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VR1-immunoreactive primary sensory neurons in the rat trigeminal ganglion
Brain Research 890 (2001) 184–188 www.elsevier.com / locate / bres
Short communication
VR1-immunoreactive primary sensory neurons in the rat trigeminal ganglion H. Ichikawa*, T. Sugimoto Second Department of Oral Anatomy and Biodental Research Center, Okayama University Dental School, 2 -5 -1 Shikata-cho, Okayama 700 -8525, Japan Accepted 14 November 2000
Abstract Immunohistochemistry for VR1, a nociceptive transducer for vanilloid compounds, protons and heat (.438C), was performed on the rat trigeminal ganglion (TG). The immunoreactivity (IR) was detected in 20% of TG cells and these neurons were mostly small- to medium-sized (mean6S.D. 4276189 mm 2 ). Twenty-six percent of the TG neurons retrogradely labeled from the facial skin exhibited VR1-IR, while the IR was detected in only 8% of those labeled from the tooth pulp. Co-expression of VR1 was common among the calcitonin gene-related peptide-immunoreactive cutaneous neurons (63%) but not among the similar tooth pulp neurons (20%). The present study indicates that primary nociceptive neurons which respond to vanilloid compounds, protons and heat are abundant in the facial skin but not in the tooth pulp. 2001 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Somatic and visceral afferents Keywords: Calcitonin gene-related peptide; Immunohistochemistry; Skin; Tooth pulp; Trigeminal ganglion; VR1
The capsaicin (vanilloid) receptor VR1 is a nociceptive transducer which can be activated by vanilloid compounds, protons and heat (.438C) [2]. In the dorsal root ganglion (DRG), VR1 is localized to small- and medium-sized neurons [1,4]. Such neurons supply their peripheral receptive fields with free nerve endings, project to the superficial dorsal horn of the spinal cord, and are considered to have unmyelinated axons [4]. The co-expression of VR1 with calcitonin gene-related peptide (CGRP), a marker for small- to medium-sized neurons in sensory ganglia [8– 10,12,13], has also been demonstrated in the DRG [4]. Recently, we demonstrated that VRL-1, a newly cloned capsaicin–receptor homologue [1], was localized to medium-sized and large neurons in the trigeminal ganglion (TG) [7]. This transducer does not respond to capsaicin, and is activated by high temperatures with a threshold .528C [1]. In addition, our retrograde tracing method *Corresponding author. Tel.: 181-86-253-6637; fax: 181-86-2356612. E-mail address: [email protected] (H. Ichikawa).
revealed that the facial skin and tooth pulp were innervated by the high threshold heat nociceptors [7]. VRL-1-containing neurons were abundant in the tooth pulp but not the facial skin. Therefore, there appears to be a difference in the content of nociceptive transducers in cutaneous and tooth pulp TG neurons. In this study, we examined the distribution and cell size of VR1-containing primary sensory neurons in the TG. The co-expression of VR1 and CGRP is also investigated in cutaneous and pulpal TG neurons. Six TGs were obtained from four male Sprague–Dawley rats (200–300 g). Rats were anesthetized with ether to the level at which respiration was markedly suppressed, and transvascularly perfused with 50 ml of saline followed by 500 ml of 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). The materials were dissected, frozen sectioned at 12 mm, and thaw-mounted on gelatin-coated glass slides. For the demonstration of VR1, ABC (avidin–biotin– horseradish peroxidase complex) method was performed. Sections were incubated with rabbit anti-VR1 serum (1:20 000, Neuromics, USA) for 24 h at room temperature,
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03253-4
