parvalbumin- and calretinin-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

ELSEVIER Brain Research 679 (1995) 205-211

BRAIN RESEARCH

Research report

Parvalbumin- and calretinin-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

Hiroyuki Ichikawa a,*, Toru Deguchi b, Tadao Nakago b, David M. Jacobowitz c, Tomosada Sugimoto a

a Second Department of Oral Anatomy, Okayama 700. Japan b Department of Orthodontics, Okayama University Dental School, Okayama 700, Japan

c Laboratory of Clinical Sciences, National Institute of Mental Health, Bethesda, MD 20892, USA

Accepted 7 February 1995

Abstract

Calcium-binding proteins and neuropeptides were examined in trigeminal neuronal cell bodies retrogradely labeled with Fast blue (FB) from the maxillary molar tooth pulp of the rat. FB-labeled cells were located in the maxillary division of the trigeminal ganglion. ~ 30 and 50% of the labeled cells were immunoreactive for parvalbumin and calcitonin gene-related peptide (CGRP), respectively. The coexpression of these substances was observed in 9.5% of FB-labeled cells. On the other hand, 2.4% of FB-labeled cells exhibited calretinin-immunoreactivity (CR-ir) and 20% tachykinin (TK)-ir. The coexpression of CR and TK was observed in 1.9% of FB-labeled cells, i.e., most of CR-ir FB-labeled neurons coexpressed TK-ir. An immuno-EM method revealed that all parvalbumin-ir nerve fibers in the root pulp were myelinated and that CGRP-ir nerve fibers were both myelinated (15%) and unmyelinated (85%). The present study indicated that primary nociceptors innervating the rat molar tooth pulp contained parvalbumin and CR and coexpressed these calcium-binding proteins and neuropeptides. It was suggested that peripheral axons of parvalbumin-ir tooth pulp primary neurons are all myelinated. Most peripheral CR-ir axons are probably unmyelinated because TK-ir myelinated axons have never been demonstrated in any peripheral organ.

Keywords: Parvalbumin; Calretinin; Trigeminal ganglion; Tooth pulp; Rat

I. Introduct ion

Parvalbumin and calretinin (CR) are widely distributed in the central and peripheral nervous systems [1,3,4,7,9-13,15,17,25,40]. In the dorsal root (DRG) and trigeminal ganglia (TG), these calcium-binding proteins (CaBPs) are colocalized to large primary neurons that exhibit a high level of carbonic anhydrase (CA) activity [10]. CA, CR and parvalbumin have been considered to be markers for the proprioceptors in the D R G [4,10,23,26,34]. However, we recently demonstrated that a substantial population of CR-containing T G neurons has small cell bodies and coexpressed tachykinin-immunoreactivity (TK-ir) rather than CA

* Corresponding author. Second Department of Oral Anatomy, Okayama University Dental School, 2-5-1 Shikata-Cho, Okayama 700, Japan. Fax: (81) (86) 222-4572.

0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 2 3 4 - 0

activity [12] and that their peripheral axons supply the oral and nasal mucosa with fine intra-epithelial endings [11,15]. Together with the fact that the proportion of proprioceptive primary neurons is small in the T G [24], these studies suggest that CaBP is not a specific marker for the proprioceptors in the TG.

The tooth pulp has been considered to be innervated by exclusively nociceptive afferents in the TG. Their neurons exhibit enzyme activity for CA or immunoreactivity for TK and calcitonin gene-related peptide (CGRP) [8,22,30-32,37,38]. Our previous studies demonstra ted that the tooth pulp received parvalbumin- and CR-ir innervation and that CR-ir pulpal nerve fibers also contained TK-ir [9]. Like CGRP- and TK-ir nerve fibers, these CaBPs-containing neurites project their fine terminals to the subodontoblastic and odontoblastic layers.

In this study, the TG ceils innervating the tooth pulp were retrogradely labeled with Fast blue (FB) and

206 H. lchikawa et al. / Brain Research 679 (1995) 205-211

Fig. 1. Double-immunofluorescent microphotographs of parvalbumin- and CGRP-irs in trigeminal neurons retrogradely labeled with FB from the first and second molar tooth pulps. One FB-labeled neuron (large arrows in A) exhibits both parvalbumin- (large arrows in B) and CGRP-irs (large arrows in C) while other three FB-labeled neurons are immunoreactive only for parvalbumin (small arrows in A-C). Arrowheads in A-C indicate a FB-labeled neuron without parvalbumin- nor CGRP-irs which is surrounded by CGRP-ir nerve fibers. In this particular field, no FB-labeled cell body exhibits CGRP-ir alone. A bar in A indicates 50 mm. Figs. A-C are at the same magnification.

i m m u n o s t a i n e d for CaBPs and neuropept ides . Possible coexpression of these substances was also examined by a doub le - immunof luorescence method. CA activity was not examined in this study since pre l iminary experi- ments indicated that the FB label in terfered with the

CA activity.

