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Endocrinology Vol. 142, No. 4 1626-1634
Copyright © 2001 by The Endocrine Society

ARTICLES

Distribution of Galanin-Like Peptide in the Rat Brain

Yoshihiro Takatsu, Hirokazu Matsumoto, Tetsuya Ohtaki, Satoshi Kumano, Chieko Kitada, Haruo Onda, Osamu Nishimura and Masahiko Fujino

Discovery Research Laboratories I, Pharmaceutical Discovery Research Division, Takeda Chemical Industries Co., Ltd., Wadai 10, Tsukuba, Ibaraki 300-4293, Japan

Address all correspondence and requests for reprints to: Dr. Tetsuya Ohtaki, Discovery Research Laboratories I, Pharmaceutical Discovery Research Division, Takeda Chemical Industries Co., Ltd., Wadai 10, Tsukuba, Ibaraki 300-4293, Japan. E-mail: ohtaki95tetsuya{at}takeda.co.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Galanin-like peptide (GALP) is a novel galanin-like peptide isolated from the porcine hypothalamus. To determine the distribution of GALP in the rat brain, we performed immunohistochemical studies using a monoclonal antibody toward the N-terminal sequence of GALP. GALP-immunoreactive neuronal cell bodies were observed only in the arcuate nucleus (Arc), which was further confirmed by in situ hybridization studies using digoxigenin-labeled antisense GALP riboprobe. Additional immunostained cells were found in the median eminence and infundibular stalk. The GALP neurons found in the Arc were further characterized by double label immunohistochemistry. More than 85% of the GALP neurons were immunostained with leptin receptor antibody. However, the GALP neurons and fibers found in the Arc were not labeled with {alpha}-MSH, somatostatin, neuropeptide Y, agouti-related protein, or galanin antibodies, indicating that GALP is found in neurons other than these known Arc neurons. Dense staining of GALP-containing fibers was found in the anterior parvicellular part of the paraventricular hypothalamic nucleus, in the ventral part of the lateral septal nucleus, and in the bed nucleus of the stria terminalis. Relatively dense staining was noted in the medial preoptic area (MPA), and weak staining was noted in the periventricular hypothalamic nucleus. Detailed double labeling studies in the paraventricular hypothalamic nucleus demonstrated that GALP-containing fibers converged in a more rostral direction than did agouti-related protein-containing fibers. Furthermore, GALP-immunoreactive fibers were in close apposition with GnRH-immunoreactive fibers in the MPA and bed nucleus of the stria terminalis, and about 6% of GnRH-positive neurons in the MPA showed close contact with the GALP-immunoreactive fibers. Our findings indicate that GALP neurons, as leptin-responsive neurons, may participate in the regulation of feeding behavior and/or reproductive functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GALANIN, WHICH WAS originally isolated from the porcine intestine (1), is a widely distributed neuropeptide (2, 3). In the central nervous system, galanin is implicated in various neuronal regulatory functions, including modulation of depression, reproduction, and feeding behavior (3, 4, 5, 6). These galanin functions are believed to be mediated by at least three recently cloned galanin receptor subtypes, GALR1 (7, 8), GALR2 (9, 10), and GALR3 (11, 12), which are all known to be distributed in the brain (13, 14, 15). However, galanin is the only known ligand of these receptors.

We recently discovered a novel galanin-like peptide (GALP) in the porcine hypothalamus with 60 amino acid residues (16). The amino acid sequence of GALP-(9–21) is identical to that of galanin-(1–13). GALP shows high affinity for the GALR2 receptor and a relatively low affinity for the GALR1 receptor, indicating that GALP is more selective for the GALR2 receptor than is galanin. However, unlike galanin, for which the cellular localization and receptors are relatively well known, the distribution of GALP is too poorly understood to deduce its physiological functions.