H. Ichikawa, T. Sugimoto / Brain Research 890 (2001) 184 – 188
followed by biotinylated goat anti-rabbit IgG and ABC complex (Vector Laboratories, USA). Following nickel ammonium sulfate-intensified diaminobenzidine reaction, the sections were dehydrated in a graded series of alcohols, cleared in xylene and cover-slipped with Entellan (Merck, Germany). For cell size analysis, the microscopic image (3215) of the cell bodies was projected over a digitizer tablet using a drawing tube. The cross-sectional area of those cell bodies that contained the nucleolus was recorded. For simultaneous visualization of VR1 and CGRP, a double immunofluorescence method was used. The sections were incubated for 24 h at room temperature with a mixture of rabbit anti-VR1 serum (1:1000) and guinea pig anti-CGRP serum (1:1000; Milab, Sweden). The sections were then treated with a mixture of lissamine rhodamine B chloride-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch Labs, USA) and fluorescein isothiocyanate-conjugated goat anti-guinea pig IgG (1:1000; Cappel, USA). For demonstration of VR1 in cutaneous and pulp neurons, four male rats (300–350 g) were used. Under deep anesthesia by i.p. injection with ethyl carbamate (650 mg / kg) and pentobarbital sodium (20 mg / kg), 0.1–0.2 ml of 1% fluorogold (FG, Fluorochrom Inc., USA) in distilled water was injected into the right first and second maxillary molar tooth pulps or infraorbital skin. After 3 days, the animals were reanesthetized with ether, and transvascularly perfused with 4% formaldehyde. The right TGs were frozen-sectioned at 12 mm, mounted on gelatin-coated glass slides, and processed for the co-expression of VR1 and CGRP as described above. For cell size analysis of tooth pulp and cutaneous neurons, the cross-sectional area of FG-labeled cell bodies that contained nuclear profiles was measured on glossy prints (3165). Because of the halo surrounding the FG-positive cells, however, accuracy of measurement was compromised. For the control, rabbit anti-VR1 serum was preabsorbed with rat VR1 (50 mg / ml, Neuromics). The specificity of guinea pig anti-CGRP serum has been described elsewhere [6].
1. The distribution and cell size of VR1immunoreactive neurons in the TG Two types of staining pattern were observed in TG neurons: In strongly-stained neurons, the reaction was detected throughout the cytoplasm (Fig. 1A). The staining was restricted to the plasma membrane and granules in lightly-stained neurons (Fig. 1A). In the control, the strong staining throughout the cytoplasm disappeared whereas the weak staining in the membrane and granules remained unchanged. Therefore, the light staining was unlikely to be specific for VR1 and only strongly-stained neurons were analyzed in this study.
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The TG contained abundant VR1-immunoreactive (-IR) neurons (Fig. 1A, B). In total, 19.9% (168 / 845) of TG neurons were immunoreactive for VR1. As shown in Fig. 2, VR1-IR neurons were mostly small- and medium-sized (range 143–1108 mm 2 ; mean6S.D 4276189 mm 2 ). A half (48.9% or 86 / 176) of TG neurons ,400 mm 2 and 22.5% (80 / 355) of those in the range 400–1000 mm 2 showed the immunoreactivity (IR). Virtually all TG neurons .1000 mm 2 were devoid of it (0.6% or 2 / 314).
2. The co-expression of VR1 and CGRP in the TG As described previously [10,11,13,14], the TG contained many CGRP-IR neurons (Fig. 1C). These neurons were small- to medium-sized and scattered throughout the TG. Our double immunofluorescence method revealed the coexpression of VR1 and CGRP (Fig. 1B, C). In total, 56.6% (391 / 691) of VR1-IR neurons exhibited CGRP-IR and 53.4% (391 / 732) of CGRP-IR TG neurons co-expressed VR1-IR.
