Expression of galanin receptor-1 (GALR1) in the rat trigeminal ganglia and molar teeth

Neuroscience Research 42 (2002) 197 /207 www.elsevier.com/locate/neures

Expression of galanin receptor-1 (GALR1) in the rat trigeminal ganglia and molar teeth Hironobu Suzuki a,b,*, Toshihiko Iwanaga c, Hiromasa Yoshie b, Jun Li d, ¨ lo Langel e, Takeyasu Maeda a Kaoru Yamabe d, Noboru Yanaihara d, U a

Department of Oral Biological Science, Division of Oral Anatomy, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan b Department of Oral Biological Science, Division of Periodontology, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan c Laboratory of Anatomy, Hokkaido University Graduate School of Veterinary Medicine, Sapporo, Japan d Yanaihara Institute, Shizuoka, Japan e Department of Neurochemistry and Neurotoxicology, University of Stockholm, Stockholm, Sweden Received 5 October 2001; accepted 12 December 2001

Abstract The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immunoreactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion, the immunoreactivity for GALR1 was recognizable in about 30% of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells. In the dental pulp, a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor, and suggests the existence of nociceptive primary afferents functioning as autocrine cells. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Galanin receptor-1; Trigeminal ganglion; Dental pulp; Immunocytochemistry; In situ hybridization

1. Introduction Galanin is a 29 amino acid peptide, originally isolated from porcine small intestine (Tatemoto et al., 1983), and has a wide distribution both in the central and peripheral nervous systems (Melander et al., 1986a, 1988; Merchenthaler et al., 1993; Ro¨kaeus et al., 1984; Skofitsch and Jacobowitz, 1985a,b), and has been suggested to possess a variety of functions (Bedecs et al., 1995; Crawley, 1995; Ho¨kfelt et al., 1996; Iismaa and

* Corresponding author. Tel.:
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Shine, 1999). Galanin is contained in some primary sensory neurons, especially small to medium-sized neurons, in the dorsal root ganglion and trigeminal ganglion (Ch’ng et al., 1985; Skofitsch and Jacobowitz, 1985b; Xu et al., 1996, 1997; Zhang et al., 1995), and believed to serve as a neuromodulator of sensory neurons; for instance, it exerts a depressive effect on pain transmission in the dorsal horn of the spinal cord (Wiesenfeld-Hallin et al., 1992). Furthermore, it has been suggested in the galanin knockout mice that galanin mediates neuronal injury and has neurotrophic effects (Holmes et al., 2000; Wynick et al., 1998). The biological effects of galanin are mediated by Gprotein-coupled receptors, galanin receptors (GALRs), on the surface of target cells (Bartfai et al., 1993;

