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The co-expression of P2X3 receptor with VR1 and VRL-1 in the rat trigeminal ganglion
Brain Research 998 (2004) 130 – 135 www.elsevier.com/locate/brainres
Short communication
The co-expression of P2X3 receptor with VR1 and VRL-1 in the rat trigeminal ganglion Hiroyuki Ichikawa *, Tomosada Sugimoto Department of Oral Function and Anatomy, and Biodental Research Center, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525, Japan Accepted 10 November 2003
Abstract The co-expression of P2X3 receptor with the vanilloid receptor subtype I (VR1) and vanilloid receptor 1-like receptor (VRL-1) was examined in the rat trigeminal ganglion (TG) by a double immunofluorescence method. P2X3 receptor-immunoreactive (ir) neurons were predominantly small to medium-sized (range = 93.8 – 1844.4 Am2, mean F S.D. = 503.8 F 286.5 Am2); 35% and 9% of P2X3 receptor-ir TG neurons were immunoreactive for VR1 and VRL-1, respectively. Small and medium-sized P2X3 receptor-ir neurons contained VR1immunoreactivity (ir), whereas medium-sized and large P2X3 receptor-ir neurons showed VRL-1-ir. The retrograde tracing and immunohistochemical methods revealed that 30% of the TG neurons retrogradely labeled from the facial skin and tooth pulp exhibited P2X3 receptor-ir. The co-expression of P2X3 receptor and VR1 was detected in 16% of cutaneous TG neurons and 6% of tooth pulp neurons. On the other hand, the co-expression of P2X3 receptor and VRL-1 was common in tooth pulp neurons (23%) and rare in cutaneous TG neurons (8%). In the tooth pulp, 95% of P2X3 receptor-ir TG neurons contained VRL-1-ir. The present study indicates that P2X3 receptor-ir TG neurons, which co-express VR-ir, are abundant in the facial skin. The tooth pulp is probably innervated by TG neurons, which contain both P2X3-and VRL-1-ir. D 2003 Published by Elsevier B.V. Theme: Sensory systems Topic: Somatic and visceral afferents Keywords: Facial skin; Immunohistochemistry; P2X3 receptor; Tooth pulp; Trigeminal ganglion; VR1; VRL-1
P2X3 receptor is one of many ligand-gated ion channels that is activated by extracellular ATP [5]. In the dorsal root ganglion (DRG) and trigeminal ganglion (TG), P2X3 receptor-containing neurons mainly have small to mediumsized cell bodies and co-express calcitonin gene-related peptide (CGRP), a marker for small to medium-sized nociceptors in the sensory ganglia [2,6,10,12,14,16,17]. In addition, the projection site of P2X3 receptor-containing DRG neurons is the superficial lamina of the spinal dorsal horn [14,15]. Therefore, P2X3 receptor-containing neurons are thought to have a function for nociceptive transduction. This idea is supported by previous findings that tooth pulp primary neurons contain the ion channel [1,5,11,13], because the tooth pulp is considered to be innervated exclusively by nociceptive afferents with cell bodies in the TG. * Coresponding author. Tel.: +81-86-235-6637; fax: +81-86-235-6612. E-mail address: [email protected] (H. Ichikawa). 0006-8993/$ - see front matter D 2003 Published by Elsevier B.V. doi:10.1016/j.brainres.2003.11.019
The capsaicin (vanilloid) receptor subtype I (VR1) is an ion channel which can be activated by vanilloid compounds, protons and heat (>43 jC) [3]. In the DRG and TG, VR1 is localized to small and medium-sized neurons with unmyelinated axons [3,7,9,13]. Vanilloid receptor 1like receptor (VRL-1), a newly cloned capsaicin receptor homologue, is activated by high temperatures with a threshold >52 jC [4]. In the sensory ganglia, VRL-1containing neurons are medium-sized to large and have myelinated axons [4,8,13]. VR1- and VRL-1-containing neurons co-express CGRP, supply the skin with free nerve endings and project to the superficial laminae of the spinal and medullary dorsal horns [4,7,8]. The tooth pulp is also innervated by VR1- and VRL-1-containing TG neurons [8,9,13]. Recently, the co-expression of VR1 and P2X3 has been demonstrated in the DRG [7]. A large proportion of DRG neurons and their terminals co-expressed these ion channels. However, the co-expression of P2X3 recep-
H. Ichikawa, T. Sugimoto / Brain Research 998 (2004) 130–135
