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Calretinin-like immunoreactivity in the Ruffini endings, slowly adapting mechanoreceptors, of the periodontal ligament of the rat incisor
Brain Research 769 Ž1997. 183–187
Short communication
Calretinin-like immunoreactivity in the Ruffini endings, slowly adapting mechanoreceptors, of the periodontal ligament of the rat incisor Kanako Ochi
a,b,)
, Satoshi Wakisaka c , Suk Hyun Youn c , Kooji Hanada b , Takeyasu Maeda
a
a
Department of Oral Anatomy, Niigata UniÕersity School of Dentistry, Niigata, Japan Department of Orthodontics, Niigata UniÕersity School of Dentistry, Niigata, Japan Department of Oral Anatomy and DeÕelopmental Biology, Osaka UniÕersity Faculty of Dentistry, Osaka, Japan b
c
Accepted 1 July 1997
Abstract The distribution and ultrastructural localization of calretinin ŽCR.-like immunoreactivity Ž-LI. were investigated in the lingual periodontal ligament of rat incisors. Some thick nerve fibers within the nerve bundle displayed CR-LI; these CR-like immunoreactive Ž-IR. nerve fibers entered the alveolar half of the lingual periodontal ligament of the incisor where dendritic terminal arborization was exhibited. Thin and beaded CR-IR nerve fibers were rarely observed in the periodontal ligament. Observations of adjacent sections immunostained with protein gene-product 9.5 ŽPGP 9.5. revealed that most, if not all, PGP 9.5-IR nerve terminals showing a dendritic arborization expressed CR-LI. Immunoelectron microscopic observations showed that electron-opaque immunoreaction products were localized in the axoplasm of the axon terminals, except for the mitochondria, which were surrounded by Schwann sheaths and multiple-layered basal lamina. Neither cell bodies, the cytoplasmic extension of terminal Schwann cells, nor other cellular elements such as periodontal fibroblasts exhibited CR-LI. The present findings suggest that Ruffini endings, an essential mechanoreceptor in the periodontal ligament and categorized as a slowly adapting mechanoreceptor, express CR-LI, and that CR may participate in the Ca2q homeostasis against external stimuli in the periodontal Ruffini endings. q 1997 Elsevier Science B.V. Keywords: Calretinin; Ruffini ending; Mechanoreceptor; Periodontal ligament; Rat
Calretinin ŽCR., a member of calcium binding proteins ŽCaBP. and first discovered as a product of a retinal cDNA clone w21x, has more than a 60% homology with calbindin D28k ŽCB. w22x. There are two types of CaBP: ‘trigger’ and ‘buffer’ CaBP Žw2x, for review.. The ‘trigger’-type CaBP act by changing shape upon binding Ca2q, and trigger the activity of neighboring enzymes. Calmodulin, for example, belongs to this category. The ‘buffering’-type CaBP simply bind Ca2q and control the intracellular Ca2q concentration. Calretinin and CB are representative CaBP of the latter type. Immunohistochemical studies have revealed that CR-LI is distributed in both central and peripheral nervous systems w3,8,9,11–14,20,22x. It is known that at least two types of nerve endings exist in the periodontal ligament among various animals:
) Corresponding author. Department of Oral Anatomy, Niigata University School of Dentistry, 2-5274 Gakkocho-dori, Niigata 951, Japan. Fax: q81 Ž25. 223-6499; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 8 4 7 - 0
mechanoreceptive Ruffini and free nerve endings. The Ruffini ending, categorized as a slowly adapting stretch mechanoreceptor w4,7,18x, has been reported to be an essential mechanoreceptor in the periodontal ligament of rodents w5,6,15–17,19,23,24x. Free nerve endings are thought to be the nociceptors. Recent studies have demonstrated that CR-LI is distributed in the free nerve ending in the oro-facial regions and in the cutaneous mechanoreceptors w8,9,11,13x. Moreover, some neurons in the trigeminal ganglion and mesencephalic trigeminal nucleus, where the somata of periodontal primary afferents are located, show CR-LI w3,12,13,20x. Taking all these together, it has been speculated that CR may be present in the periodontal primary afferents. In the present study, we examined the distribution and ultrastructural localization of CR-LI in the lingual periodontal ligament of the rat incisor. The distribution of CR-LI was further compared to that of protein gene-product 9.5 ŽPGP 9.5.-LI, one of the general neuronal markers w10,27x. Adult male Sprague–Dawley rats, weighing 200–250 g,