0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 1 ) 0 0 3 2 3 - 6

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Merchenthaler et al., 1993; Skofitsch et al., 1986). A series of pharmacological studies have succeeded in cloning three types of receptors, designated as GALR1, GALR2, GALR3 (Burgevin et al., 1995; Habert-Ortoli et al., 1994; Parker et al., 1995), and northern blot analysis and in situ hybridization histochemistry have shown that GALR1 is predominantly expressed in the brain and spinal cord (Burgevin et al., 1995; Parker et al., 1995), while GALR2 and GALR3 show a broader distribution pattern than GALR1 (Sullivan et al., 1997; Waters and Krause, 2000). Recent immunocytochemical studies have revealed the distribution of GALR1 in the colon (Matkowskyj et al., 2000) as well as in the hypothalamus (Burazin et al., 2001). Furthermore, Burazin et al. (2000) investigated the distribution of GALRs in rat developing brain by in situ hybridization. However, there has been no report available about the distribution of GALR1 in the peripheral sensory system including the trigeminal ganglion. The dental pulp receives a dense sensory nerve innervation from the trigeminal ganglion (Byers, 1984) and has been regarded as a favorite model to investigate mechanisms of pain sensation since most pulpal afferents are nociceptive. Nerve fibers in the dental pulp gather beneath the odontoblast cell layer to form a subodontoblastic nerve plexus of Raschkow (cf. Frank and Nalbandian, 1989); some of them enter the predentin and dentin showing a complex running pattern (Gunji, 1982). All nerve fibers in the predentin/dentin terminate as free nerve endings which are ultrastructurally characterized by accumulation of mitochondria and lack of Schwann cell covering (Gunji, 1982). The sensory nerves in dental pulp contain various neuropeptides including galanin, substance P, and calcitonin gene-related peptide (CGRP) (Akai and Wakisaka, 1990; Silverman and Kruger, 1987; Wakisaka et al., 1996). Wakisaka et al. (1996) demonstrated that a small population of pulpal nerves shows galanin-like immunoreactivity, and that they are originated from small to medium-sized neurons in the trigeminal ganglion. However, the functional significance of galanin system in the teeth remains unknown, partially due to lack of information about GALR both in trigeminal ganglion cells and pulpal nerves. Using two specific antisera, we examined the existence of GALR1 in rat trigeminal ganglion cells and pulpal nerves. The GALR1 expression was confirmed by in situ hybridization using synthetic oligonucleotide probes for GALR1 mRNA.

2. Materials and methods All animal experiments were performed under guidelines by the Institutional Animal and Care Committee at

the Niigata University Graduate School of Medical and Dental Sciences.

2.1. Animals Ten Wistar rats, weighing about 200 g, were used for immunohistochemical observation. Under deep anesthesia by an intraperitoneal injection of chloral hydrate (40 mg/kg), animals were perfused transcardially either with 4% paraformaldehyde in phosphate buffer (PB, pH 7.4) for light microscopy (n /7), or with a mixture of 4% paraformaldehyde and 0.0125% glutaraldehyde in 0.067 M PB for immunoelectron microscopy (n/3). Following perfusion fixation, the trigeminal ganglia and the upper jaws including molar teeth were removed and further immersed in the same fixative for an additional 2 days at 4 8C. The jaw tissues were decalcified with 10% EDTA /2Na solution for 2 weeks at 4 8C. The trigeminal ganglia and decalcified jaws were equilibrated a 30% sucrose solution overnight for cryoprotection, then rapidly frozen in liquid nitrogen. The trigeminal ganglia and upper jaws were sectioned sagittally in 20/40 and 15 mm thick, respectively, with a freezing microtome, and collected in phosphate-buffered saline (PBS, pH 7.4). Some trigeminal ganglia were cut in 10 mm thick in a cryostat for investigating co-expression of galanin and GALR1 in adjacent sections. For in situ hybridization analysis, an additional three rats were decapitated under deep anesthesia in the same way. The fresh trigeminal ganglia were removed, rapidly frozen in liquid nitrogen. Cryostat sections, 10 mm in thickness, were prepared and mounted onto glass slides pre-coated with 3-aminopropyltriethoxy silane.

2.2. Immuno-peroxidase procedure Free floating sections both of trigeminal ganglia and upper jaws were processed for the avidin /biotin /complex (ABC) method according to Hsu et al. (1981). After the blocking of endogenous peroxidase activity with methanol containing 0.3% H2O2 for 30 min, they were incubated with rabbit polyclonal antisera for 16 /18 h at room temperature. The antisera used in this study were shown in Table 1. Sections were then incubated with biotinylated goat anti-rabbit IgG (1:1000; Vector lab. Inc., Burlingame, CA) and subsequently with ABC complex (Vector) for 60 min each at room temperature. The antigen /antibody reaction sites were made visible by incubation with 0.04% 3-3? diaminobenzidine and 0.003% H2O2 in 0.05 M Tris /HCl, pH 7.6. The immunostained sections were counter-stained with 0.03% methylene blue. They were dehydrated through an ascending series of ethanol, and cover-slipped with Permount (Fisher Scientific, NJ).