3. VR1-IR neurons innervating the facial skin and tooth pulp Three days after FG application to the upper molar tooth pulps or infraorbital skin, many cell bodies were labeled in the TG (Fig. 1D, G, J). They were mostly located in the maxillary division of the ganglion. As described previously [7,15,16], cutaneous TG neurons (range 126–2736 mm 2 ; mean6S.D. 6676434 mm 2 ) mostly had smaller cell bodies than tooth pulp neurons (range5193–2621 mm 2 , mean6S.D.58246432 mm 2 ) (Fig. 1). Retrograde tracing and immunohistochemical methods revealed that cutaneous and tooth pulp TG neurons contained VR1-IR (Fig. 1D, E, G, H, J, K). In total, 25.9% (44 / 170) and 7.9% (10 / 126) of TG neurons innervating the facial skin (Fig. 1D, E, G, H) and tooth pulp (Fig. 1J, K), respectively, were immunoreactive for VR1. A half (48.9% or 22 / 45) of small (,400 mm 2) and 22.7% (22 / 97) of medium-sized (400–1000 mm 2 ) cutaneous neurons showed the IR. On the other hand, only 16.7% (3 / 18) of small- (,400 mm 2 ) and 9.9% (7 / 71) of medium-sized (400–1000 mm 2 ) tooth pulp neurons were immunoreactive for VR1 (Fig. 1). Large neurons (.1000 mm 2 ) were devoid of the IR among cutaneous (0 / 28) and tooth pulp neurons (0 / 37). A double immunofluorescence method revealed the coexpression of VR1 and CGRP in cutaneous and tooth pulp TG neurons. In total, 68.2% (30 / 44) of VR1-IR cutaneous neurons showed CGRP-IR and 62.5% (30 / 48) of CGRPIR cutaneous neurons exhibited VR1-IR (Fig. 1D–I) while 17.7% (30 / 170) of all cutaneous neurons co-expressed VR1- and CGRP-IR. On the other hand, all VR1-IR tooth
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H. Ichikawa, T. Sugimoto / Brain Research 890 (2001) 184 – 188
Fig. 1. Microphotographs for VR1 (A, B, E, H, K), CGRP (C, F, I, L) and FG (D, G, J) in the TG. The cytoplasmic matrix in many small- and medium-sized TG neurons is strongly stained (arrows in A), whereas the membrane and granules in large neurons are lightly stained (arrowheads in A). The strongly-stained neurons are considered to be immunoreactive for VR1. Panels B and C show the same field of view. A double immunofluorescence method reveals that VR1-IR TG neurons co-express CGRP-IR (arrows in B, C). Neurons which exhibit either VR1- (double arrows) or CGRP-IR (arrowheads) are also observed in the TG (B, C). Panels D–F, G–I and J–L show the same fields of view, respectively. TG neurons retrogradely labeled from the skin (arrows in D) and molar tooth pulp (arrows in J) with FG co-express VR1 (arrows in E, K) and CGRP (arrows in F, L). TG neurons which express VR1-IR alone (double arrows in H, I) innervate the facial skin (double arrows in G). Arrowheads point to cutaneous (G) and tooth pulp (J) neurons which contain CGRP-IR alone (H, I, K, L). Bars5100 mm (A, B). Panels B–L are at the same magnification.
H. Ichikawa, T. Sugimoto / Brain Research 890 (2001) 184 – 188
187
ences may reflect a larger proportion of unmyelinated CGRP-IR neurons in the skin than in the tooth pulp. In conclusion, we have described VR1-IR in the TG. The IR was detected in 20% of TG cells. Virtually all VR1-IR TG cells were small- to medium-sized (,1000 mm 2 ). The proportion of VR1-IR neurons among cutaneous TG neurons was larger than among tooth pulp neurons. CGRP-IR TG neurons which co-expressed VR1IR were common in the facial skin but not in the tooth pulp. The present study indicates that small- to mediumsized nociceptors responding to vanilloid compounds, protons or heat are abundant in the facial skin but not in the tooth pulp.
References Fig. 2. A histogram showing the cell size spectrum of VR1-positive and -negative neurons in the TG. The data were obtained from 845 TG neurons.