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were deeply anesthetized with chloral hydrate and perfused transcardically with 0.02 M phosphate-buffered saline ŽPBS. followed by 4% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4.. Some animals were perfused with a mixture of 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer for immunoelectron microscopy. Mandibles were removed en bloc and further fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for an additional 2–3 days at 48C. They were decalcified with 7.5% ethylene diaminetetraacetic acid ŽEDTA. at 48C for 4–6 weeks with gentle shaking. Following cryoprotection in PBS containing 20% sucrose and 5% glycerol, serial sections were sagittally cut at a thickness of 18 mm in a cryostat and thaw-mounted onto poly-L-lysine-coated glass slides for light microscopy. For immunoelectron microscopy, cryostat sections at a thickness of 50–60 mm were prepared, collected in PBS and treated as free-floating sections. Following treatment with methanol containing 0.3% H 2 O 2 for 30 min to block endogenous peroxidase activity, sections were incubated with PBS containing 3% normal swine serum ŽDako, Glostrup, Denmark. and 1% bovine serum albumin ŽSigma, St. Louis, MO. for 30 min at room temperature. Sections were incubated either with a polyclonal anti-CR antiserum Ž1 : 5000; Chemicon International Inc., Temecula, CA. or with a polyclonal anti-PGP 9.5 antiserum Ž1 : 7000; Ultraclone, Cambridge, UK. for 16–18 h at room temperature. Following several rinses in PBS, they were incubated with a biotinylated anti-rabbit IgG Ž1 : 300; Dako., and subsequently with ABC complex ŽVector, Burlingame, CA. for 90 min each at room temperature. Horseradish peroxidase ŽHRP. was developed by an incubation in 0.05 M Tris-HCl buffer containing 0.04% DAB and 0.003% H 2 O 2 with nickel ammonium sulfate ŽNAS. intensification Ž0.08–0.1% .. Sections were counter-stained with methyl green, dehydrated through an ascending series of ethanol, cleared by xylene and coverslipped with Permount ŽFisher Scientific, NJ.. The protocol for electron microscopy was almost identical to that for light microscopy, except that HRP was developed without NAS intensification. Following DAB reaction, sections were fixed with 1% OsO4 reduced with 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for 60 min, dehydrated through an ascending series of ethanol and embedded in epoxy resin ŽEpon 812; Taab.. Ultrathin sections were prepared with a diamond knife and examined with an H-7000 transmission electron microscope ŽHitachi Co., Tokyo, Japan. under an operating voltage of 75 kV after slight staining with uranyl acetate and lead citrate. The specificity of the primary antibody against CR was examined by preabsorption. The primary antibody was preabsorbed with a recombinant human CR ŽSWant; Bellinzona, Switzerland; 1 mgr1 ml of diluted primary antibody.. Sections incubated with the preabsorbed primary antibody did not show any immunoreactions. The
origin, characterization, and specificity of anti-PGP 9.5 antiserum have been frequently reported elsewhere w10,27x. Thus, we considered the immunoreactions for CR and PGP 9.5 observed in the present study to be specific for CR and PGP 9.5, respectively. Thick PGP 9.5-IR nerve fibers were observed in the nerve bundle, probably representing the inferior alveolar nerve. In the alveolus-related portion of the lingual periodontal ligament of the incisor, PGP 9.5-IR nerve fibers ramified repeatedly, and showed a dendritic terminal arborization ŽFig. 1A,C.. Relatively thin nerve fibers also displayed PGP 9.5-LI ŽFig. 1C.. No PGP 9.5-IR nerve fibers were observed in the tooth-related portion of the lingual periodontal ligament ŽFig. 1A,C.. The distribution of CR-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers ŽFig. 1B,D; compared to Fig. 1A,C, respectively.. Thick CR-IR nerve fibers lay within the nerve bundle, with some of them entering the lingual periodontal ligament. They terminated in a dendritic fashion; this terminal formation resembled that shown by PGP 9.5-immunocytochemistry ŽFig. 1D,E.. Thin CR-IR nerve fibers were rarely observed. Under the electron microscope, the periodontal Ruffini endings were easily identified as expanded axon terminals filled with rich mitochondria, and further surrounded several Schwann sheaths and thick basal lamina ŽFig. 2A.. Electron-opaque immunoreaction products for CR were distributed throughout the ground substances of the axon terminals except for cell organellae such as mitochondria ŽFig. 2B.. All axon terminals of the periodontal Ruffini endings contained CR-immunoreactive materials ŽFig. 2A.. The finger-like projections of the axoplasm, regarded as axonal spines, were filled with CR-immunopositive materials ŽFig. 2B.. The covering Schwann sheaths did not display CR-immunoreactivity ŽFig. 2B.. Further, the cell bodies of the terminal Schwann cells, an analog to lamellar cells of the cutaneous mechanoreceptors, lacked CR-immunoreaction products ŽFig. 2A.. Other cellular elements such as fibroblasts and endothelial cells were immunonegative in the middle of the periodontal ligament ŽFig. 2A.. The present study clearly demonstrated the existence of CR-LI in the lingual portion of the periodontal ligament of the rat. For examination of the colocalization between PGP 9.5- and CR-LI, the double-immunofluorescence method is typically necessary, but we employed the ABC method to the serially cut sections since this method has greater sensitivity than the immunofluorescence for CR. We compared the distributions of PGP 9.5- and CR-LI, and found that most thick nerve fibers displaying a dendritic ramification showed CR-LI. In the immunoelectron microscopic analysis, CR-LI was localized in the expanded axon terminals covered with Schwann cell sheaths, indicating that CR-IR axon terminals should be regarded as periodontal Ruffini endings. Previous studies have shown that CR-LI is localized in the cutaneous mechanoreceptors, such as Pacinian corpuscles, subepithelial lamellar corpuscles in