2. Materials and methods

Five adult male S p r a g u e - D a w l e y rats (200-300 g) were used for the re t rograde- t rac ing and immunof luo-

rescence methods. U n d e r deep anesthesia by i.p. injec- t ion with ethyl carbamate (650 m g / k g ) and pento- barb i ta i sodium (20 mg/kg ) , 2% FB (Sigma, USA) in distilled water was appl ied to the left first and second maxillary molar pulps. The method for the tracer appli- cat ion was descr ibed in detail elsewhere [31,32]. After 3 days, the animals were reanes the t ized with e ther to

the level at which respira t ion was markedly suppressed and transvascularly perfused with 50 ml saline followed by 500 ml 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). The left t r igeminal gangl ion and the part of the bra in stem, including the mesencephal ic t r igeminal tract nucleus, were postfixed with the same fixative for 30 min, immersed in phosphate -buf fe red 20% sucrose solut ion overnight, horizontal ly f rozen-sect ioned at 12

/xm and m o u n t e d on gela t in-coated glass slides. The bra in stem of the rats, that had received tracer application, was cryosectioned at 50 /xm and examined with f luorescent i l luminat ion for possible FB-labeling.

A doub le - immunof luorescence method was performed for the histochemical demons t ra t ion of CaBPs and neuropept ides . Sections were incuba ted with a mixture of mouse monoc lona l an t i -parva lbumin antibody (1:5000; Sigma) and rabbi t a n t i - C G R P serum (1:1000; Cambr idge Research Biochemicals, USA) or of rabbi t an t i -CR serum (1:1000) [40] and rat mono-

Fig. 2. Electron micrographs of parvalbumin- (A and B) and CGRP-irs (C and D) in horizontal sections of the molar radices. Parvalbumin-ir products are all localized in the axoplasm of myelinated fibers that were mostly medium to large fibers (small arrows in A). One parvalbumin-ir profile lacking myelin sheath (large arrow in A) is extremely large for an unmyelinated axon and considered to be a terminal specialization of an otherwise myelinated axon. A closer view (B) of the paranodal segment of parvalbumin-ir axon (arrowheads in A and B) shows a marked accentuation of the axolemma compared with the internodal segment (an arrow in B). The serial section analysis shows the parvalbumin-ir paranodal axon to acquire the compact myelin in a different sectioning plane (B, inset). CGRP-ir products are mostly localized in the axoplasm of unmyelinated fibers (small arrows in C). Although small myelinated axons occasionally exhibit CGRP-ir, large myelinated axons are always devoid of it. At a higher magnification, strong reaction can be observed in a thin myelinated fiber (a large arrow in D) and some unmyelinated fibers (small arrows in D). Bars in C and D indicate 5 and 1 /xm, respectively. Figs. A and C and B and D, respectively, are at the same magnifications.

H. Ichikawa et al. / Brain Research 679 (1995) 205-211 207

clonal anti-TK antibody (1:1000; Sera Lab., UK) for 24 h at 4 ° C. Subsequently, sections were incubated in a mixture of fluorescein isothiocyanate (FITC)-con-

jugated donkey anti-mouse IgG (1:100; Jackson Im- munoResearch Labs, USA) and lissamine rhodamine-B chloride (LRITC)-conjugated donkey anti-rabbit IgG

k,,

208 H. lchikawa et al. / Brain Research 679 (1995) 205-211

(1:500; Jackson ImmunoResearch Labs) for parvalbumin and CGRP or in a mixture of LRITC-conjugated donkey anti-rabbit IgG (1:500) and FITC-conjugated goat anti-rat IgG (1:500; Cappel, USA) for CR and TK. The sections were viewed with an Olympus epifluores- cent microscope with a U filter for FB, a B filter for FITC and a G filter for LRITC. There was generally little or no cross-over fluorescence between FB, FITC and LRITC. However, cells which were heavily labeled with FITC and LRITC showed a faint green and red cross-over fluorescence with a U filter. This was easily distinguished from the blue fluorescence of FB.