tor with VR1 or VRL-1 has never been reported in the TG. In this study, we examined the TG neurons innervating the facial skin and the tooth pulp for possible co-expression P2X3 and other ion channels. Five TGs were obtained from three 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 and post-fixed with the same fixative for 30 min, soaked in a phosphate-buffered 20% sucrose solution overnight, frozen sectioned at 12 Am, and thaw-mounted on gelatincoated glass slides. For simultaneous visualization of P2X3 receptor with VR1 and VRL-1, a double immunofluorescence method was used. The sections were incubated for 24 h at room temperature with a mixture of rabbit anti-P2X3 receptor serum (1:500, Neuromics, USA) and goat anti-VR1 serum (1:200, Santa Cruz Biotechnology, USA) or with a mixture of guinea pig anti-P2X3 receptor serum (1:500, Neuromics) and rabbit anti-VRL-1 serum (1:1500). The sections were then treated with a mixture of lissamine rhodamine B chloride-conjugated donkey anti-rabbit IgG (1:500 for P2X3 receptor or VRL-1, Jackson ImmunoResearch Labs, USA) and either fluorescein isothiocyanate-conjugated donkey anti-goat IgG (1:100 for VR1, Jackson ImmunoResearch Labs) or fluorescein isothiocyanate-conjugated
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donkey anti-guinea pig IgG (1:100 for P2X3, Jackson ImmunoResearch Labs). For demonstration of P2X3 receptor-ir TG neurons innervating the facial skin and tooth pulp, 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 Al of 1% fluorogold (FG, Fluorochrom, Englewood, CO, USA) in distilled water was injected into the infraorbital skin or right first maxillary molar tooth pulp. After 3 days, the animals were reanesthetized with ether, and transvascularly perfused with 4% formaldehyde. The right TGs were frozen-sectioned at 12 Am, mounted on gelatin-coated glass slides, and processed for the co-expression of P2X3 receptor with VR1 or VRL-1 as described above. The brainstem of the rats, which had received tracer application from the tooth pulp, was also cryosectioned at 50 Am and examined with fluorescent illumination for possible FG labeling. All these FG-labeled cells are considered to be primary neurons innervating the tooth pulp and the possibility of periodontal spread of FG through the apical foramen is negligible, because the labeled neuron was not observed in the mesencephalic trigeminal tract nucleus. For cell size analysis of P2X3 receptor-ir neurons and FG-labeled neurons, the cross-sectional area of fluorescent cell bodies that contained nuclear profiles was measured on glossy prints (165). For the control, rabbit and guinea-pig anti-P2X3 receptor sera were preabsorbed with the protein (20 Ag/ml,
Fig. 1. Immunofluorescent microphotographs for P2X3 receptor (A, C), VR1 (B), and VRL-1 (D) in the TG. Small to medium-sized P2X3 receptor-ir neurons contain VR1-ir (arrows in A, B), whereas medium-sized to large P2X3 receptor-ir neurons show VRL-1-ir (arrows in C, D). Arrowheads in A – D point to P2X3 receptor-ir neurons, which are devoid of VR1- and VRL-1-ir. Bar = 100 Am (A). All panels are at the same magnification.
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H. Ichikawa, T. Sugimoto / Brain Research 998 (2004) 130–135
Neuromics). No staining was observed in the control. The specificities of other antibodies have been described elsewhere [4,9].
1. The distribution and cell size of P2X3 receptor-ir neurons in the TG The TG contained abundant P2X3 receptor-ir neurons (Fig. 1A, C). These neurons were scattered throughout the ganglion. The immunoreactivity (ir) was detected in the cytoplasm but not the nucleus. P2X3 receptor-ir TG neurons were predominantly small to medium-sized (range = 93.8 – 1844.4 Am2, mean F S.D. = 503.8 F 286.5 Am2); 44.5% (360/809) of P2X3 receptor-ir TG neurons were smaller
Fig. 3. Histograms showing the cell size spectrum of cutaneous (A) and tooth pulp (B) TG neurons with or without P2X3 receptor-ir. The data were obtained from 328 cutaneous neurons and 163 tooth pulp neurons.
Fig. 2. Histograms showing the cell size spectra of P2X3 receptor-ir neurons with or without VR1-ir (A) and VRL-1-ir in the TG. The data for VR1-ir and VRL-1-ir were obtained from 401 and 409 P2X3 receptor-ir TG neurons, respectively.