K. Ochi et al.r Brain Research 769 (1997) 183–187
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Fig. 1. Microphotographs of PGP 9.5- ŽA,C. and CR-IR nerve fibers ŽB,D,E. in the lingual periodontal ligament of the incisor. A,B: lower magnifications of PGP 9.5-LI ŽA. and CR-LI ŽB.. A: PGP 9.5-IR nerve fibers are detected within the nerve bundle Žarrowhead.. In the alveolus-related part ŽARP. of the lingual periodontal ligament, PGP-9.5-IR nerve fibers terminate in a dendritic fashion Žsmall and long arrows.. No PGP 9.5-IR nerve fibers are observed in the tooth-related part ŽTRP.. B: thick CR-IR nerve fibers are observed within the nerve bundle Žarrowhead.. The terminal morphology of CR-IR nerve fibers in the ARP is similar to that of PGP 9.5-IR nerve fibers, but less expanded. Short and long arrows indicate the same terminals shown in A. AB, alveolar bone. Scale bar Žin B. s 100 mm. A and B are at the same magnification. C,D: higher magnifications of PGP 9.5-LI ŽC. and CR-LI ŽD. indicated by long arrows in A and B, respectively. C: PGP 9.5-IR nerve fibers show dendritic terminal arborization. PGP 9.5-LI is also observed in the relatively thin nerve fibers. D: CR-IR nerve fibers also display similar terminal arborization to that of PGP 9.5-IR nerve fibers, but CR-LI is detected in the thick nerve fibers only. Asterisks indicate the same blood vessel. TRP, tooth-related part. E: another CR-IR nerve fiber shows branched terminal morphology. Scale bar Žin E. s 50 mm. C–E are at the same magnification.
the glabrous skin of the rat, and Herbst corpuscles associated with feathers in hindlimbs of the chick; all of these mechanoreceptors are physiologically categorized as rapidly adapting mechanoreceptors, but not the Ruffini endings, which are slowly adapting mechanoreceptors w8,9x. To our knowledge, this paper is the first to report the presence of CR-LI in the Ruffini ending, a slowly adapting mechanoreceptor. We might explain the differences in the expression of CR-LI between the skin and the periodontal ligament by the different environmental conditions. The periodontal ligament is continuously exposed to occlusal forces, and active and constant remodeling of periodontal collagen fibers occurs; the turnover of collagen fibers in the periodontal ligament is known to be faster than that in other connective tissues of the body w25x. As the Ruffini
ending is the only mechanoreceptor in the lingual periodontal ligament of the rodents, and is responsible for all external mechanical stimuli, it is speculated that the periodontal Ruffini ending may have physiological characteristics similar to rapidly adapting mechanoreceptors as well as slowly adapting mechanoreceptors, resulting in the expression of CR-LI in the periodontal Ruffini endings. It is known that the periodontal ligament also contains free nerve endings in addition to Ruffini endings. Recently Ichikawa et al. w13x reported that, in the rat molar tooth pulp, CR-IR nerve fibers appeared thin and beaded. These CR-IR intrapulpal nerve fibers are thought to be unmyelinated nerve fibers, since they co-expressed substance P-LI w13x. Careful observations, however, revealed that thin CR-IR nerve fibers were rarely observed in the periodontal
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Fig. 2. Immunoelectron microphotographs of CR-immunoreactions in the lingual periodontal ligament ŽPL.. A: a low magnification of the alveolus-related part of the rat periodontal ligament. The expanded axon terminals Žasterisk. of the Ruffini endings are filled with CR-immunoreaction products. These terminals are covered with immunonegative Schwann sheaths, and further surrounded by thick multi-layered basal lamina Žarrowheads.. The cell body of terminal Schwann cell ŽTS. lacks CR-immunoreactions. F, fibroblasts. =3000. Scale bar s 5 mm. B: a higher magnification of the axon terminals ŽAT. shown by a thick arrow in A. Electron-dense immunoreactive products are distributed in the ground substance. The axonal spine positive for CR Žarrow. extends through the slits of the immunonegative Schwann sheaths ŽSS.. BL, basal lamina. =11 000. Scale bar s 1 mm.