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Table 1 Characterization of primary antibodies used in this study

2.5. Immunocytochemical controls

Antiserum

The specificity of two kinds of anti-GALR1 sera was checked by an absorption test. When the primary antisera were preabsorbed with an excess amount of the corresponding antigens (10 mg/ml diluted antibody) overnight 4 8C, they did not show any specific immunoreaction. The specificity of the antisera against galanin and PGP 9.5 has been reported in the literature (Glunbenkian et al., 1987; Nakakura-Ohshima et al., 1993).

Supplier

Rat GALR1 (137 /150) (RY569), Yanaihara In137 stitute, ShizuoVHSRRSSSLRVSRN150 ka, Japan Rat GALR1 (267 /277) (RY599), Yanaihara In267 stitute, ShizuoWAEFGAFPLTP277 ka, Japan Rat galanin Yanaihara Institute, Shizuoka, Japan Peninsula, USA Human protein gene product 9.5 Ultraclone, (PGP 9.5) England

Dilution 4000

4000 /5000

5000

1000 10000 (polyclonal), 1000 (monoclonal)

2.3. Double immunostaining with GALR1 and protein gene product 9.5 (PGP 9.5) For clarification of relation between GALR1-expressing nerves versus nerves labeled with PGP 9.5, a general neuronal marker, in the dental pulp, double immunostaining was performed. Frozen, Free-floating sections of upper molars were simultaneously incubated with the rabbit anti-GALR1 serum (RY599, 1:4000) and a mouse monoclonal antibody against PGP 9.5 (1:1000; 31A3, Ultraclone, Cambridge, UK). After incubation with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (1:100; Vector) for GALR1, the sections were reacted with rhodamine isothiocyanate (RITC)-conjugated anti-mouse IgG (1:100; Vector) for PGP 9.5. Following rinses in PBS, the double-labeled sections were thaw-mounted onto gelatin-coated glass slides, and cover-slipped with Vectashield (Vector) and examined with a confocal microscope (LSM 510; Carl Zeiss, Oberkochen, Germany) in FITC-RITC mode.

2.6. In situ hybridization analysis in trigeminal ganglion The cryostat sections for in situ hybridization were fixed with 4% paraformaldehyde for 10 min and acetylated for 10 min with 9.25% acetic anhydride in 0.1 M triethanolamine /HCl (pH 8.0). Synthetic oligodeoxynucleotides, which are complementary to nucleotide residues 4/51, 506 /553, 784 /831 and 975/1022 of the rat GALR1 cDNA (Burazin et al., 2000; Merchenthaler et al., 1993), were purchased from Nihon Gene Research Laboratories Inc. (Sendai, Japan). The oligonucleotides were labeled with 35S-dATP, using terminal deoxyribonucleotitidyl transferase (BRL, Gaithersburg, MD) at a specific activity 0.5 /109 dpm/mg DNA. The sections were prehybridized for 2 h in a buffer containing 50% formamide, 0.1 M Tris /HCl (pH 7.5), 4 /SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.6M NaCl, 0.25% sodium dodecyl sulfate (SDS), 200 mg/ml tRNA, 1mM EDTA, and 10% dextran sulfate. Hybridization was performed at 42 8C for 10 h in the prehybridization buffer supplemented with 10,000 cpm/ml of 35S-labeled oligonucleotide probes. The slides were washed at room temperature for 20 min in 2 /SSC containing 0.1% sarkosyl and twice at 55 8C for 40 min in 0.1 /SSC containing 0.1% sarkosyl. After drying, the sections were dipped in Kodak NTB2 nuclear track emulsion and exposed for 2 months.