than 400 Am2 and 48.5% (392/809) fell in the range of 400– 1000 Am2. Only 7.1% (57/809) of P2X3 receptor-ir TG neurons were larger than 1000 Am2. As described previously [7,8,9,13], the TG contained many VR1- and VRL-1-ir neurons (Fig. 1B, D). VR1-ir neurons were mostly small to medium-sized, and VRL-1-ir neurons were medium-sized to large. Our double immunofluorescence method revealed the co-expression of P2X3 receptor with VR1 and VRL-1; 34.7% (139/401) and 8.6% (35/408) of P2X3 receptor-ir neurons were immunoreactive for VR1 and VRL-1, respectively (Fig. 1A –D). Conversely, 39.8% (139/349) of VR1-ir neurons and 48.6% (35/72) of VRL-1-ir neurons showed P2X3 receptor-ir. About 40% (70/177) of small P2X3 receptor-ir neurons and 34.3% (68/ 198) of medium-sized P2X3 receptor-ir neurons exhibited VR1-ir (Fig. 2A). Large P2X3 receptor-ir neurons were mostly devoid of VR1-ir (3.8% or 1/26). On the other hand, small P2X3 receptor-ir neurons were devoid of VRL-1-ir
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(Fig. 2B). About 10% (22/194) of medium-sized P2X3 receptor-ir neurons and 42% (13/31) of large P2X3 receptor-ir neurons co-expressed VRL-1-ir.
2. P2X3 receptor-ir TG neurons innervating the facial skin and tooth pulp At 3 days after FG application to the infraorbital skin or upper molar tooth pulp, many cell bodies were labeled in the TG. They were located in the maxillary division of the ganglion. Cutaneous TG neurons were predominantly small to medium-sized (small: 29.0% or 95/328, medium-sized: 52.1% or 171/328, large: 18.9% or 62/328, Fig. 3A), whereas tooth pulp TG neurons were mostly medium-sized to large (small: 15.3% or 25/163, medium-sized: 58.3% or 95/163, large: 26.4% or 43/163, Fig. 3B). Retrograde tracing and immunohistochemical methods revealed that 32.3% (106/328) and 31.3% (51/163) of TG neurons innervating the facial skin and tooth pulp, respectively, contained P2X3 receptor-ir. As shown in Fig. 3A, 40% (38/95) of small, 31.0% (53/171) of medium-sized, and 24.2% (15/62) of large cutaneous neurons were immunoreactive for the ion channel. On the other hand, 20% (5/25) of small and 34.7% (33/95) of medium-sized tooth pulp
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neurons showed P2X3 receptor-ir (Fig. 3B). Thirty percent (13/43) of large tooth pulp neurons exhibited the ir. The double immunofluorescence method demonstrated that 15.8% (23/146) of cutaneous neurons co-expressed P2X3 receptor- and VR1-ir (Fig. 4A –C). About 40% (23/ 63) of P2X3 receptor-ir cutaneous neurons contained VR1ir. Twenty-nine percent (42/146) of cutaneous neurons contained VR1-ir and a half (54.8% or 23/42) of VR1-ir cutaneous neurons showed P2X3 receptor-ir. On the other hand, only 6.0% (11/182) of cutaneous neurons coexpressed P2X3 receptor- and VRL-1-ir (Fig. 4D – F). Twenty-six percent (11/43) of P2X3 receptor-ir cutaneous neurons exhibited VRL-1-ir. VRL-1-ir was detected in 12.6% (23/182) of cutaneous neurons and a half (47.8% or 11/23) of VRL-1-ir cutaneous neurons expressed P2X3 receptor-ir. About 8% (6/79) and 22.6% (19/84) of tooth pulp neurons co-expressed P2X3 receptor-ir with VR1-ir and VRL-1-ir, respectively (Fig. 5A –D). About 20% (6/31) of P2X3 receptor-ir tooth pulp neurons showed VR1-ir. Ten percent (8/79) of tooth pulp neurons contained VR1-ir and 75% (6/8) of VR1-ir tooth pulp neurons contained P2X3 receptor-ir. On the other hand, P2X3 receptor-ir tooth pulp neurons mostly co-expressed VRL-1-ir (95% or 19/20, Fig. 4D – F). Half (46.4% or 39/84) of tooth
Fig. 4. Immunofluorescent microphotographs for FG retrogradely transported from the facial skin (A, D), P2X3 receptor (B, E), VR1 (C), and VRL-1 (F) in the TG. Panels A – C and D – F show the same fields of view, respectively. FG-labeled TG neurons (arrows in A, D) co-express P2X3 receptor-ir with VR1-ir (arrows in B, C) and VRL-1-ir (arrows in E, F). Arrowheads in A – F point to P2X3 receptor-ir cutaneous neurons which are devoid of VR1-ir (A – C) and VRL1 (D – F). A bar = 100 Am (A). All panels are at the same magnification.