ligament of rat incisor. Therefore, it is concluded that CR-LI is localized in the Ruffini endings and not in the free nerve endings in the periodontal ligament. It is generally accepted that the somata of primary afferent neurons innervating the periodontal ligament are located in the trigeminal ganglion and mesencephalic trigeminal nucleus. An electron microscopic study combined with an autoradiographic technique w6x has revealed that periodontal Ruffini endings in the rat incisor were labeled following the injection of w 3 Hxthymidine into the trigeminal ganglion, suggesting that the somata of some periodontal Ruffini endings in the incisor are located in the trigeminal ganglion. It has been shown that CR-IR neurons can be detected in the mesencephalic trigeminal nucleus w3,20x. Further combined neuronal tracing and immunohistochemistry will determine whether CR-IR periodontal Ruffini endings have their somata in the mesencephalic trigeminal nucleus. The functional significance of CR in the periodontal Ruffini endings remains to be elucidated. It is believed that
Ca2q plays an important role in mechano-electric transduction w1x. An experimental study showed that mechanical stimuli cause the deformation of the axon terminals of the mechanoreceptor and induce rapid Ca2q entry into the axon terminals as demonstrated by histochemistry of highaffinity Ca2q-ATPase w26x. As one of the possible physiological roles of CR is a buffering of the intracellular Ca2q concentration, CR in the periodontal Ruffini endings may be involved in the Ca2q homeostasis against external stimuli such as constant occlusal forces and remodeling of the periodontal collagen fibers.
Acknowledgements We thank Mr. M. Hoshino for his excellent photographic assistance. This study was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture and Science of Japan ŽNo. 09671849 to S.W. and No. 08457478 to T.M...
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References w1x G.N. Akoev, N.P. Alekseev, B.V. Krylov, Mechanoreceptors, Their Functional Organization, Springer, Berlin, 1988, pp. 1–198. w2x C. Andressen, I. Blumcke, M.R. Celio, Calcium-binding proteins: ¨ selective markers for nerve cells, Cell Tissue Res. 271 Ž1993. 181–208. w3x R. Arai, L. Winsky, L. Arai, D.M. Jacobowitz, Immunohistochemical localization of calretinin the rat hindbrain, J. Comp. Neurol. 310 Ž1991. 21–44. w4x D. Biemesderfer, B.L. Munger, J. Binck, R. Dubner, The pilo-Ruffini complex: a non-sinus hair and associated slowly-adapting mechanoreceptor in primate skin, Brain Res. 165 Ž1978. 197–222. w5x M.R. Byers, Sensory innervation of periodontal ligament of rat molars consists of unencapsulated Ruffini-like mechanoreceptors and free nerve endings, J. Comp. Neurol. 231 Ž1985. 500–518. w6x M.R. Byers, W. Dong, Comparison of trigeminal receptor location and structure in the periodontal ligament of different types of teeth from the rat, cat, and monkey, J. Comp. Neurol. 279 Ž1989. 117–127. w7x M.R. Chambers, K.H. Andres, M. During, A. Iggo, Structure and function of the slowly adapting type II mechanoreceptor in hairy skin, Q. J. Exp. Physiol. 57 Ž1972. 417–445. w8x C. Duc, I. Barakat-Walter, B. Droz, Peripheral projections of calretinin-immunoreactive primary sensory neurons in chick hindlimbs, Brain Res. 622 Ž1993. 321–324. w9x C. Duc, I. Barakat-Walter, B. Droz, Innervation of putative rapidly adapting mechanoreceptors by calbindin- and calretinin-immunoreactive primary sensory neurons in the rat, Eur. J. Neurosci. 6 Ž1994. 264–271. w10x S. Gulbenkian, J. Wharton, J.M. Polak, The visualization of cardiovascular innervation in the guinea pig using an antiserum to protein gene product 9.5 ŽPGP 9.5., J. Auton. Nerv. Syst. 18 Ž1987. 235–247. w11x H. Ichikawa, D.M. Jacobowitz, T. Sugimoto, Calretinin-immunoreactivity in the oro-facial and pharyngeal regions of the rat, Neurosci. Lett. 146 Ž1992. 155–158. w12x H. Ichikawa, D.M. Jacobowitz, T. Sugimoto, Calretinin-immunoreactive neurons in the trigeminal and dorsal root ganglia of the rat, Brain Res. 617 Ž1993. 96–102. w13x H. Ichikawa, T. Deguchi, S. Mitani, T. Nakago, D.M. Jacobowitz, T. Yamaai, T. Sugimoto, Neural parvalbumin and calretinin in the tooth pulp, Brain Res. 647 Ž1994. 124–130. w14x D.M. Jacobowitz, L. Winsky, Immunocytochemical localization of