In the initial study using RT-PCR1, we found a considerable amount of GALP transcript in the dissected hypothalamic tissues from rat brains and relatively little in the cerebral cortex, hippocampus, striatum, mesencephalon, or medulla oblongata. In the peripheral tissues, GALP was expressed only in the pituitary and testis. Recently, it was shown by in situ hybridization studies that GALP expression is restricted to the arcuate hypothalamic nucleus (Arc) in the rat brain (17, 18). Furthermore, Juréus et al. reported that expression of GALP in the Arc is under the control of leptin (17). Since their characterization, GALP has been studied as a possible leptin-regulated peptide, and a more complete understanding of the GALP neuronal system is actively being pursued.

In the present study we performed histochemical analysis to determine the distribution of GALP neurons and neural fibers in the rat brain using immunostaining and in situ hybridization techniques. Moreover, GALP neurons were characterized by double label immunohistochemistry for leptin receptor and several hypothalamic peptides. We considered the physiological significance of GALP based on its observed distribution.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Wistar rats (230–300 g; Charles River Laboratories, Inc., Wilmington, MA) were housed in a light (12-h light, 12-h dark cycle; lights on at 0700 h)- and temperature (23 ± 2 C)-controlled environment. Food and water were provided ad libitum. The rats were anesthetized with an ip injection of sodium pentobarbital (75 mg/kg; Dainippon Pharmaceutical, Osaka, Japan) and perfused transcardially with 100 ml 2% sodium nitrite in saline followed by 400 ml Mildform 10 N (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The brain was removed, blocked, and immersed in the same fixative (for 2 h for the immunohistochemistry or for 8 h for the in situ hybridization). They were immersed in 12%, 15%, and 18% sucrose for 4, 8, and 24 h, respectively, at 4 C, then rapidly frozen on dry ice. Coronal sections (15 µm for immunohistochemistry or 8 µm for in situ hybridization) were cut on a cryostat at -17 C and mounted onto SuperFrost slide glasses (Matsunami Glass Ind., Ltd., Osaka, Japan).

Preparation of monoclonal antibodies
For immunogens, keyhole limpet hemocyanin (60 nmol) was maleimidated with N-({gamma}-maleimidobutyryloxy) succinimide and then conjugated with [Cys10]GALP-(1–10)NH2 (3.7 µmol). The immunogen (40 µg/mouse) together with complete or incomplete Freund’s adjuvant were sc injected into BALB/c mice (female, 8 weeks old) at 3-week intervals. Four days after iv injection of immunogen (100 µg), spleen cells were fused with mouse myeloma cells P3-X63Ag8-U1 as described previously (19). Monoclonal antibody GR2–1N (IgG1, {kappa}) was selected and purified from ascites fluid with a protein A-immobilized column (IPA-300, Repligen, Cambridge, MA).

Single labeling immunohistochemistry
Sections were treated with a 1% H2O2-methanol solution for 15 min, and then washed in PBS containing 0.3% Triton X-100. After preincubation in 10% normal horse serum for 30 min, sections were incubated in GR2–1N mouse monoclonal antibody at 5 µg/ml overnight at 4 C. The sections were washed in PBS three times and incubated in biotinylated antimouse IgG (1:200; Vector Laboratories, Inc., Burlingame, CA) for 30 min at room temperature. After three washes in PBS, the sections were incubated in avidin-biotin-peroxidase complex (ABC Elite, Vector Laboratories, Inc.) for 30 min at room temperature, followed by a 10-min incubation in tyramide signal amplification solution according to the manufacturer’s instructions (TSA-Indirect kit, NEN Life Science Products, Boston, MA). The immunolabeling was visualized with a mixture of diaminobenzidine and H2O2 in 0.05 M Tris-buffered saline solution. The sections were dehydrated and mounted with Canada Balsam (Sigma). In preadsorption studies, GR2–1N antibody was incubated with 2 x 10-5 M synthetic porcine GALP or rat galanin for 1 h at room temperature.