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Fig. 5. Immunofluorescent microphotographs for FG retrogradely transported from the tooth pulp (A, D), P2X3 receptor (B, E), VR1 (C), and VRL-1 (F) in the TG. Panels A – C and D – F show the same fields of view, respectively. In tooth pulp TG neurons, the co-expression of P2X3 receptor-ir and VR1-ir was rare. However, P2X3 receptor-ir tooth pulp neurons were usually immunoreactive for VRL-1. Arrowheads in A – C point to P2X3 receptor-ir tooth pulp neurons which are devoid of VR1-ir. Arrows and arrowheads in D – F point to VRL-1-ir tooth pulp neuron with and without P2X3 receptor-ir, respectively. Bar = 100 Am (A). All panels are at the same magnification.
pulp neurons showed VRL-1 and half (48.7% or 19/39) of VRL-1-ir tooth pulp neurons had P2X3 receptor-ir. The present study demonstrated that the P2X3 receptor was localized to primary sensory neurons in the TG. These neurons were predominantly small to medium-sized. Our double immunofluorescence study also revealed the co-expression of P2X3 receptor with VR1 and VRL-1 in the TG. Small and medium-sized P2X3 receptor-ir neurons showed VR1-ir, whereas medium-sized and large P2X3-ir neurons contained VRL-1-ir. A previous study demonstrated that the co-expression was very rare in the TG [14]. Therefore, P2X3 receptor-ir TG neurons may coexpress either VR-1-ir or VRL-1-ir. VR-1-and VRL-1-ir TG neurons are thought to be of unmyelinated and myelinated axons, respectively [3,4,7,8]. Therefore, P2X3 receptor-ir TG neurons probably send their unmyelinated and myelinated axons to the oro-facial tissues. The present study investigated the distribution of P2X3 receptor in cutaneous and tooth pulp TG neurons. The proportion of P2X3 receptor-ir neurons among cutaneous TG neurons (32%) was similar to that among tooth pulp neurons (31%). However, the co-expression of P2X3 receptor and VR1 was abundant in cutaneous TG neurons (16%) and relatively rare in tooth pulp neurons (6%). On
the other hand, the co-expression of P2X3 receptor and VRL-1 was common in tooth pulp neurons (23%) but not in cutaneous TG neurons (8%). Previous and present studies demonstrated that tooth pulp neurons are mostly larger than cutaneous TG neurons [8,9]. Therefore, it is likely that the co-expression of P2X3 receptor with VR1 and VRL-1 is associated with the difference of their cell size spectra. P2X3 receptor-ir TG neurons which have umyelinated axons are probably abundant in the facial skin. On the other hand, the tooth pulp is thought to be innervated by myelinated P2X3 receptor-ir neurons, because P2X3-ir tooth pulp mostly co-expressed VRL-1-ir. In conclusion, we have described the co-expression of P2X3 receptor with VR1 and VRL-1 in the TG. Small to medium-sized P2X3 receptor-ir neurons exhibited VR1-ir, whereas medium-sized to large P2X3-ir neurons showed VRL-1-ir. The co-expression of P2X3 with VR1 and VRL-1 was common in cutaneous and tooth pulp TG neurons, respectively. In the tooth pulp, P2X3 receptor-ir neurons mostly contained VRL-1-ir. These findings indicate that P2X3 receptor-ir TG neurons which co-express VR1-ir are abundant in the facial skin. The tooth pulp is probably innervated by TG neurons which have both P2X3 receptorand VRL-1-ir.
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Acknowledgements We greatly appreciate Dr. David Julius at the Department of Cellular and Molecular Pharmacology, University of California, for his kind gift of the anti-VRL-1-serum. This work was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology to H.I. (No. 14571733). References [1] A.M. Alavi, G.R. Dubyak, G. Burnstock, Immunohistochemical evidence for ATP receptors in human dental pulp, J. Dent. Res. 80 (2001) 476 – 483. [2] E.J. Bradbury, G. Burnstock, S.B. McMahon, The expression of P2X3 purinoreceptors in sensory neurons: effects of axotomy and glial-derived neurotrophic factor, Mol. Cell. Neurosci. 12 (1998) 256 – 268. [3] 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. [4] 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. [5] S.P. Cook, L. Vulchanova, K.M. Hargreaves, R. Elde, E.W. McCleskey, Distinct ATP receptors on pain-sensing and stretch-sensing neurons, Nature 387 (1997) 505 – 508. [6] J. Eriksson, U. Bongenhielm, E. Kidd, B. Matthews, K. Fried, Distribution of P2X3 receptors in the rat trigeminal ganglion after inferior alveolar nerve injury, Neurosci. Lett. 254 (1998) 37 – 40. [7] 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.