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calretinin in the forebrain of the rat, J. Comp. Neurol. 304 Ž1991. 198–218. K. Kannari, O. Sato, T. Maeda, T. Iwanaga, T. Fujita, A possible mechanism of mechanoreception in Ruffini endings in the periodontal ligament of hamster incisors, J. Comp. Neurol. 313 Ž1991. 368–376. T. Maeda, T. Iwanaga, T. Fujita, Y. Takahashi, S. Kobayashi, Distribution of nerve fibers immunoreactive to neurofilament protein of rat molars and periodontium, Cell Tissue Res. 249 Ž1987. 13–23. T. Maeda, O. Sato, S. Kobayashi, T. Iwanaga, T. Fujita, The ultrastructure of Ruffini endings in the periodontal ligament of rat incisors with special reference to the terminal Schwann cells ŽKcells., Anat. Rec. 223 Ž1989. 95–103. B.L. Munger, C. Ide, The structure and function of cutaneous sensory receptors, Arch. Histol. Cytol. 51 Ž1988. 1–34. K. Nakakura-Ohshima, T. Maeda, O. Sato, Y. Takano, Postnatal development of periodontal innervation in rat incisors: an immunohistochemical study using protein gene product 9.5 antibody, Arch. Histol. Cytol. 56 Ž1993. 385–398. A. Resibois, J.H. Rogers, Calretinin in rat brain: an immunohisto´ chemical study, J. Comp. Neurol. 46 Ž1992. 101–134. J.H. Rogers, Calretinin: a gene for a novel calcium-binding protein expressed principally in neurons, J. Cell Biol. 105 Ž1987. 1343–1353. J.H. Rogers, Immunoreactivity for calretinin and other calcium-binding proteins in cerebellum, Neuroscience 31 Ž1989. 711–721. O. Sato, T. Maeda, S. Kobayashi, T. Iwanaga, T. Fujita, Y. Takahashi, Innervation of periodontal ligament and dental pulp in the rat incisor: an immunohistochemical investigation of neurofilament protein and glia-specific S-100 protein, Cell Tissue Res. 251 Ž1988. 13–21. O. Sato, T. Maeda, S. Kobayashi, T. Iwanaga, Innervation of the incisors and periodontal ligament in several rodents: an immunohistochemical study of neurofilament protein and glia-specific S-100 protein, Acta Anat. 134 Ž1989. 94–99. J. Sodek, Comparison of the rates of synthesis and turnover of collagen and non-collagen proteins in adult rat periodontal tissues and skin using a microassay, Arch. Oral Biol. 22 Ž1977. 655–665. T. Tachibana, T. Nawa, Ultrastructural localization of Caqq-ATPase in Meissner’s corpuscle of the Mongolian gerbil, Arch. Histol. Cytol. 55 Ž1992. 375–379. R.J. Thompson, J.F. Doran, P. Jackson, A.P. Dhillon, J. Rode, PGP 9.5 – a new marker for vertebrate neurons and neuroendocrine cells, Brain Res. 278 Ž1983. 224–228.
Short communication
Calretinin-like immunoreactivity in the Ruffini endings, slowly adapting mechanoreceptors, of the periodontal ligament of the rat incisor Kanako Ochi
a,b,)
, Satoshi Wakisaka c , Suk Hyun Youn c , Kooji Hanada b , Takeyasu Maeda
a
a
Department of Oral Anatomy, Niigata UniÕersity School of Dentistry, Niigata, Japan Department of Orthodontics, Niigata UniÕersity School of Dentistry, Niigata, Japan Department of Oral Anatomy and DeÕelopmental Biology, Osaka UniÕersity Faculty of Dentistry, Osaka, Japan b
c
Accepted 1 July 1997
Abstract The distribution and ultrastructural localization of calretinin ŽCR.-like immunoreactivity Ž-LI. were investigated in the lingual periodontal ligament of rat incisors. Some thick nerve fibers within the nerve bundle displayed CR-LI; these CR-like immunoreactive Ž-IR. nerve fibers entered the alveolar half of the lingual periodontal ligament of the incisor where dendritic terminal arborization was exhibited. Thin and beaded CR-IR nerve fibers were rarely observed in the periodontal ligament. Observations of adjacent sections immunostained with protein gene-product 9.5 ŽPGP 9.5. revealed that most, if not all, PGP 9.5-IR nerve terminals showing a dendritic arborization expressed CR-LI. Immunoelectron microscopic observations showed that electron-opaque immunoreaction products were localized in the axoplasm of the axon terminals, except for the mitochondria, which were surrounded by Schwann sheaths and multiple-layered basal lamina. Neither cell bodies, the cytoplasmic extension of terminal Schwann cells, nor other cellular elements such as periodontal fibroblasts exhibited CR-LI. The present findings suggest that Ruffini endings, an essential mechanoreceptor in the periodontal ligament and categorized as a slowly adapting mechanoreceptor, express CR-LI, and that CR may participate in the Ca2q homeostasis against external stimuli in the periodontal Ruffini endings. q 1997 Elsevier Science B.V. Keywords: Calretinin; Ruffini ending; Mechanoreceptor; Periodontal ligament; Rat