pulp neurons contained CGRP-IR (10 / 10) and 20.4% (10 / 49) of CGRP-IR tooth pulp neurons showed VR1-IR (Fig. 1J–L). The present study demonstrated that VR1-IR neurons were small- to medium-sized in the TG (,1000 mm 2 ) and that virtually all large TG neurons (.1000 mm 2 ) were VR1-immunonegative. In addition, the proportion of VR1IR neurons among cutaneous neurons (26%) was larger than among tooth pulp TG neurons (8%). This is in contrast with our previous observation that 37% of the TG neurons retrogradely labeled from the tooth pulp exhibited VRL-1-IR while the IR was detected in only 9% of those labeled from the facial skin [7]. Thus, the content of nociceptive transducers in TG neurons may depend on the variety of their receptive fields. Previous and present studies have demonstrated that small primary sensory neurons are common in the skin compared with the tooth pulp [7,15,16]. The difference of the cell size spectra may be associated with the content of VR1. It is likely that the small- to medium-sized nociceptive primary neurons are abundant in the facial skin but not in the tooth pulp. The present double immunofluorescence study also revealed the co-expression of VR1 and CGRP in the TG. Although CGRP is mainly localized to small primary neurons with unmyelinated axons, a substantial subpopulation of the CGRP-IR cells emit myelinated axons [5,8]. CGRP-IR TG neurons which co-express VR1 probably have unmyelinated axons, because VR1-IR neurons are considered to be unmyelinated in the sensory ganglia [1,2,4]. In this study, the co-expression of VR1 was common among the CGRP-IR cutaneous neurons (63%) but not the similar tooth pulp neurons (20%). This finding is probably consistent with the previous findings that CGRP-IR nerve fibers in the skin were more sensitive to capsaicin than in the tooth pulp [3,11,12]. These differ-
[1] M.J. Caterina, T.A. Rosen, M. Tominaga, A.J. Brake, D. Julius, A capsaicin-receptor homologue with a high threshold for noxious heat, Nature 398 (1999) 436–441. [2] M.J. Caterina, M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, D. Julius, The capsaicin receptor: a heat-activated ion channel in the pain pathway, Nature 389 (1997) 816–824. [3] P. Geppetti, S. Frilli, D. Renzi, P. Santicioli, C.A. Maggi, E. Theodorsson, M. Fanciullacci, Distribution of calcitonin gene-related peptide-like immunoreactivity in various rat tissues: correlation with substance P and other tachykinins and sensitivity to capsaicin, Regul. Pept. 23 (1988) 289–298. [4] A. Guo, L. Vulchanova, J. Wang, X. Li, R. Elde, Immunocytochemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites, Eur. J. Neurosci. 11 (1999) 946–958. [5] H. Ichikawa, T. Deguchi, T. Nakago, D.M. Jacobowitz, T. Sugimoto, Parvalbumin- and calretinin-immunoreactive trigeminal neurons innervating the rat molar tooth pulp, Brain Res. 679 (1995) 205–211. [6] H. Ichikawa, C.J. Helke, Distribution, origin and plasticity of galanin-immunoreactivity in the rat carotid body, Neuroscience 52 (1993) 757–767. [7] H. Ichikawa, T. Sugimoto, VRL-1-immunoreactive primary sensory neurons in the rat trigeminal nervous system, Neuroscience, in press. [8] A. Ishida-Yamamoto, E. Senba, M. Tohyama, Distribution and fine structure of calcitonin gene-related peptide-like immunoreactive nerve fibers in the rat skin, Brain Res. 491 (1989) 93–101. ¨ [9] G. Ju, T. Hokfelt, E. Brodin, J. Fahrenkrug, J.A. Fischer, P. Frey, R.P. Elde, J.C. Brown, Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells, Cell Tissue Res. 247 (1987) 417–431. [10] L. Kruger, J.D. Silverman, P.W. Mantyh, C. Sternini, N.C. Brecha, Peripheral patterns of calcitonin gene-related peptide general somatic sensory innervation: cutaneous and deep terminations, J. Comp. Neurol. 280 (1989) 291–302. [11] H. Mori, A. Ishida-Yamamoto, E. Senba, Y. Ueda, M. Tohyama, Calcitonin gene-related peptide containing sensory neurons innervating tooth pulp and buccal mucosa of the rat: an immunohistochemical analysis, J. Chem. Neuroanat. 3 (1990) 155–163. [12] J.D. Silverman, L. Kruger, An interpretation of dental innervation based upon the pattern of calcitonin gene-related peptide (CGRP) immunoreactive thin sensory axons, Somatosens. Res. 5 (1987) 157–175. [13] J.D. Silverman, L. Kruger, Calcitonin gene-related-peptide-immuno-
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reactive innervation of the rat head with emphasis on specialized sensory structures, J. Comp. Neurol. 280 (1989) 303–330. [14] G. Skofitsch, D.M. Jacobowitz, Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat, Peptides 6 (1985) 747–754. [15] T. Sugimoto, M. Takemura, Tooth pulp primary neurons: Cell size
analysis, central connection, and carbonic anhydrase activity, Brain Res. Bull. 30 (1993) 221–226. [16] T. Sugimoto, M. Takemura, S. Wakisaka, Cell size analysis of primary neurons innervating the cornea and tooth pulp of the rat, Pain 32 (1988) 375–381.