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[8] H. Ichikawa, T. Sugimoto, VRL-1-immunoreactive primary sensory neurons in the rat trigeminal nervous system, Neuroscience 101 (2000) 719 – 725. [9] H. Ichikawa, T. Sugimoto, VR1-immunoreactive primary sensory neurons in the rat trigeminal ganglion, Brain Res. 890 (2001) 184 – 188. [10] H. Ichikawa, T. Fukunaga, H.W. Jin, M. Fujita, T. Takano-Yamamoto, T. Sugimoto, VR1-, VRL-1- and P2X3 receptor-immunoreactive innervation of the rat temporomandibular joint, Brain Res. (Submitted for publication). [11] J. Jiang, J. Gu, Expression of adenosine triphosphate P2X3 receptors in rat molar pulp and trigeminal ganglia, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endo. 94 (2002) 622 – 626. [12] S.D. Novakovic, L.C. Kassotakis, I.B. Oglesby, J.A. Smith, R.M. Eglen, A.P. Ford, J.C. Hunter, Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury, Pain 80 (1999) 273 – 282. [13] T. Renton, Y. Yiangou, P.A. Baecker, A.P. Ford, P. Anand, Capsaicin receptor VR1 and ATP purinoceptor P2X3 in painful and nonpainful human tooth pulp, J. Orofac. Pain 17 (2003) 245 – 250. [14] E. Stenholm, U. Bongenhielm, M. Ahlquist, K. Fried, VRl- and VRL-l-like immunoreactivity in normal and injured trigeminal dental primary sensory neurons of the rat, Acta Odontol. Scand. 60 (2002) 72 – 79. [15] L. Vulchanova, M.S. Riedl, S.J. Shuster, G. Buell, A. Surprenant, R.A. North, R. Elde, Immunohistochemical study of the P2X2 and P2X3 receptor subunits in rat and monkey sensory neurons and their central terminals, Neuropharmacology 36 (1997) 1229 – 1242. [16] L. Vulchanova, M.S. Riedl, S.J. Shuster, L.S. Stone, K.M. Hargreaves, G. Buell, A. Surprenant, R.A. North, R. Elde, P2X3 is expressed by DRG neurons that terminate in inner lamina II, Eur. J. Neurosci. 10 (1998) 3470 – 3478. [17] Z. Xiang, X. Bo, G. Burnstock, Localization of ATP-gated P2X receptor immunoreactivity in rat sensory and sympathetic ganglia, Neurosci. Lett. 256 (1998) 105 – 108.
Short communication
The co-expression of P2X3 receptor with VR1 and VRL-1 in the rat trigeminal ganglion Hiroyuki Ichikawa *, Tomosada Sugimoto Department of Oral Function and Anatomy, and Biodental Research Center, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525, Japan Accepted 10 November 2003
Abstract The co-expression of P2X3 receptor with the vanilloid receptor subtype I (VR1) and vanilloid receptor 1-like receptor (VRL-1) was examined in the rat trigeminal ganglion (TG) by a double immunofluorescence method. P2X3 receptor-immunoreactive (ir) neurons were predominantly small to medium-sized (range = 93.8 – 1844.4 Am2, mean F S.D. = 503.8 F 286.5 Am2); 35% and 9% of P2X3 receptor-ir TG neurons were immunoreactive for VR1 and VRL-1, respectively. Small and medium-sized P2X3 receptor-ir neurons contained VR1immunoreactivity (ir), whereas medium-sized and large P2X3 receptor-ir neurons showed VRL-1-ir. The retrograde tracing and immunohistochemical methods revealed that 30% of the TG neurons retrogradely labeled from the facial skin and tooth pulp exhibited P2X3 receptor-ir. The co-expression of P2X3 receptor and VR1 was detected in 16% of cutaneous TG neurons and 6% of tooth pulp neurons. On the other hand, the co-expression of P2X3 receptor and VRL-1 was common in tooth pulp neurons (23%) and rare in cutaneous TG neurons (8%). In the tooth pulp, 95% of P2X3 receptor-ir TG neurons contained VRL-1-ir. The present study indicates that P2X3 receptor-ir TG neurons, which co-express VR-ir, are abundant in the facial skin. The tooth pulp is probably innervated by TG neurons, which contain both P2X3-and VRL-1-ir. D 2003 Published by Elsevier B.V. Theme: Sensory systems Topic: Somatic and visceral afferents Keywords: Facial skin; Immunohistochemistry; P2X3 receptor; Tooth pulp; Trigeminal ganglion; VR1; VRL-1
P2X3 receptor is one of many ligand-gated ion channels that is activated by extracellular ATP [5]. In the dorsal root ganglion (DRG) and trigeminal ganglion (TG), P2X3 receptor-containing neurons mainly have small to mediumsized cell bodies and co-express calcitonin gene-related peptide (CGRP), a marker for small to medium-sized nociceptors in the sensory ganglia [2,6,10,12,14,16,17]. In addition, the projection site of P2X3 receptor-containing DRG neurons is the superficial lamina of the spinal dorsal horn [14,15]. Therefore, P2X3 receptor-containing neurons are thought to have a function for nociceptive transduction. This idea is supported by previous findings that tooth pulp primary neurons contain the ion channel [1,5,11,13], because the tooth pulp is considered to be innervated exclusively by nociceptive afferents with cell bodies in the TG. * Coresponding author. Tel.: +81-86-235-6637; fax: +81-86-235-6612. E-mail address: [email protected] (H. Ichikawa). 0006-8993/$ - see front matter D 2003 Published by Elsevier B.V. doi:10.1016/j.brainres.2003.11.019