2.4. Immuno-electron microscopy For immunocytochemistry at the electron microscopic level, immunostained sections for GALR1 were post-fixed with 1% osmium tetroxide reduced with 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for 3 h at 4 8C, dehydrated in ascending ethanols, and finally embedded in epoxy resin (Epon 812, Taab, Berkshire, UK). One micrometer-thick sections were stained with 0.03% methylene blue. Ultrathin sections were prepared with an ultramicrotome, and examined with a Hitachi H-7000 transmission electron microscope (Hitachi Co. Ltd., Tokyo, Japan), following brief staining with uranyl acetate and lead citrate.

2.7. Quantitative analysis on GALR1-positive (neurons) and 35S-labeled neurons in trigeminal ganglion The ratio of immunolabeled neurons and 35S-labeled neurons to the total number of neurons in rat trigeminal ganglion was measured by counting the number of cells under a light microscope (duplicate in a blind) (three sections in each animal; total n /9). The surface area of labeled neurons was also measured by use of a digital image analyzer (KS-300, Karl Zeiss, Germany). These data were presented as mean9/standard deviation (S.D.).

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3. Results 3.1. Immunoreactivity for GALR1 and/or galanin in the trigeminal ganglion The antisera against GALR1 displayed intense immunoreactivity in a small population of rat trigeminal ganglion cells (Fig. 1a /c). The intensity of staining was much weaker for the cytosome than for the cell membrane, which was shown in 1 mm thick plastic sections (Fig. 1b). In addition to immunoreaction in the somata, some thin nerve fibers displayed immunoreactivity for the antiserum (RY599) that recognizes the amino acid sequence of GALR1 267-277 (Fig. 1a). The quantitative analysis of GALR1-immunoreactive ganglion cells showed that the percentage of the positive cells to the total number of ganglion cells was 36.39/ 10.2% (n /182). In size distribution, the GALR1immunoreactive cells ranged from 200 to 1600 mm2 with peak at 400/600 mm2 (mean9/SD; 636.49/271.1 mm2) (Fig. 2). The occurrence of immunoreactivity in cells larger than 1200 mm2 was very rare. Observations

of serially cut sections alternatively stained with GALR1 (Fig. 1c) and galanin (Fig. 1d) antisera showed exhibited immunoreactivity for both GALR1 and galanin. A very few neurons showed either GALR1- or galanin-immunoreactivity. Note that trigeminal ganglion contained neurons immunonegative for both GALR1 and/or galanin (Fig. 1c,d). 3.2. In situ hybridization analysis on the trigeminal ganglion In situ hybridization analysis, trimgeminal ganglion cells displayed intense hybridization signal for GALR1 mRNA but not for satellite cells (Fig. 3a,b). The percentage of 35S-labeled cells to the total number of cells was estimated to be 31.79/8.2% (n /224). In the size analysis, the mean cross-sectional area of cell bodies that expressed GALR1 mRNA was 597.59/260.9 mm2 with peak at 400/600 mm2. Most of the cells were in small to medium-sized (Fig. 2). The specificity of in situ hybridization was confirmed by both the consistent detection of signal for four non-overlapping antisense

Fig. 1. Photomicrographs showing immunoreactivity for GALR1 (a /c) and galanin (d) in the rat trigeminal ganglion. (a) The GALR1 antiserum (RY599) intensely stains a small population of ganglion cells (arrows) and nerve fibers (+).145. (b) The GALR1 antiserum (RY569) stains positively certain neurons (arrows), with more intense reactivity in the cell membrane, in the rat trigeminal ganglion. Myelinated fibers are darkly stained with methylene blue. One micrometer-thick plastic section.  145. (c, d) Photomicrographs of serial cryostat sections immunostained for GALR1 (RY599, c) and galanin (d). Note that the neurons pointing out by arrows coexpress GALR1 and galanin.  130. Scale bars 50 mm in (a), 50 mm in (b), 50 mm in (c, d).