Short communication
VR1-immunoreactive primary sensory neurons in the rat trigeminal ganglion H. Ichikawa*, T. Sugimoto Second Department of Oral Anatomy and Biodental Research Center, Okayama University Dental School, 2 -5 -1 Shikata-cho, Okayama 700 -8525, Japan Accepted 14 November 2000
Abstract Immunohistochemistry for VR1, a nociceptive transducer for vanilloid compounds, protons and heat (.438C), was performed on the rat trigeminal ganglion (TG). The immunoreactivity (IR) was detected in 20% of TG cells and these neurons were mostly small- to medium-sized (mean6S.D. 4276189 mm 2 ). Twenty-six percent of the TG neurons retrogradely labeled from the facial skin exhibited VR1-IR, while the IR was detected in only 8% of those labeled from the tooth pulp. Co-expression of VR1 was common among the calcitonin gene-related peptide-immunoreactive cutaneous neurons (63%) but not among the similar tooth pulp neurons (20%). The present study indicates that primary nociceptive neurons which respond to vanilloid compounds, protons and heat are abundant in the facial skin but not in the tooth pulp. 2001 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Somatic and visceral afferents Keywords: Calcitonin gene-related peptide; Immunohistochemistry; Skin; Tooth pulp; Trigeminal ganglion; VR1
The capsaicin (vanilloid) receptor VR1 is a nociceptive transducer which can be activated by vanilloid compounds, protons and heat (.438C) [2]. In the dorsal root ganglion (DRG), VR1 is localized to small- and medium-sized neurons [1,4]. Such neurons supply their peripheral receptive fields with free nerve endings, project to the superficial dorsal horn of the spinal cord, and are considered to have unmyelinated axons [4]. The co-expression of VR1 with calcitonin gene-related peptide (CGRP), a marker for small- to medium-sized neurons in sensory ganglia [8– 10,12,13], has also been demonstrated in the DRG [4]. Recently, we demonstrated that VRL-1, a newly cloned capsaicin–receptor homologue [1], was localized to medium-sized and large neurons in the trigeminal ganglion (TG) [7]. This transducer does not respond to capsaicin, and is activated by high temperatures with a threshold .528C [1]. In addition, our retrograde tracing method *Corresponding author. Tel.: 181-86-253-6637; fax: 181-86-2356612. E-mail address: [email protected] (H. Ichikawa).
revealed that the facial skin and tooth pulp were innervated by the high threshold heat nociceptors [7]. VRL-1-containing neurons were abundant in the tooth pulp but not the facial skin. Therefore, there appears to be a difference in the content of nociceptive transducers in cutaneous and tooth pulp TG neurons. In this study, we examined the distribution and cell size of VR1-containing primary sensory neurons in the TG. The co-expression of VR1 and CGRP is also investigated in cutaneous and pulpal TG neurons. Six TGs were obtained from four male Sprague–Dawley rats (200–300 g). Rats were anesthetized with ether to the level at which respiration was markedly suppressed, and transvascularly perfused with 50 ml of saline followed by 500 ml of 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). The materials were dissected, frozen sectioned at 12 mm, and thaw-mounted on gelatin-coated glass slides. For the demonstration of VR1, ABC (avidin–biotin– horseradish peroxidase complex) method was performed. Sections were incubated with rabbit anti-VR1 serum (1:20 000, Neuromics, USA) for 24 h at room temperature,
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03253-4
H. Ichikawa, T. Sugimoto / Brain Research 890 (2001) 184 – 188
followed by biotinylated goat anti-rabbit IgG and ABC complex (Vector Laboratories, USA). Following nickel ammonium sulfate-intensified diaminobenzidine reaction, the sections were dehydrated in a graded series of alcohols, cleared in xylene and cover-slipped with Entellan (Merck, Germany). For cell size analysis, the microscopic image (3215) of the cell bodies was projected over a digitizer tablet using a drawing tube. The cross-sectional area of those cell bodies that contained the nucleolus was recorded. For simultaneous visualization of VR1 and CGRP, a double immunofluorescence method was used. The sections were incubated for 24 h at room temperature with a mixture of rabbit anti-VR1 serum (1:1000) and guinea pig anti-CGRP serum (1:1000; Milab, Sweden). The sections were then treated with a mixture of lissamine rhodamine B chloride-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch Labs, USA) and fluorescein isothiocyanate-conjugated goat anti-guinea pig IgG (1:1000; Cappel, USA). For demonstration of VR1 in cutaneous and pulp neurons, four male rats (300–350 g) were used. Under deep anesthesia by i.p. injection with ethyl carbamate (650 mg / kg) and pentobarbital sodium (20 mg / kg), 0.1–0.2 ml of 1% fluorogold (FG, Fluorochrom Inc., USA) in distilled water was injected into the right first and second maxillary molar tooth pulps or infraorbital skin. After 3 days, the animals were reanesthetized with ether, and transvascularly perfused with 4% formaldehyde. The right TGs were frozen-sectioned at 12 mm, mounted on gelatin-coated glass slides, and processed for the co-expression of VR1 and CGRP as described above. For cell size analysis of tooth pulp and cutaneous neurons, the cross-sectional area of FG-labeled cell bodies that contained nuclear profiles was measured on glossy prints (3165). Because of the halo surrounding the FG-positive cells, however, accuracy of measurement was compromised. For the control, rabbit anti-VR1 serum was preabsorbed with rat VR1 (50 mg / ml, Neuromics). The specificity of guinea pig anti-CGRP serum has been described elsewhere [6].