Double labeling immunohistochemistry
After preincubation in 10% normal donkey serum for 30 min, sections were incubated with a first primary antibody of polyclonal goat antibody to the peptide corresponding to amino acids 877–894 mapping at the carboxyl-terminus of leptin receptor (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal goat antibody to CRF (1:100; Santa Cruz Biotechnology, Inc.), polyclonal rabbit antiserum to rat galanin-(15–29) (1:200; Yanaihara Institute, Inc., Fujinomiya, Japan), neuropeptide Y (NPY; 1:200; Peninsula Laboratories, Inc., Belmont, CA), {alpha}-MSH (1:1,000; Peninsula Laboratories, Inc.), agouti-related protein (AGRP; 1:10,000; Phoenix Pharmaceuticals, Inc., Mountain View, CA), LHRH (1:500; Cosmobio, Inc., Tokyo, Japan), or somatostatin (1:1,000; Yanaihara Institute, Inc.) for 3 days at 4 C. The sections were treated with biotinylated donkey antigoat or antirabbit IgG (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and incubated with a mouse monoclonal antibody against GALP at 5 µg/ml overnight at 4 C. The sections were then incubated in peroxidase- conjugated antimouse IgG (1:500; Jackson ImmunoResearch Laboratories, Inc.) for 2 h at room temperature, followed by a 10-min incubation in tyramide signal amplification solution according to the manufacturer’s instructions [TSA-direct (green) kit, NEN Life Science Products]. Biotin labeling was visualized with Texas Red dye-conjugated streptavidin (1:100; Jackson ImmunoResearch Laboratories, Inc.). The sections were mounted using Vectashield (Vector Laboratories, Inc.) and examined on a Leica Corp. TCS confocal microscope (Rockleigh, NJ).

Probe labeling for in situ hybridization
Plasmid DNA containing 700 bp rat GALP complementary DNA (16) was linearized by digestion with EcoRI or XhoI. Digoxigenin-labeled probes were generated by labeling with digoxigenin-11-UTP (Roche Molecular Biochemicals, Indianapolis, IN) using T7 or T3 RNA polymerase (Nippon Gene Co., Ltd., Japan) in a 20-µl transcription mixture containing 1.8 µg linearized plasmid; 2 µl 10 x transcription buffer; 1 µl 0.2 mM dithiothreitol; 1 µl of 10-mM stocks of ATP, GTP, and CTP; 6.5 µl 1 mM UTP; 3.5 µl of 10-mM stocks of digoxigenin-11-UTP; and 1 µl ribonuclease inhibitor (Nippon Gene Co., Ltd., Osaka, Japan). The transcription mixtures were incubated for 1 h at 37 C. The labeled probes were hydrolyzed to approximately 150-base fragments.

In situ hybridization
In situ hybridization was performed according to the manufacturer’s instructions (ISHR Kit, Nippon Gene Co., Ltd.). In brief, sections were dehydrated in ethanol, delipidated in chloroform, and rehydrated in graded concentrations of ethanol. The slides were incubated with proteinase K (5 µg/ml) for 10 min at 37 C before treatment with glycine (2 mg/ml), then treated with 0.1 M triethanolamine for 5 min, followed by 0.25% acetic anhydrate for 15 min. The sections were hybridized in an oven at 42 C for 16 h in diluted digoxigenin-labeled probes (1 µg/ml)/hybridization buffer containing 50% formamide, 2 x SSC (standard saline citrate), 1 µg/µl transfer RNA, 1 µg/µl salmon sperm DNA, 1 µg/µl BSA, and 10% dextran sulfate. The slides were washed three times with 4 x SSC for 20 min each time at 42 C. After treatment with ribonuclease A (20 µg/ml) for 30 min at 37 C, the sections were rinsed in 0.1 x SSC for 20 min at 42 C. After three washes in 0.05% Tween-20, 0.1 M Tris-HCl, and 0.15 M NaCl, the sections were preincubated in 0.5% blocking reagent, 0.1 M Tris-HCl, and 0.15 M NaCl for 30 min and incubated with peroxidase-conjugated antidigoxigenin Fab for 30 min (1:200; Roche Molecular Biochemicals). The sections were washed in 0.05% Tween-20, 0.1 M Tris-HCl, and 0.15 M NaCl three times and incubated in biotinylated tyramide for 20 min (TSA-Indirect kit, NEN Life Science Products). Biotin labeling was visualized with Texas Red dye-conjugated streptavidin (1:100; Jackson ImmunoResearch Laboratories, Inc.) and examined on a Leica Corp. TCS confocal microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of GALP-positive cell bodies in the Arc
Immunohistochemistry was performed with anti-GALP monoclonal antibody (GR2–1N clone) toward the rat GALP-(1–9) sequence. This sequence is specific to GALP and is not shared with galanin; therefore, the antibody shows no cross-reactivity for galanin. Immunohistochemical staining revealed that GALP-positive neuronal cell bodies are localized in the Arc and are especially dense in the medial posterior part (ArcMP; Fig. 1Go, A and B). The specificity of immunostaining was confirmed by preadsorption studies in which the immunostaining was blocked with GALP (Fig. 1CGo), but not with galanin (Fig. 1DGo). This result was supported by in situ hybridization studies using digoxigenin-labeled GALP antisense riboprobe transcribed from the full-length rat GALP complementary DNA (Fig. 1Go, E and F). No immunostained neural cell bodies were found in any other hypothalamic nuclei or other brain loci, but specifically stained cell bodies were found in the median eminence (ME; Fig. 2Go, A and B) and infundibular stalk (InfS; Fig. 2Go, E and F).