The capsaicin (vanilloid) receptor subtype I (VR1) is an ion channel which can be activated by vanilloid compounds, protons and heat (>43 jC) [3]. In the DRG and TG, VR1 is localized to small and medium-sized neurons with unmyelinated axons [3,7,9,13]. Vanilloid receptor 1like receptor (VRL-1), a newly cloned capsaicin receptor homologue, is activated by high temperatures with a threshold >52 jC [4]. In the sensory ganglia, VRL-1containing neurons are medium-sized to large and have myelinated axons [4,8,13]. VR1- and VRL-1-containing neurons co-express CGRP, supply the skin with free nerve endings and project to the superficial laminae of the spinal and medullary dorsal horns [4,7,8]. The tooth pulp is also innervated by VR1- and VRL-1-containing TG neurons [8,9,13]. Recently, the co-expression of VR1 and P2X3 has been demonstrated in the DRG [7]. A large proportion of DRG neurons and their terminals co-expressed these ion channels. However, the co-expression of P2X3 recep-
H. Ichikawa, T. Sugimoto / Brain Research 998 (2004) 130–135
tor with VR1 or VRL-1 has never been reported in the TG. In this study, we examined the TG neurons innervating the facial skin and the tooth pulp for possible co-expression P2X3 and other ion channels. Five TGs were obtained from three 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 and post-fixed with the same fixative for 30 min, soaked in a phosphate-buffered 20% sucrose solution overnight, frozen sectioned at 12 Am, and thaw-mounted on gelatincoated glass slides. For simultaneous visualization of P2X3 receptor with VR1 and VRL-1, a double immunofluorescence method was used. The sections were incubated for 24 h at room temperature with a mixture of rabbit anti-P2X3 receptor serum (1:500, Neuromics, USA) and goat anti-VR1 serum (1:200, Santa Cruz Biotechnology, USA) or with a mixture of guinea pig anti-P2X3 receptor serum (1:500, Neuromics) and rabbit anti-VRL-1 serum (1:1500). The sections were then treated with a mixture of lissamine rhodamine B chloride-conjugated donkey anti-rabbit IgG (1:500 for P2X3 receptor or VRL-1, Jackson ImmunoResearch Labs, USA) and either fluorescein isothiocyanate-conjugated donkey anti-goat IgG (1:100 for VR1, Jackson ImmunoResearch Labs) or fluorescein isothiocyanate-conjugated
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donkey anti-guinea pig IgG (1:100 for P2X3, Jackson ImmunoResearch Labs). For demonstration of P2X3 receptor-ir TG neurons innervating the facial skin and tooth pulp, 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 Al of 1% fluorogold (FG, Fluorochrom, Englewood, CO, USA) in distilled water was injected into the infraorbital skin or right first maxillary molar tooth pulp. After 3 days, the animals were reanesthetized with ether, and transvascularly perfused with 4% formaldehyde. The right TGs were frozen-sectioned at 12 Am, mounted on gelatin-coated glass slides, and processed for the co-expression of P2X3 receptor with VR1 or VRL-1 as described above. The brainstem of the rats, which had received tracer application from the tooth pulp, was also cryosectioned at 50 Am and examined with fluorescent illumination for possible FG labeling. All these FG-labeled cells are considered to be primary neurons innervating the tooth pulp and the possibility of periodontal spread of FG through the apical foramen is negligible, because the labeled neuron was not observed in the mesencephalic trigeminal tract nucleus. For cell size analysis of P2X3 receptor-ir neurons and FG-labeled neurons, the cross-sectional area of fluorescent cell bodies that contained nuclear profiles was measured on glossy prints (165). For the control, rabbit and guinea-pig anti-P2X3 receptor sera were preabsorbed with the protein (20 Ag/ml,
Fig. 1. Immunofluorescent microphotographs for P2X3 receptor (A, C), VR1 (B), and VRL-1 (D) in the TG. Small to medium-sized P2X3 receptor-ir neurons contain VR1-ir (arrows in A, B), whereas medium-sized to large P2X3 receptor-ir neurons show VRL-1-ir (arrows in C, D). Arrowheads in A – D point to P2X3 receptor-ir neurons, which are devoid of VR1- and VRL-1-ir. Bar = 100 Am (A). All panels are at the same magnification.
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H. Ichikawa, T. Sugimoto / Brain Research 998 (2004) 130–135
Neuromics). No staining was observed in the control. The specificities of other antibodies have been described elsewhere [4,9].