1. The distribution and cell size of VR1immunoreactive neurons in the TG Two types of staining pattern were observed in TG neurons: In strongly-stained neurons, the reaction was detected throughout the cytoplasm (Fig. 1A). The staining was restricted to the plasma membrane and granules in lightly-stained neurons (Fig. 1A). In the control, the strong staining throughout the cytoplasm disappeared whereas the weak staining in the membrane and granules remained unchanged. Therefore, the light staining was unlikely to be specific for VR1 and only strongly-stained neurons were analyzed in this study.
185
The TG contained abundant VR1-immunoreactive (-IR) neurons (Fig. 1A, B). In total, 19.9% (168 / 845) of TG neurons were immunoreactive for VR1. As shown in Fig. 2, VR1-IR neurons were mostly small- and medium-sized (range 143–1108 mm 2 ; mean6S.D 4276189 mm 2 ). A half (48.9% or 86 / 176) of TG neurons ,400 mm 2 and 22.5% (80 / 355) of those in the range 400–1000 mm 2 showed the immunoreactivity (IR). Virtually all TG neurons .1000 mm 2 were devoid of it (0.6% or 2 / 314).
2. The co-expression of VR1 and CGRP in the TG As described previously [10,11,13,14], the TG contained many CGRP-IR neurons (Fig. 1C). These neurons were small- to medium-sized and scattered throughout the TG. Our double immunofluorescence method revealed the coexpression of VR1 and CGRP (Fig. 1B, C). In total, 56.6% (391 / 691) of VR1-IR neurons exhibited CGRP-IR and 53.4% (391 / 732) of CGRP-IR TG neurons co-expressed VR1-IR.
3. VR1-IR neurons innervating the facial skin and tooth pulp Three days after FG application to the upper molar tooth pulps or infraorbital skin, many cell bodies were labeled in the TG (Fig. 1D, G, J). They were mostly located in the maxillary division of the ganglion. As described previously [7,15,16], cutaneous TG neurons (range 126–2736 mm 2 ; mean6S.D. 6676434 mm 2 ) mostly had smaller cell bodies than tooth pulp neurons (range5193–2621 mm 2 , mean6S.D.58246432 mm 2 ) (Fig. 1). Retrograde tracing and immunohistochemical methods revealed that cutaneous and tooth pulp TG neurons contained VR1-IR (Fig. 1D, E, G, H, J, K). In total, 25.9% (44 / 170) and 7.9% (10 / 126) of TG neurons innervating the facial skin (Fig. 1D, E, G, H) and tooth pulp (Fig. 1J, K), respectively, were immunoreactive for VR1. A half (48.9% or 22 / 45) of small (,400 mm 2) and 22.7% (22 / 97) of medium-sized (400–1000 mm 2 ) cutaneous neurons showed the IR. On the other hand, only 16.7% (3 / 18) of small- (,400 mm 2 ) and 9.9% (7 / 71) of medium-sized (400–1000 mm 2 ) tooth pulp neurons were immunoreactive for VR1 (Fig. 1). Large neurons (.1000 mm 2 ) were devoid of the IR among cutaneous (0 / 28) and tooth pulp neurons (0 / 37). A double immunofluorescence method revealed the coexpression of VR1 and CGRP in cutaneous and tooth pulp TG neurons. In total, 68.2% (30 / 44) of VR1-IR cutaneous neurons showed CGRP-IR and 62.5% (30 / 48) of CGRPIR cutaneous neurons exhibited VR1-IR (Fig. 1D–I) while 17.7% (30 / 170) of all cutaneous neurons co-expressed VR1- and CGRP-IR. On the other hand, all VR1-IR tooth
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H. Ichikawa, T. Sugimoto / Brain Research 890 (2001) 184 – 188