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Fig. 2. Quantitative analysis on the sectional surface area of trigeminal ganglion cells expressing GALR1 mRNA (dark column) and GALR1 protein (bright column). X and Y axes indicate the section area (mm2) and the cell number (%), respectively. Both show a similar pattern in sectional area of positive neurons.

probes and the disappearance of signal when an excess dose of corresponding cold oligonucleotides was added to the hybridization fluid. 3.3. Distribution of PGP 9.5- and galanin-immunoreative nerve fibers in molar teeth The distribution of PGP 9.5-positive nerve fibers in the dental pulp of the molar teeth was similarly

organized to that shown in a previous report (cf. Byers, 1984). Namely, PGP 9.5-immunoreactive nerve bundles with limited branching ascended via the center of the root pulp. As soon as the nerve bundles reached the coronal pulp, they began to arborize extensively and then distributed throughout the pulp chamber (Fig. 4a,b). Compared to PGP 9.5-positive nerve fibers, galanin-positive nerves, mostly beaded, were more sparsely distributed (Fig. 4c). The PGP 9.5-immunor-

Fig. 3. Micrographs showing GALR1 mRNA expression in rat trigeminal ganglion. In situ hybridization using an oligonucleotide probe under bright (a) and dark field (b). 35S-labeled signals for GALR1 mRNA are localized in some neuronal somata (arrows).  180. Scale bars 50 mm.

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Fig. 4

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eactive nerve fibers gathered beneath the odontoblast cell layer to form the subodontoblastic nerve plexus of Raschkow (cf. Frank and Nalbandian, 1989) (Fig. 4b), whereas galanin-positive nerve fibers were much less abundant than PGP 9.5-positive nerves (Fig. 4c). Some PGP 9.5-positive nerve bundles climbed up perpendicularly through the odontoblast cell layer toward the pulpo-dentinal border, where some branches penetrated into the predentin, and further into dentin (Fig. 4b). In contrast, no galanin-positive nerve braches were recognizable in the predentin and dentin (Fig. 4c). 3.4. Immunocytochemistry for GALR1 in molar teeth Pulpal nerves displayed immunoreactivity for GALR1 (RY599) (Fig. 4d) but not for another antiserum (RY569). A small population of GALR1-immunoreactive fibers ascended through the root pulp to the coronal pulp, where they branched into comparatively smooth and thin nerve fibers and they were widely distributed. A considerable number of the GALR1immunoreactive nerve fibers gathered beneath the odontoblast cell layer, which formed terminal arbors in the vicinity of the odontoblasts (Fig. 4e). Some of the immunoreactive nerve branches passed through the odontoblast cell layer, but they never entered the predentin and dentin. Confocal images of dental nerves in the molar tooth show relations of GALR1- and PGP 9.5-positive nerve fibers (Fig. 5). Namely, the subodontoblastic nerve plexus consisted of double-labeled nerve fibers (yellow) and nerve fibers immunoreactive for PGP 9.5 only (red). Nerves immunoreactive for GALR1 only (green) were not found in the rat molar teeth. Double-labeled nerve fibers localized in the vicinity of the odontoblast cell layer, never advanced into the predentin and dentin (Fig. 5). For immunoelectron microscopic observations, immunoreactive product for GALR1 was identified as electron dense deposits in the pulpal nerve fibers (Fig. 6a). They were diffusely distributed in the axoplasm of unmyelinated nerve fibers. We did not find any immunoreaction in myelinated nerves and other cellular elements including Schwann cells and pulpal fibroblasts. In the odontoblast cell layer, it was frequent that immunoreactive nerve endings, being lost Schwann cell covering completely and including many mitochodrion,

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were adjacent to the cell body of odontoblasts (Fig. 6b,c). However, a distinct synaptic formation was not recognized in this study (Fig. 6d). On the other hand, the odontoblast cell processes and nerve endings in the predentin and dentin lacked immunoreactivity for GALR1 (Fig. 6e).