1. The distribution and cell size of P2X3 receptor-ir neurons in the TG The TG contained abundant P2X3 receptor-ir neurons (Fig. 1A, C). These neurons were scattered throughout the ganglion. The immunoreactivity (ir) was detected in the cytoplasm but not the nucleus. P2X3 receptor-ir TG neurons were predominantly small to medium-sized (range = 93.8 – 1844.4 Am2, mean F S.D. = 503.8 F 286.5 Am2); 44.5% (360/809) of P2X3 receptor-ir TG neurons were smaller
Fig. 3. Histograms showing the cell size spectrum of cutaneous (A) and tooth pulp (B) TG neurons with or without P2X3 receptor-ir. The data were obtained from 328 cutaneous neurons and 163 tooth pulp neurons.
Fig. 2. Histograms showing the cell size spectra of P2X3 receptor-ir neurons with or without VR1-ir (A) and VRL-1-ir in the TG. The data for VR1-ir and VRL-1-ir were obtained from 401 and 409 P2X3 receptor-ir TG neurons, respectively.
than 400 Am2 and 48.5% (392/809) fell in the range of 400– 1000 Am2. Only 7.1% (57/809) of P2X3 receptor-ir TG neurons were larger than 1000 Am2. As described previously [7,8,9,13], the TG contained many VR1- and VRL-1-ir neurons (Fig. 1B, D). VR1-ir neurons were mostly small to medium-sized, and VRL-1-ir neurons were medium-sized to large. Our double immunofluorescence method revealed the co-expression of P2X3 receptor with VR1 and VRL-1; 34.7% (139/401) and 8.6% (35/408) of P2X3 receptor-ir neurons were immunoreactive for VR1 and VRL-1, respectively (Fig. 1A –D). Conversely, 39.8% (139/349) of VR1-ir neurons and 48.6% (35/72) of VRL-1-ir neurons showed P2X3 receptor-ir. About 40% (70/177) of small P2X3 receptor-ir neurons and 34.3% (68/ 198) of medium-sized P2X3 receptor-ir neurons exhibited VR1-ir (Fig. 2A). Large P2X3 receptor-ir neurons were mostly devoid of VR1-ir (3.8% or 1/26). On the other hand, small P2X3 receptor-ir neurons were devoid of VRL-1-ir
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(Fig. 2B). About 10% (22/194) of medium-sized P2X3 receptor-ir neurons and 42% (13/31) of large P2X3 receptor-ir neurons co-expressed VRL-1-ir.
2. P2X3 receptor-ir TG neurons innervating the facial skin and tooth pulp At 3 days after FG application to the infraorbital skin or upper molar tooth pulp, many cell bodies were labeled in the TG. They were located in the maxillary division of the ganglion. Cutaneous TG neurons were predominantly small to medium-sized (small: 29.0% or 95/328, medium-sized: 52.1% or 171/328, large: 18.9% or 62/328, Fig. 3A), whereas tooth pulp TG neurons were mostly medium-sized to large (small: 15.3% or 25/163, medium-sized: 58.3% or 95/163, large: 26.4% or 43/163, Fig. 3B). Retrograde tracing and immunohistochemical methods revealed that 32.3% (106/328) and 31.3% (51/163) of TG neurons innervating the facial skin and tooth pulp, respectively, contained P2X3 receptor-ir. As shown in Fig. 3A, 40% (38/95) of small, 31.0% (53/171) of medium-sized, and 24.2% (15/62) of large cutaneous neurons were immunoreactive for the ion channel. On the other hand, 20% (5/25) of small and 34.7% (33/95) of medium-sized tooth pulp
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neurons showed P2X3 receptor-ir (Fig. 3B). Thirty percent (13/43) of large tooth pulp neurons exhibited the ir. The double immunofluorescence method demonstrated that 15.8% (23/146) of cutaneous neurons co-expressed P2X3 receptor- and VR1-ir (Fig. 4A –C). About 40% (23/ 63) of P2X3 receptor-ir cutaneous neurons contained VR1ir. Twenty-nine percent (42/146) of cutaneous neurons contained VR1-ir and a half (54.8% or 23/42) of VR1-ir cutaneous neurons showed P2X3 receptor-ir. On the other hand, only 6.0% (11/182) of cutaneous neurons coexpressed P2X3 receptor- and VRL-1-ir (Fig. 4D – F). Twenty-six percent (11/43) of P2X3 receptor-ir cutaneous neurons exhibited VRL-1-ir. VRL-1-ir was detected in 12.6% (23/182) of cutaneous neurons and a half (47.8% or 11/23) of VRL-1-ir cutaneous neurons expressed P2X3 receptor-ir. About 8% (6/79) and 22.6% (19/84) of tooth pulp neurons co-expressed P2X3 receptor-ir with VR1-ir and VRL-1-ir, respectively (Fig. 5A –D). About 20% (6/31) of P2X3 receptor-ir tooth pulp neurons showed VR1-ir. Ten percent (8/79) of tooth pulp neurons contained VR1-ir and 75% (6/8) of VR1-ir tooth pulp neurons contained P2X3 receptor-ir. On the other hand, P2X3 receptor-ir tooth pulp neurons mostly co-expressed VRL-1-ir (95% or 19/20, Fig. 4D – F). Half (46.4% or 39/84) of tooth
Fig. 4. Immunofluorescent microphotographs for FG retrogradely transported from the facial skin (A, D), P2X3 receptor (B, E), VR1 (C), and VRL-1 (F) in the TG. Panels A – C and D – F show the same fields of view, respectively. FG-labeled TG neurons (arrows in A, D) co-express P2X3 receptor-ir with VR1-ir (arrows in B, C) and VRL-1-ir (arrows in E, F). Arrowheads in A – F point to P2X3 receptor-ir cutaneous neurons which are devoid of VR1-ir (A – C) and VRL1 (D – F). A bar = 100 Am (A). All panels are at the same magnification.