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  		Figure 1. Photomicrographs showing GALP- immunoreactive cell bodies (A and B) and GALP mRNA-expressing cells in the ArcMP (E and F). A, GALP-immunoreactive neural cells found only in the Arc, especially in its caudal part. B, High power enlargement of A. Arrows indicate GALP-immunoreactive perikarya. C, Preabsorption with 2 x 10-5 M synthetic porcine GALP blocked the staining reaction. D, Preabsorption with 2 x 10-5 M rat galanin did not block the staining reaction. E, GALP mRNA-expressing cell bodies were found in the Arc as immunohistochemical staining (compared with A). F, High magnification of E. Arrows indicate GALP mRNA-expressing perikarya. G, A negative control with digoxigenin- labeled GALP sense riboprobe was evident as an absence of staining. MRe, Mamillary recess of the third ventricle. Magnification: A, C, D, E, G, x40; B and F, x150. All micrographs were obtained from coronal sections.

 


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  		Figure 2. Photomicrographs showing GALP-immunoreactive cell bodies in the median eminence and infundibular stalk. A, Almost no immunoreactive GALP fibers were observed in the external zone of the ME, but occasionally immunoreactive cells were found in the ME. B, High power enlargement of A. Arrowheads indicate GALP-immunoreactive cell bodies. C, Preabsorption with 2 x 10-5 M synthetic porcine GALP blocked the staining reaction. D, High power enlargement of C. E, Immunoreactive cells were found in the InfS. F, High power enlargement of E. Arrowheads indicate GALP-immunoreactive cell bodies. G, Preabsorption with 2 x 10-5 M synthetic porcine GALP blocked the staining reaction. H, High power enlargement of G. 3V, Third ventricle. Magnification: A, C, E, and G, x40; B, D, E, and F, x80. All micrographs were obtained from coronal sections.

 
The Arc is one of the hypothalamic nuclei showing the highest concentration of immunoreactivity for leptin receptor (Ob-R) (20, 21, 22, 23). Several neurons that reside in the Arc including POMC/cocaine- and amphetamine-regulated transcript (CART), NPY/agouti-related protein (AGRP), and galanin neurons, were shown to coexpress Ob-R (22, 24, 25, 26). Then, we performed confocal microscopic analyses for double label immunohistochemistry with anti-Ob-R antibody to characterize GALP neurons in the Arc. As shown in Fig. 3Go, Ob-R-positive cell bodies were distributed throughout the Arc, whereas GALP-positive cell bodies were localized in the periventricular area. Approximately 88% (44 of 50) of the GALP-positive neurons were double labeled with anti-Ob-R antibody. The GALP-positive neurons accounted for less than 5% of the Ob-R- positive cells found in the ArcMP.



