area has been analyzed separately. This partitioning generally facilitates quantitative comparisons between animals (33). It was also based on previous reports indicating that the POMC populations of the arcuate nucleus are heterogeneous, in terms of projections (2) and sensibility to hormonal treatments (2, 8, 28, 29, 30).

As previously reported (22, 24), GalR1 and GalR2 mRNA are expressed in numerous cells of the arcuate nucleus, indicating that a large number of the different cell types present in the nucleus might be able to express galanin receptor mRNAs. Interestingly, the distribution of GalR2 mRNA-expressing cells does not completely overlap with that of GalR1 mRNA-expressing cells, indicating that some cells selectively express either GalR1 or GalR2 mRNA. From a technical point of view, this observation confirms the absence of cross-reactivity between the two riboprobes, which is also supported by the fact that some GalR1 mRNA-expressing cells were observed in hypothalamic nuclei that did not express GalR2 mRNA, e.g. the ventromedial nucleus.

Examination of dual-labeled neurons revealed that a subpopulation of POMC neurons expresses the GalR1 and/or GalR2 subtypes of galanin receptor mRNAs, and POMC/GalR1- or POMC/GalR2-labeled neurons were not preferentially distributed in a locoregional subdivision of the arcuate nucleus. These observations strongly suggest that galanin can directly regulate the activity of POMC neurons via at least two receptor subtypes, GalR1 and GalR2. These results raise the question of the localization of the action of galanin on POMC neurons: cell body or nerve terminals? Actually, the presence of receptor mRNAs does not automatically imply the existence of functional galanin receptor proteins at the level of the cell body. However, a high density of galanin binding sites has been detected in the arcuate nucleus (25), suggesting that POMC neurons may bear galanin receptor proteins on their cell body membranes. It has also been shown that galanin-immunoreactive fibers make synaptic contacts with ß-END cell bodies (13). These nerve terminals may originate from intrinsic neurons, because destruction of the arcuate nucleus with monosodium glutamate treatment results in a significant decrease in galanin immunoreactive fibers (34), and because deafferentation of the arcuate nucleus does not alter the staining for galanin in the nucleus (13). Altogether, these data suggest that an arcuate galaninergic pathway might directly modulate the activity of POMC neurons at their cell body level.

The number of POMC neurons expressing GalR1 or GalR2 galanin receptor messenger exhibited a gradient across the four rostro-caudal subdivisions of the arcuate nucleus, with more numerous double-labeled neurons in the most rostral areas. Because the most rostral POMC neurons project preferentially toward the preoptic area where the GnRH cell bodies are located (2), our results strongly suggest that POMC neurons expressing galanin receptor mRNAs are preferentially involved in the regulation of the activity of GnRH neurons. Concurrently, because both POMC-derived peptides, such as ß-endorphin and -MSH, and galanin are also known to regulate feeding behavior (3), a direct regulatory influence of galanin on POMC neurons may also play a role in the control of food intake.

The number of POMC neurons detected by the digoxigenin-labeling method was not affected by castration or testosterone replacement, indicating that androgens do not regulate the number of POMC-expressing neurons. Conversely, in general, castration decreased the amounts of GalR1 or GalR2 mRNA expressed in POMC neurons, and these effects were abolished by testosterone replacement in all rostro-caudal subdivisions of the arcuate nucleus. These data show that androgens stimulate the expression of GalR1 and GalR2 genes, as already reported for glutamate receptors in hypothalamic neurons (35). Consequently, it seems that testosterone has a stimulatory effect on galanin receptor mRNA expression in POMC neurons. Because both the POMC neuronal system and testosterone are strongly implicated in the regulation of the GnRH axis, it seems that testosterone can modulate the GnRH neuroendocrine axis, in part, via the regulation of galanin receptor expression in POMC neurons. The observation that castration decreased the expression levels of galanin receptor mRNAs in POMC neurons is consistent with previous studies that showed that POMC neurons are regulated by gonadal steroids (2, 7, 8, 9). However, the effects of testosterone deprivation on galanin receptor mRNA expression are not restricted to POMC neurons, because castration also decreased the number of non-POMC cells expressing galanin receptors and/or the levels of galanin receptor message in non-POMC cells of the arcuate nucleus. In agreement with this finding, it has been observed that androgen receptor mRNA-expressing cells are widely distributed in the arcuate nucleus (36).

To our knowledge, this is the first report describing the effects of castration on the expression of GalR1 and GalR2 mRNA in the arcuate nucleus. Up to now, few studies have been conducted to investigate the effect of testosterone on the expression of galanin receptors. It has been found that, in male rat, the density of galanin binding sites increases during puberty in some brain areas, in correlation with the variations of sex steroid hormone concentration (25). We have recently shown that gonadal steroids regulate the expression of GalR1 mRNA in the preoptic area of the female rat (37). It has also been reported that male gonadal steroids enhance the expression of the galanin precursor gene (38, 39, 40). Thus, male gonadal steroids seem to modulate components of the galaninergic pathway, including the expression of galanin receptors in target cells.

The fact that POMC neurons express GalR1 or GalR2 mRNA suggests that galanin may exert complex effects on POMC neuron activity, depending, for instance, on the relative affinity of each receptor subtype. In support of this hypothesis, it has been shown that GalR1 is coupled with a G_iß protein, whereas GalR2 is coupled with a G_o or G_q/G₁₁ protein (41), indicating that galanin may have either a stimulatory or an inhibitory influence, according to the receptor subtype which is activated.

In summary, this study has demonstrated that, in the arcuate nucleus, galanin might directly modulate the activity of POMC neurons via an action on GalR1 or GalR2 galanin receptors. Because these receptors are preferentially expressed by the most rostral POMC neurons, known to project predominantly to the preoptic area, and since galanin receptor expression in POMC neurons is affected by testosterone, we can speculate that POMC neurons, which express galanin receptor genes, may play a key role in the regulation of the activity of GnRH neurons.

	Acknowledgments

 
The authors thank Mrs. G. Mortreux for excellent technical assistance with the RIAs.

	Footnotes

 
¹ This work was supported by the Lille-Amiero-Rouen-Caen Neuroscience Network and the University of Lille 2.

Received September 24, 1999.

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