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Fig. 5. Immunofluorescent microphotographs for FG retrogradely transported from the tooth pulp (A, D), P2X3 receptor (B, E), VR1 (C), and VRL-1 (F) in the TG. Panels A – C and D – F show the same fields of view, respectively. In tooth pulp TG neurons, the co-expression of P2X3 receptor-ir and VR1-ir was rare. However, P2X3 receptor-ir tooth pulp neurons were usually immunoreactive for VRL-1. Arrowheads in A – C point to P2X3 receptor-ir tooth pulp neurons which are devoid of VR1-ir. Arrows and arrowheads in D – F point to VRL-1-ir tooth pulp neuron with and without P2X3 receptor-ir, respectively. Bar = 100 Am (A). All panels are at the same magnification.
pulp neurons showed VRL-1 and half (48.7% or 19/39) of VRL-1-ir tooth pulp neurons had P2X3 receptor-ir. The present study demonstrated that the P2X3 receptor was localized to primary sensory neurons in the TG. These neurons were predominantly small to medium-sized. Our double immunofluorescence study also revealed the co-expression of P2X3 receptor with VR1 and VRL-1 in the TG. Small and medium-sized P2X3 receptor-ir neurons showed VR1-ir, whereas medium-sized and large P2X3-ir neurons contained VRL-1-ir. A previous study demonstrated that the co-expression was very rare in the TG [14]. Therefore, P2X3 receptor-ir TG neurons may coexpress either VR-1-ir or VRL-1-ir. VR-1-and VRL-1-ir TG neurons are thought to be of unmyelinated and myelinated axons, respectively [3,4,7,8]. Therefore, P2X3 receptor-ir TG neurons probably send their unmyelinated and myelinated axons to the oro-facial tissues. The present study investigated the distribution of P2X3 receptor in cutaneous and tooth pulp TG neurons. The proportion of P2X3 receptor-ir neurons among cutaneous TG neurons (32%) was similar to that among tooth pulp neurons (31%). However, the co-expression of P2X3 receptor and VR1 was abundant in cutaneous TG neurons (16%) and relatively rare in tooth pulp neurons (6%). On
the other hand, the co-expression of P2X3 receptor and VRL-1 was common in tooth pulp neurons (23%) but not in cutaneous TG neurons (8%). Previous and present studies demonstrated that tooth pulp neurons are mostly larger than cutaneous TG neurons [8,9]. Therefore, it is likely that the co-expression of P2X3 receptor with VR1 and VRL-1 is associated with the difference of their cell size spectra. P2X3 receptor-ir TG neurons which have umyelinated axons are probably abundant in the facial skin. On the other hand, the tooth pulp is thought to be innervated by myelinated P2X3 receptor-ir neurons, because P2X3-ir tooth pulp mostly co-expressed VRL-1-ir. In conclusion, we have described the co-expression of P2X3 receptor with VR1 and VRL-1 in the TG. Small to medium-sized P2X3 receptor-ir neurons exhibited VR1-ir, whereas medium-sized to large P2X3-ir neurons showed VRL-1-ir. The co-expression of P2X3 with VR1 and VRL-1 was common in cutaneous and tooth pulp TG neurons, respectively. In the tooth pulp, P2X3 receptor-ir neurons mostly contained VRL-1-ir. These findings indicate that P2X3 receptor-ir TG neurons which co-express VR1-ir are abundant in the facial skin. The tooth pulp is probably innervated by TG neurons which have both P2X3 receptorand VRL-1-ir.
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