Calretinin ŽCR., a member of calcium binding proteins ŽCaBP. and first discovered as a product of a retinal cDNA clone w21x, has more than a 60% homology with calbindin D28k ŽCB. w22x. There are two types of CaBP: ‘trigger’ and ‘buffer’ CaBP Žw2x, for review.. The ‘trigger’-type CaBP act by changing shape upon binding Ca2q, and trigger the activity of neighboring enzymes. Calmodulin, for example, belongs to this category. The ‘buffering’-type CaBP simply bind Ca2q and control the intracellular Ca2q concentration. Calretinin and CB are representative CaBP of the latter type. Immunohistochemical studies have revealed that CR-LI is distributed in both central and peripheral nervous systems w3,8,9,11–14,20,22x. It is known that at least two types of nerve endings exist in the periodontal ligament among various animals:
) Corresponding author. Department of Oral Anatomy, Niigata University School of Dentistry, 2-5274 Gakkocho-dori, Niigata 951, Japan. Fax: q81 Ž25. 223-6499; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 8 4 7 - 0
mechanoreceptive Ruffini and free nerve endings. The Ruffini ending, categorized as a slowly adapting stretch mechanoreceptor w4,7,18x, has been reported to be an essential mechanoreceptor in the periodontal ligament of rodents w5,6,15–17,19,23,24x. Free nerve endings are thought to be the nociceptors. Recent studies have demonstrated that CR-LI is distributed in the free nerve ending in the oro-facial regions and in the cutaneous mechanoreceptors w8,9,11,13x. Moreover, some neurons in the trigeminal ganglion and mesencephalic trigeminal nucleus, where the somata of periodontal primary afferents are located, show CR-LI w3,12,13,20x. Taking all these together, it has been speculated that CR may be present in the periodontal primary afferents. In the present study, we examined the distribution and ultrastructural localization of CR-LI in the lingual periodontal ligament of the rat incisor. The distribution of CR-LI was further compared to that of protein gene-product 9.5 ŽPGP 9.5.-LI, one of the general neuronal markers w10,27x. Adult male Sprague–Dawley rats, weighing 200–250 g,
184
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were deeply anesthetized with chloral hydrate and perfused transcardically with 0.02 M phosphate-buffered saline ŽPBS. followed by 4% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4.. Some animals were perfused with a mixture of 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer for immunoelectron microscopy. Mandibles were removed en bloc and further fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for an additional 2–3 days at 48C. They were decalcified with 7.5% ethylene diaminetetraacetic acid ŽEDTA. at 48C for 4–6 weeks with gentle shaking. Following cryoprotection in PBS containing 20% sucrose and 5% glycerol, serial sections were sagittally cut at a thickness of 18 mm in a cryostat and thaw-mounted onto poly-L-lysine-coated glass slides for light microscopy. For immunoelectron microscopy, cryostat sections at a thickness of 50–60 mm were prepared, collected in PBS and treated as free-floating sections. Following treatment with methanol containing 0.3% H 2 O 2 for 30 min to block endogenous peroxidase activity, sections were incubated with PBS containing 3% normal swine serum ŽDako, Glostrup, Denmark. and 1% bovine serum albumin ŽSigma, St. Louis, MO. for 30 min at room temperature. Sections were incubated either with a polyclonal anti-CR antiserum Ž1 : 5000; Chemicon International Inc., Temecula, CA. or with a polyclonal anti-PGP 9.5 antiserum Ž1 : 7000; Ultraclone, Cambridge, UK. for 16–18 h at room temperature. Following several rinses in PBS, they were incubated with a biotinylated anti-rabbit IgG Ž1 : 300; Dako., and subsequently with ABC complex ŽVector, Burlingame, CA. for 90 min each at room temperature. Horseradish peroxidase ŽHRP. was developed by an incubation in 0.05 M Tris-HCl buffer containing 0.04% DAB and 0.003% H 2 O 2 with nickel ammonium sulfate ŽNAS. intensification Ž0.08–0.1% .. Sections were counter-stained with methyl green, dehydrated through an ascending series of ethanol, cleared by xylene and coverslipped with Permount ŽFisher Scientific, NJ.. The protocol for electron microscopy was almost identical to that for light microscopy, except that HRP was developed without NAS intensification. Following DAB reaction, sections were fixed with 1% OsO4 reduced with 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for 60 min, dehydrated through an ascending series of ethanol and embedded in epoxy resin ŽEpon 812; Taab.. Ultrathin sections were prepared with a diamond knife and examined with an H-7000 transmission electron microscope ŽHitachi Co., Tokyo, Japan. under an operating voltage of 75 kV after slight staining with uranyl acetate and lead citrate. The specificity of the primary antibody against CR was examined by preabsorption. The primary antibody was preabsorbed with a recombinant human CR ŽSWant; Bellinzona, Switzerland; 1 mgr1 ml of diluted primary antibody.. Sections incubated with the preabsorbed primary antibody did not show any immunoreactions. The