4. Discussion Recent immunocytochemical study has revealed the distribution of GALRs in the colon (Matkowskyj et al., 2000) as well as in the brain including hypothalamus (Burazin et al., 2000, 2001), but no information has been available about the distribution of GALR in trigeminal ganglion cells and pulpal nerves that contain galaninimmunoreactivity (Matsuda et al., 1994; Moore, 1989; Stro¨mberg et al., 1987; Wakisaka et al., 1996). In this study, we demonstrated the expression of GALR1 in a small population of trigeminal ganglion cells: about 30% were identified to be positive by both immunocytochemistry and in situ hybridization. Although the expression of GALR1 mRNA has been reported in the rat dorsal root ganglion, the percentage is slightly higher for the trigeminal ganglion (Xu et al., 1996). Most GALR1immunoreactive cells in the trigeminal ganglion presented here were in small to medium-sized, ranged from 200 to 1600 mm2 with peak at 400 /600 mm2. This is almost identical to the result observed in the dorsal root ganglion (Xu et al., 1996). These data provide strong evidence for the consistent existence of GALR1 in the sensory ganglia of spinal and cranial nerves. In the present study, we demonstrated the presence of GALR1-immunoreactivity in nerve fibers in the dental pulp. To our knowledge this is the first report. It is apparent that GALR1-immunoreactive nerve fibers are originated from the trigeminal ganglion, because 1) the distribution pattern in the dental pulp was similar to that of galanin-containing nerve fibers (Wakisaka et al., 1996), and 2) interruption of the inferior alveolar nerve causes the complete disappearance of galanin-positive nerves in the dental pulp of rat molar teeth (Wakisaka et al., 1996). Although we could not perform double immunostaining for GALR1 and galanin in pulpal nerves, co-expression of galanin and GALR1 was clearly demonstrated in trigeminal ganglion cells. The coexistence of 125I-galanin binding sites with galanin-like

Fig. 4. Immunocytochemistry for PGP 9.5 (a, b), galanin (c) and GALR1 (d, e) in the rat molar tooth. Counter-stained with methylene blue. (a) PGP 9.5-immunoreactive nerve bundles ascend root pulp with the blood vessels. When they reach the coronal pulp, they arborize to distribute throughout the pulp chamber  38. (b) Higher magnification of the boxed area in Fig. 4a. PGP 9.5-positive nerves gather beneath the odontoblast cell layer to form a nerve plexus of Raschkow (+). Some nerve fibers (arrows) penetrate into the predentin beyond the odontoblast cell layer.  130. (c) The galanin-positive nerves are sparsely distributed in the coronal pulp. Note the difference in number between PGP 9.5 (b) and galanin (c) immunoreactive nerves.  130 (d) A small number of pulpal nerves react to the GALR1 antiserum (RY599).  38. (e) Higher magnification of the boxed area in Fig. 4d. GALR1-immunoractive nerves also concentrate near the odontoblast cell layer, and almost of them terminate in the vicinity of the odontoblasts. No nerve fiber entering the predentin is recognizable.  130. Scale bars 500 mm in (a), 50 mm in (b), 50 mm in (c), 500 mm in (d), 50 mm in (e). AB, alveolar bone; PD, predentin; D, dentin.

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Fig. 5. Confocal microscopic image of a double stained section by use of antisera against PGP 9.5 (colored red) and GALR1 (colored green) in coronal pulp of the rat molar tooth. Double positive neurons, colored as yellow, terminate in the vicinity of the odontoblast cell layer.  260. Scale bar 50 mm. PD, predentin; D, dentin.