Fig. 1. Microphotographs for VR1 (A, B, E, H, K), CGRP (C, F, I, L) and FG (D, G, J) in the TG. The cytoplasmic matrix in many small- and medium-sized TG neurons is strongly stained (arrows in A), whereas the membrane and granules in large neurons are lightly stained (arrowheads in A). The strongly-stained neurons are considered to be immunoreactive for VR1. Panels B and C show the same field of view. A double immunofluorescence method reveals that VR1-IR TG neurons co-express CGRP-IR (arrows in B, C). Neurons which exhibit either VR1- (double arrows) or CGRP-IR (arrowheads) are also observed in the TG (B, C). Panels D–F, G–I and J–L show the same fields of view, respectively. TG neurons retrogradely labeled from the skin (arrows in D) and molar tooth pulp (arrows in J) with FG co-express VR1 (arrows in E, K) and CGRP (arrows in F, L). TG neurons which express VR1-IR alone (double arrows in H, I) innervate the facial skin (double arrows in G). Arrowheads point to cutaneous (G) and tooth pulp (J) neurons which contain CGRP-IR alone (H, I, K, L). Bars5100 mm (A, B). Panels B–L are at the same magnification.
H. Ichikawa, T. Sugimoto / Brain Research 890 (2001) 184 – 188
187
ences may reflect a larger proportion of unmyelinated CGRP-IR neurons in the skin than in the tooth pulp. In conclusion, we have described VR1-IR in the TG. The IR was detected in 20% of TG cells. Virtually all VR1-IR TG cells were small- to medium-sized (,1000 mm 2 ). The proportion of VR1-IR neurons among cutaneous TG neurons was larger than among tooth pulp neurons. CGRP-IR TG neurons which co-expressed VR1IR were common in the facial skin but not in the tooth pulp. The present study indicates that small- to mediumsized nociceptors responding to vanilloid compounds, protons or heat are abundant in the facial skin but not in the tooth pulp.
References Fig. 2. A histogram showing the cell size spectrum of VR1-positive and -negative neurons in the TG. The data were obtained from 845 TG neurons.
pulp neurons contained CGRP-IR (10 / 10) and 20.4% (10 / 49) of CGRP-IR tooth pulp neurons showed VR1-IR (Fig. 1J–L). The present study demonstrated that VR1-IR neurons were small- to medium-sized in the TG (,1000 mm 2 ) and that virtually all large TG neurons (.1000 mm 2 ) were VR1-immunonegative. In addition, the proportion of VR1IR neurons among cutaneous neurons (26%) was larger than among tooth pulp TG neurons (8%). This is in contrast with our previous observation that 37% of the TG neurons retrogradely labeled from the tooth pulp exhibited VRL-1-IR while the IR was detected in only 9% of those labeled from the facial skin [7]. Thus, the content of nociceptive transducers in TG neurons may depend on the variety of their receptive fields. Previous and present studies have demonstrated that small primary sensory neurons are common in the skin compared with the tooth pulp [7,15,16]. The difference of the cell size spectra may be associated with the content of VR1. It is likely that the small- to medium-sized nociceptive primary neurons are abundant in the facial skin but not in the tooth pulp. The present double immunofluorescence study also revealed the co-expression of VR1 and CGRP in the TG. Although CGRP is mainly localized to small primary neurons with unmyelinated axons, a substantial subpopulation of the CGRP-IR cells emit myelinated axons [5,8]. CGRP-IR TG neurons which co-express VR1 probably have unmyelinated axons, because VR1-IR neurons are considered to be unmyelinated in the sensory ganglia [1,2,4]. In this study, the co-expression of VR1 was common among the CGRP-IR cutaneous neurons (63%) but not the similar tooth pulp neurons (20%). This finding is probably consistent with the previous findings that CGRP-IR nerve fibers in the skin were more sensitive to capsaicin than in the tooth pulp [3,11,12]. These differ-
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