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  		Figure 3. Immunofluorescence photomicrographs of coronal section of the Arc. A, GALP-positive neurons. B, Leptin receptor (Ob-R)-like immunoreactivity. C, Overlay of GALP (green) and Ob-R (red) indicated that nearly all of the GALP-positive neurons were stained with antileptin receptor (Ob-R) antibody (indicated by arrows), but not vice versa (indicated by arrowheads). 3V, Third ventricle. Magnification, x150.

 
Next, we tested whether GALP is expressed in known Ob-R-positive neurons or in other distinct neurons. No GALP cell bodies were double labeled with {alpha}-MSH (a mature peptide processed from POMC) antibodies (Fig. 4Go, A and B). The POMC ({alpha}-MSH) neurons were distributed in the lateral part of the Arc, which is in contrast to the distribution of the GALP-positive neurons (Fig. 4AGo). Although nerve fibers containing NPY, AGRP, and galanin were observed in the Arc, the cell bodies of these neurons were not obviously stained using immunohistochemical methods. Most of the GALP-immunoreactive fibers were not double labeled with NPY antibody (Fig. 4EGo), AGRP antibody (data not shown), or galanin antibody (Fig. 4FGo) in the Arc, suggesting that GALP-positive neurons are distinct from the known neural populations stained by these antibodies. As Ram and Gundlach recently reported that an area of somatostatin messenger RNA (mRNA) expression overlaps the distribution of GALP mRNA in the posterior and ventromedial regions of the Arc (18), we tested whether GALP is coexpressed in the somatostatin neurons. As shown in Fig. 4Go, C and D, somatostatin-positive cells were found very close to GALP-positive cells; however, no GALP-positive cells were double labeled with antisomatostatin antibody.



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  		Figure 4. Immunofluorescence photomicrographs of coronal section of the Arc. A, GALP immunoreactivity (green) and {alpha}-MSH immunoreactivity (red). B, High magnification of the Arc. The GALP-positive neurons (arrows) were localized in the medial part in contrast to the {alpha}-MSH neurons (arrowheads), which were localized in the lateral part. No GALP cell bodies were double labeled with {alpha}-MSH antibodies (B). C, GALP immunoreactivity (green) and somatostatin-immunoreactivity (red). D, High magnification of the Arc. No GALP cell bodies (arrows) coincided with somatostatin-positive cells (arrowheads; D). E and F, GALP immunoreactivity (green) and NPY (E; red) or galanin immunoreactivity (F; red). Most GALP nerve fibers were negative against NPY antibody (E) or galanin antibody (F) and vice versa. 3V, Third ventricle. Magnification: A, C, E, and F, x40; B and D, x150.

 
Distribution of GALP-containing nerve fibers in the hypothalamic paraventricular nucleus (PVH)
The PVH contains a neural mechanism that affects feeding behavior (27). Many kinds of peptidergic neurons regulated by leptin project to the PVH from the Arc (26, 28, 29), and several peptides that affect feeding and body weight, such as CRF and galanin, are localized in the PVH (27, 30). Therefore, we made a detailed analysis of the distribution of GALP-immunoreactive fibers in the PVH. Relatively dense staining of GALP-containing nerve fibers was observed in the parvicellular division of the PVH (Fig. 5Go, A and B). As summarized in Table 1Go, the rostro-caudal distribution of GALP-containing fibers in the PVH was obviously different from that of other neuronal fibers and cell bodies. GALP-containing fibers were observed most abundantly in the anterior parvicellular part of the PVH (PaAP), whereas AGRP- containing fibers (Fig. 5Go, D–F), galanin-containing fibers (data not shown), and CRF neurons (Fig. 5Go, G–I) were distributed abundantly in the medial part of the PVH, but were seen only occasionally in the anterior parvicellular part (Fig. 5Go). We observed no remarkable projection of GALP-containing fibers into CRF neurons. The lateral hypothalamus is another important area for feeding behavior (27) and contains many neural terminals of the leptin-regulated Arc neurons (26, 31, 32). Nevertheless, no GALP-immunoreactive fibers were found in the lateral hypothalamus (data not shown).