origin, characterization, and specificity of anti-PGP 9.5 antiserum have been frequently reported elsewhere w10,27x. Thus, we considered the immunoreactions for CR and PGP 9.5 observed in the present study to be specific for CR and PGP 9.5, respectively. Thick PGP 9.5-IR nerve fibers were observed in the nerve bundle, probably representing the inferior alveolar nerve. In the alveolus-related portion of the lingual periodontal ligament of the incisor, PGP 9.5-IR nerve fibers ramified repeatedly, and showed a dendritic terminal arborization ŽFig. 1A,C.. Relatively thin nerve fibers also displayed PGP 9.5-LI ŽFig. 1C.. No PGP 9.5-IR nerve fibers were observed in the tooth-related portion of the lingual periodontal ligament ŽFig. 1A,C.. The distribution of CR-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers ŽFig. 1B,D; compared to Fig. 1A,C, respectively.. Thick CR-IR nerve fibers lay within the nerve bundle, with some of them entering the lingual periodontal ligament. They terminated in a dendritic fashion; this terminal formation resembled that shown by PGP 9.5-immunocytochemistry ŽFig. 1D,E.. Thin CR-IR nerve fibers were rarely observed. Under the electron microscope, the periodontal Ruffini endings were easily identified as expanded axon terminals filled with rich mitochondria, and further surrounded several Schwann sheaths and thick basal lamina ŽFig. 2A.. Electron-opaque immunoreaction products for CR were distributed throughout the ground substances of the axon terminals except for cell organellae such as mitochondria ŽFig. 2B.. All axon terminals of the periodontal Ruffini endings contained CR-immunoreactive materials ŽFig. 2A.. The finger-like projections of the axoplasm, regarded as axonal spines, were filled with CR-immunopositive materials ŽFig. 2B.. The covering Schwann sheaths did not display CR-immunoreactivity ŽFig. 2B.. Further, the cell bodies of the terminal Schwann cells, an analog to lamellar cells of the cutaneous mechanoreceptors, lacked CR-immunoreaction products ŽFig. 2A.. Other cellular elements such as fibroblasts and endothelial cells were immunonegative in the middle of the periodontal ligament ŽFig. 2A.. The present study clearly demonstrated the existence of CR-LI in the lingual portion of the periodontal ligament of the rat. For examination of the colocalization between PGP 9.5- and CR-LI, the double-immunofluorescence method is typically necessary, but we employed the ABC method to the serially cut sections since this method has greater sensitivity than the immunofluorescence for CR. We compared the distributions of PGP 9.5- and CR-LI, and found that most thick nerve fibers displaying a dendritic ramification showed CR-LI. In the immunoelectron microscopic analysis, CR-LI was localized in the expanded axon terminals covered with Schwann cell sheaths, indicating that CR-IR axon terminals should be regarded as periodontal Ruffini endings. Previous studies have shown that CR-LI is localized in the cutaneous mechanoreceptors, such as Pacinian corpuscles, subepithelial lamellar corpuscles in
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Fig. 1. Microphotographs of PGP 9.5- ŽA,C. and CR-IR nerve fibers ŽB,D,E. in the lingual periodontal ligament of the incisor. A,B: lower magnifications of PGP 9.5-LI ŽA. and CR-LI ŽB.. A: PGP 9.5-IR nerve fibers are detected within the nerve bundle Žarrowhead.. In the alveolus-related part ŽARP. of the lingual periodontal ligament, PGP-9.5-IR nerve fibers terminate in a dendritic fashion Žsmall and long arrows.. No PGP 9.5-IR nerve fibers are observed in the tooth-related part ŽTRP.. B: thick CR-IR nerve fibers are observed within the nerve bundle Žarrowhead.. The terminal morphology of CR-IR nerve fibers in the ARP is similar to that of PGP 9.5-IR nerve fibers, but less expanded. Short and long arrows indicate the same terminals shown in A. AB, alveolar bone. Scale bar Žin B. s 100 mm. A and B are at the same magnification. C,D: higher magnifications of PGP 9.5-LI ŽC. and CR-LI ŽD. indicated by long arrows in A and B, respectively. C: PGP 9.5-IR nerve fibers show dendritic terminal arborization. PGP 9.5-LI is also observed in the relatively thin nerve fibers. D: CR-IR nerve fibers also display similar terminal arborization to that of PGP 9.5-IR nerve fibers, but CR-LI is detected in the thick nerve fibers only. Asterisks indicate the same blood vessel. TRP, tooth-related part. E: another CR-IR nerve fiber shows branched terminal morphology. Scale bar Žin E. s 50 mm. C–E are at the same magnification.