immunoreactivity was reported in the dorsal root ganglion (Melander et al., 1986a,b, 1988; Skofitsch et al., 1986). GALR1 as well as galanin is transported centrifugally through peripheral and central branches of primary afferent neurons (Xu et al., 1996). By use of double in situ hybridization technique, Xu et al. (1996) demonstrated the coexistence of GALR1 and calcitonin gene-related peptide (CGRP) mRNAs in the dorsal root ganglion of adult rats, and suggested that GALR1-expressing neurons belong to either unmyelinated C-fibers or thin myelinated Ad-fibers. An electrophysiological study showed that the dental pulp

contains Ad- and C-fibers; the former is exclusively distributed in the predentin and dentin whereas the latter is distributed from the pulpo-dentinal border zone to the inner pulp (Byers, 1984). Current immunoelectron microscopic observations revealed that nerve terminals positive for GALR1, lacking Schwann cell sheath completely or partially, were found near the odontoblasts but not in the predentin and dentin. Taken together, it is likely that pulpal nerve fibers immunoreactive for GALR1 belong to unmyelinated C-fibers. However, we cannot exclude the possibility that GALR1 is contained in thin myelinated nerve fibers, because we

Fig. 6. Immunoelectron microscopic observation of GALR1-immunoreactive nerves in the rat molar tooth. (a) Immunoreactive products for GALR1 are confined to the axoplasm of unmyelinated nerve fibers. An associated Schwann cell (S) and a pulpal fibroblast (F) are devoid of immunoreaction.  5100. BV, blood vessel. (b) Nerve terminals at the distal portion of the odontoblast cell layer. Three immunoreactive nerve fibers lose Schwann cell covering to directly contact with odontoblast (OB). One nerve ending indicated by an arrow are negative in reaction.  6000. (c) Immunoreactive nerve fibers climb up and end in the odontoblast cell layer (OB).  4500. (d) Higher magnification of a nerve ending indicated by an arrow in Fig. 6c. No apparent synaptic formation is seen between odontoblasts and the immunoreactive nerve endings.  9900. (e) Immunoelectron micrograph of a predentinal nerve ending. Neither predetinal nerve ending nor odontoblast cell process (OB) shows GALR1-immunoreactivity.  18,900. Scale bars 2 mm in (a), 2 mm in (b), 2 mm in (c), 1 mm in (d), 0.5 mm in (e).

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Fig. 6

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did not use Triton X-100, which enables antibody to penetrate into sections, in immunoelectron microscopy. Indeed, we experienced good staining of myelinated nerves by use of Triton X-100, but the ultrastructures were highly damaged (Sodeyama et al., 1996). Galanin has a broad range as a neuromodulator, hormone or paracrine factor (for reviews Bedecs et al., 1995; Crawley, 1995; Iismaa and Shine, 1999), and several studies have performed to clarify functional roles of galanin mainly in the spinal cord (cf. Ho¨kfelt et al., 1996; Iismaa and Shine, 1999; Marti et al., 1987). The existence of galanin in trigeminal and dorsal root ganglia suggests the involvement of this peptide in sensory processing (Matsuda et al., 1994; Moore, 1989; Stro¨mberg et al., 1987). Physiological and histochemical studies suggested that the main effect of galanin in the dorsal horn is postsynaptic in normal rats (WiesenfeldHallin et al., 1990, 1992). Experimental studies using administrations of galanin and galanin receptor antagonists such as galantide and M35 (Reimann et al., 1994; Wiesenfeld-Hallin et al., 1990, 1992) suggested that galanin represents an endogenous antinociceptive messenger molecule. These suggestions combined with present data render prediction that galanin released from primary afferents exerts analgesic effects by suppressing excitability of both primary afferents themselves and second-order neurons through second messenger molecules. However, we can not role out possibility that activation of galanin receptor enhances the release of anti-nociceptive substances from primary afferents, because endogenous morphine-like peptides including enkephalin and endorphin are contained in primary afferents innervating tooth pulps (Casasco et al., 1990; Gronblad et al., 1984; Robinson et al., 1989). In addition to the possible involvement of galanin in the sensory transduction and transmission, other functions such as trophic effects should be considered in trigeminal primary afferents.

Acknowledgements The authors thank M. Hoshino and K. Takeuchi for their technical assistance. This study was supported by Grant-in-Aid for Scientific Research and Multi-disciplinary Research from the Ministry of Education, Culture, Science, Sports, Science and Technology, Japan.

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