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  		Figure 5. Photomicrographs showing GALP-immunoreactive fibers in the PVH. A, Abundant GALP-immunoreactive neural fibers in the PaAP. B, High power enlargement of A. C, Paucity of GALP-immunoreactive fibers in the paraventricular hypothalamic nucleus, medial parvicellular (PaMP; caudal portion). D–I, GALP immunoreactivity (green), AGRP immunoreactivity (D–F; red), and CRF immunoreactivity (G–I; red) found in the PaAP (D and G), in the rostral portion of PaMP (E and H), and in the medial portion of PaMP (F and I). The GALP-immunoreactive neural fibers (green) were relatively dense in the PaAP, but were relatively sparse in the rostral to medial portion of the PaMP, whereas AGRP and CRF immunoreactivity was light in the PaAP, but heavy in the rostral to medial portion of the PaMP. 3V, Third ventricle. Magnification: A and C, x20; D–I, x40. All micrographs were obtained from coronal sections.

 

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  		Table 1. Distribution of GALP immunoreactivity and comparison with immunoreactivities of galanin, NPY, AGRP, and CRF in the male rat hypothalamus

 
Distribution of GALP-containing nerve fibers in other brain regions
GALP-immunoreactive fibers were observed in several other brain regions. As shown in Fig. 6Go, the anterodorsal preoptic nucleus, the bed nucleus of the stria terminalis (BST) including the dorsal and ventral parts, and the ventral part of the lateral septal nucleus (LSV) showed dense GALP-containing fibers. It should be noted that the median preoptic nucleus (MnPO) was almost devoid of GALP-containing fibers (Fig. 6Go). The medial preoptic area (MPA) also showed relatively abundant GALP-containing fibers (Fig. 7Go, A and B). The periventricular hypothalamic nucleus (Pe) contained a small number of GALP-immunoreactive fibers (Fig. 7Go, C and D). As shown in Fig. 2Go, no remarkable staining of GALP-containing fibers was found in the external zone of the ME, suggesting that the GALP produced in the Arc is unlikely to serve as a hypophysiotropic hormone.



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  		Figure 6. Photomicrographs showing GALP-immunoreactive fibers in the area surrounding the anterior commissure. A, Anterodorsal preoptic nucleus and the dorsal portion of the BST (high power enlargement is shown in B) and the ventral part of the BST and lateral septal nucleus (high power enlargement is shown in C) contained abundant GALP-immunoreactive neural fibers. 3V, Third ventricle; ac, anterior commissure; f, fornix. Magnification: A, x20; B and C, x40. All micrographs were obtained from coronal sections.

 


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  		Figure 7. Photomicrographs showing GALP-immunoreactive fibers in the medial preoptic nucleus and the periventricular hypothalamic nucleus. A, Medial preoptic nucleus with relatively abundant GALP fibers. B, High power enlargement of A. C, Periventricular hypothalamic nucleus with a few GALP-immunoreactive fibers. D, High power enlargement of C. 3V, Third ventricle. Magnification: A and C, x20; B and D, x40. All micrographs were obtained from coronal sections.

 
As GnRH neurons are well known to localize in the MPA (33), we tested whether GALP-containing fibers have some neural contact with the GnRH neurons. As shown in Fig. 8Go, double label immunostaining revealed that GALP-containing fibers were in close contact with GnRH perikarya (Fig. 8Go, C and D) and dendrites (Fig. 8EGo) in the MPA, especially in the ventrolateral part. Similar contact with the perikarya was observed in about 6% (9 of 156) of the GnRH neurons counted in the MPA. Furthermore, GALP-immunoreactive fibers were often seen in close apposition with GnRH-immunoreactive fibers in the MPA and BST (Fig. 8BGo), and some of these fibers made contact or were twisted with GnRH-immunoreactive fibers (Fig. 8FGo). These observations suggest that the GALP neurons may provide direct input to the GnRH neurons in the MPA and may modulate the release of neurotransmitters from the nerve terminals of the GnRH-immunoreactive fibers.