the glabrous skin of the rat, and Herbst corpuscles associated with feathers in hindlimbs of the chick; all of these mechanoreceptors are physiologically categorized as rapidly adapting mechanoreceptors, but not the Ruffini endings, which are slowly adapting mechanoreceptors w8,9x. To our knowledge, this paper is the first to report the presence of CR-LI in the Ruffini ending, a slowly adapting mechanoreceptor. We might explain the differences in the expression of CR-LI between the skin and the periodontal ligament by the different environmental conditions. The periodontal ligament is continuously exposed to occlusal forces, and active and constant remodeling of periodontal collagen fibers occurs; the turnover of collagen fibers in the periodontal ligament is known to be faster than that in other connective tissues of the body w25x. As the Ruffini
ending is the only mechanoreceptor in the lingual periodontal ligament of the rodents, and is responsible for all external mechanical stimuli, it is speculated that the periodontal Ruffini ending may have physiological characteristics similar to rapidly adapting mechanoreceptors as well as slowly adapting mechanoreceptors, resulting in the expression of CR-LI in the periodontal Ruffini endings. It is known that the periodontal ligament also contains free nerve endings in addition to Ruffini endings. Recently Ichikawa et al. w13x reported that, in the rat molar tooth pulp, CR-IR nerve fibers appeared thin and beaded. These CR-IR intrapulpal nerve fibers are thought to be unmyelinated nerve fibers, since they co-expressed substance P-LI w13x. Careful observations, however, revealed that thin CR-IR nerve fibers were rarely observed in the periodontal
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Fig. 2. Immunoelectron microphotographs of CR-immunoreactions in the lingual periodontal ligament ŽPL.. A: a low magnification of the alveolus-related part of the rat periodontal ligament. The expanded axon terminals Žasterisk. of the Ruffini endings are filled with CR-immunoreaction products. These terminals are covered with immunonegative Schwann sheaths, and further surrounded by thick multi-layered basal lamina Žarrowheads.. The cell body of terminal Schwann cell ŽTS. lacks CR-immunoreactions. F, fibroblasts. =3000. Scale bar s 5 mm. B: a higher magnification of the axon terminals ŽAT. shown by a thick arrow in A. Electron-dense immunoreactive products are distributed in the ground substance. The axonal spine positive for CR Žarrow. extends through the slits of the immunonegative Schwann sheaths ŽSS.. BL, basal lamina. =11 000. Scale bar s 1 mm.
ligament of rat incisor. Therefore, it is concluded that CR-LI is localized in the Ruffini endings and not in the free nerve endings in the periodontal ligament. It is generally accepted that the somata of primary afferent neurons innervating the periodontal ligament are located in the trigeminal ganglion and mesencephalic trigeminal nucleus. An electron microscopic study combined with an autoradiographic technique w6x has revealed that periodontal Ruffini endings in the rat incisor were labeled following the injection of w 3 Hxthymidine into the trigeminal ganglion, suggesting that the somata of some periodontal Ruffini endings in the incisor are located in the trigeminal ganglion. It has been shown that CR-IR neurons can be detected in the mesencephalic trigeminal nucleus w3,20x. Further combined neuronal tracing and immunohistochemistry will determine whether CR-IR periodontal Ruffini endings have their somata in the mesencephalic trigeminal nucleus. The functional significance of CR in the periodontal Ruffini endings remains to be elucidated. It is believed that
Ca2q plays an important role in mechano-electric transduction w1x. An experimental study showed that mechanical stimuli cause the deformation of the axon terminals of the mechanoreceptor and induce rapid Ca2q entry into the axon terminals as demonstrated by histochemistry of highaffinity Ca2q-ATPase w26x. As one of the possible physiological roles of CR is a buffering of the intracellular Ca2q concentration, CR in the periodontal Ruffini endings may be involved in the Ca2q homeostasis against external stimuli such as constant occlusal forces and remodeling of the periodontal collagen fibers.
Acknowledgements We thank Mr. M. Hoshino for his excellent photographic assistance. This study was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture and Science of Japan ŽNo. 09671849 to S.W. and No. 08457478 to T.M...
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