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  		Figure 8. Immunofluorescence photomicrographs of coronal section of the MPA and BST. A and B, GALP (green) and GnRH (red) immunoreactivities were stained in the MPA (A) and BST (B). The arrow shows a GnRH-positive perikarya, which were in close contact with the GALP-immunoreactive fiber. C–F, The cross-contact of GALP-containing fibers to GnRH-positive perikarya (two examples are indicated in C and D) and dendrite (E) observed in the MPA and that to GnRH-positive fibers (F) observed in the BST. The cross-contact was confirmed after examining the individual image in a series of 0.7-µm optical sections. ox, Optic chiasm. Magnification: A and B, x40; C–F, x150. All micrographs were obtained from coronal sections.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The immunohistochemistry and in situ hybridization analyses in the present study show that GALP is specifically expressed in neural cell bodies located in the Arc. This is consistent with previous in situ hybridization studies (17, 18). The localization of GALP is markedly different from that of galanin, which is expressed in various hypothalamic nuclei, including the dorsomedial hypothalamic nucleus (DM), preoptic area, supraoptic nucleus, Arc, Pe, lateral hypothalamus, and PVH, as well as in other brain loci, such as the locus ceruleus (2, 3). On the other hand, we found GALP-containing fibers in the Arc, Pe, MPA, PVH, BST, and LSV. These neural fibers probably originate in the Arc, as it is the only nucleus to express GALP. We hypothesize that there are at least two major GALP neural pathways; one is from the Arc to the PVH, and the other is from the Arc to the MPA, BST, and LSV. Additional immunostained cells were found in the ME and InfS, which should be studied in further investigations. Juréus et al. also found evidence of GALP mRNA-containing cells in the ME and InfS (17). Very recently, Kerr et al. reported that GALP mRNA was detected in the rat pituitary posterior lobe, but not in the anterior or intermediate lobes (34). The result was schematically summarized in Fig. 9Go.



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  		Figure 9. Schematic drawings (A–H) showing the distribution of GALP-containing fibers (light lines) and cells (•) in the septum and hypothalamus according to the Paxinos and Watson rat brain atlas (51 ). Coordinates in mm from bregma: A, +0.2; B, -0.3; C, -0.8; D, -1.4; E, -1.8; F, -2.8; G, -3.6; and H, -4.3. Abbreviations used are according to the Paxinos and Watson rat brain atlas (51 ).

 
It has been reported that leptin deficiency during fasting decreases the number of GALP-expressing cells in the Arc, and that leptin administration in fasted rats increased GALP cell number beyond the level observed in rats fed a normal diet (17). As the double labeling immunohistochemistry used in the present study clearly demonstrates that the arcuate GALP neurons coexpress leptin receptors, it is conceivable that the GALP neurons are directly regulated by leptin. GALP neurons were further shown to be different from the known leptin receptor-expressing neurons localized in the Arc, such as the NPY/AGRP (22, 24), galanin (22), and POMC/CART (25, 26) neurons. The NPY/AGRP and POMC/CART neurons are well characterized leptin-responsive neurons; the orexigenic NPY/AGRP neurons are known to increase NPY mRNA transcription in leptin-deficient conditions (35, 36), and the anorectic POMC/CART neurons are known to increase POMC mRNA in response to elevated leptin levels (36, 37, 38). The GALP neurons are similar to the POMC/CART neurons in that both neurons are up-regulated by leptin (17), although there is a marked difference in the distribution of these two neural populations. The localization of GALP neurons, especially in the periventricular area of the Arc, is similar to that of the NPY/AGRP neurons (32, 39); however, the NPY/AGRP neurons are regulated by leptin in the opposite direction. As the profile of the GALP neurons is thus different from that of the NPY/AGRP and POMC/CART neurons, we propose that the GALP neurons constitute a novel leptin-responsive population localized in the periventricular area of the Arc.

The NPY/AGRP and POMC/CART neurons send neural projections to several overlapping regions such as the PVH, paraventricular thalamic nucleus, MPO, BST, and LS (40). This overlapped projection supports the hypothesis that AGRP serves as a locally delivered endogenous antagonist for the melanocortin receptor (41). Of these neural projections, the one to the PVH is presumed to be the most important pathway for regulating feeding behavior and energy expenditure (27, 28). Cowley et al. observed that {alpha}-MSH (i.e. POMC)-containing fibers converge in the vicinity of the NPY/AGRP-containing fibers in the medial PVH (28). In contrast to these closely related neurons with opposite leptin sensitivities and opposite effects on feeding behavior, the GALP-containing fibers were found in the more rostral part, i.e. in the PaAP nucleus. This slight difference suggests that the GALP neurons may have unique regulatory functions other than or in addition to the regulation of feeding behavior.

Another important physiological role of leptin is the regulation of reproductive functions. Leptin is known to reverse the food restriction-induced delay of puberty onset in female rats and infertility of genetically obese (ob/ob) mice (42, 43). However, the mechanisms of leptin actions on reproduction are not fully understood. Previous studies have revealed that one important mechanism is the control of pulsatile GnRH secretion in the hypothalamus. The administration of leptin antiserum into the lateral ventricle of rats caused a marked decrease in LH pulsatility compared with that in rats treated with normal rabbit serum (44). Furthermore, leptin administration to the male monkey or the ovariectomized female rat reversed the suppression of LH pulsatility during fasting (43, 45). As it has shown little coexpression of GnRH and Ob-R in the hypothalamus (22), leptin may induce GnRH secretion by acting transsynaptically (43). The NPY/AGRP and POMC/CART neurons are candidates for a signal transduction pathway that mediates leptin signals to the GnRH neuron. Indeed, the GnRH neurons in the MPA are known to have innervations from both NPY and POMC terminals, which are known to originate from the Arc (46, 47, 48). It is also known that intracerebroventricularly administered NPY reduces the secretion of LH in ovariectomized monkeys (49). However, there is no known mechanism that positively regulates GnRH neuronal activity under the regulation of leptin. In the present study we showed that the GnRH-positive neurons in the MPA were in close contact with GALP-immunoreactive fibers, suggesting that the GALP terminals form synapses with the GnRH neurons. As these GALP terminals probably originate from the Arc GALP neurons, we hypothesize that GALP mediates the regulatory activity of leptin. Considering that the GALP neurons are up-regulated by leptin in contrast to NPY, we expect that GALP may activate the GnRH neurons to LH secretion.

We also considered the distribution of the GALR2 receptor compared with that of GALP-containing fibers. Mitchell et al. found that GALR2 mRNA is expressed in various hypothalamic nuclei, such as the MPO, suprachiasmatic nucleus, Pe, PVH, Arc, DM, medial mammillary nucleus, and MnPO (14). In the present study we found GALP-containing fibers in some of those nuclei, including the MPA, Pe, PVH, and Arc, but we found very little in the DM, medial mammillary nucleus, and MnPO. In the hippocampus, intense labeling of GALR2 mRNA was reported in the dentate gyrus (50), where we found no staining of GALP-containing fibers. Conversely, we found many GALP-containing fibers in the LSV, which was reported to contain little GALR2 receptor mRNA. These regional differences between GALR2 mRNA and GALP immunoreactivity suggest the possibility that GALP does not serve as a physiological ligand of the GALR2 receptor, and the possible existence of a hitherto unknown GALP-specific receptor. Further investigations should be undertaken to characterize GALP-binding sites in the rat brain.

In summary, we determined the distribution of GALP neurons and GALP-containing fibers in the rat hypothalamus. The GALP neurons locate specifically in the Arc and coexpress leptin receptors. The GALP-containing fibers are distributed predominantly in the PVH, MPA, BST, and LSV and have cross-contact with the GnRH neurons in the MPA. Our findings indicate that GALP neurons may be directly regulated by leptin and participate in the regulation of feeding behavior and/or reproductive functions.


   Acknowledgments
 
The authors are grateful to Dr. K. Inoue at Saitama University for helpful discussions on brain histology and immunohistochemistry. We also thank Drs. S. Kobayashi, K. Miyazaki, and M. Asaoka at the University of Tsukuba for technical advice regarding double labeling immunohistochemistry.

Received September 13, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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