For an immuno-EM method, five animals were deeply anesthetized with pentobarbital sodium (40 mg/kg i.p.) and transvascularly perfused with saline following by 0.1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). Maxillae containing molar teeth were dissected and decalcified with 4.13% ethylene diaminetetraacetic acid disodium salt in 0.1 M phosphate buffer (pH 7.4) for 3 weeks at 4 ° C. Then, these materials were soaked in a phosphate-buffered 20% sucrose solution overnight. Un- frozen 50 mm-thick horizontal sections through the molar radices were cut with a Microslicer (Dosaka EM, Japan). An ABC (avidin-biotin-horseradish peroxidase complex) method was performed for parvalbumin or CGRP stain. Sections were incubated with mouse mon- oclonal anti-parvalbumin antibody (1:50,000) or rabbit anti-CGRP serum (1:30,000) for 3-5 days at 4°C followed by incubation with biotinylated anti-mouse or anti-rabbit IgG and ABC complex (Vector Labs., USA). Immunoreaction products were visualized with di- aminobenzidin. Parvalbumin- or CGRP-immuno- stained sections were postfixed in 1% osmium tetrox- ide in 0.1 M phosphate buffer (pH 7.4), dehydrated through a graded series of ethanols and embedded in Polybed 812. Some sections were examined without further stain while others after staining with lead cit- rate for 10-40 s.

The specificities of primary antibodies used in this study have been described elsewhere [9,14,16,40].

3. Results

3.1. Retrograde-tracing study

At 3 days after FB application to the maxillary molar tooth pulps, many cell bodies were labeled in the TG (up to 26 cells/section). They had various intensi- ties of FB fluorescence in the cytoplasm and nucleus and were localized to the maxillary division of the ganglion. All these FB-labeled cells were considered to be primary neurons innervating the tooth pulp and the possibility of periodontal spread of FB through the apical foramen is negligible because the labeled neu-

rons were not observed in the mesencephalic trigeminal tract nucleus.

The immunohistochemical method revealed that FB-labeled trigeminal primary neurons contained CaBPs and neuropeptides. ~ 30% (68/221) and 50% (113/221) of the labeled cells were immunoreactive for parvalbumin and CGRP, respectively. The coexpression of these substances was observed in 9.5% (21/221) of FB-labeled cells (Fig. 1A-C). 30.1% (21/68) of parvalbumin-ir FB-labeled cells showed CGRP-ir while 18.6% (21/113) of CGRP-ir perikarya were immunoreactive for parvalbumin. 21.3% (47/221) of the labeled cells exhibited parvalbumin alone and 41.6% (92/221) were immunoreactive only for CGRP.

2.4% (5/211) and 20% (43/211) of FG-labeled cells exhibited CR- and TK-irs, respectively (photomicro- graphs not shown). The coexpression of CR and TK was observed in 1.9% (4/211) of FB-labeled cells. Most CR-ir FB-labeled neurons coexpressed with TK-ir (4/5 or 80%) while ~ 10% (4/43) of TK-ir ones also exhibited CR-ir. Only one FB-labeled cell (< 1%, 1/211) exhibited CR-ir but lacked TK-ir in all the examined ganglia. 18.5% (39/211) of FB-labeled cells contained TK-ir alone.

3.2. Ultrastructure of parvalbumin- and CGRP-ir nerve fibers in root pulp

Parvalbumin- and CGRP-ir nerve fibers were easily identified with an EM at a low magnification by electron dense granular precipitates in their axoplasm (Fig. 2A,C). The total subpopulations of nerve fibers containing parvalbumin- and CGRP-irs were 16.8% (22/131 in three nerve bundles) and 37.7% (26/66 in two bundles), respectively. The distribution of the immunoreaction products in the axoplasm was similar for both parvalbumin- and CGRP-irs. At a higher magnification, the immunoreaction products were extensively distributed over the neurofilaments, microtubles and the membrane. Among the membranes, the outer membrane of mitochondria, axolemma and agranular reticulum exhibited the immunoreactivity. Cristae of mitochondria were always devoid of the reaction product. No reaction product was detected in the Schwann cell, including the myelin. The parvalbumin-ir axons were all myelinated (22/22) and mostly belonged to the largest class of the pulpal fibers observed in this study (mostly, 3-5 t~m in short diameter, including the myelin sheath thickness) (Fig. 2A,B). Besides the myelinated fibers, we found one extremely large (5 tzm in short diameter) unmyelinated profile that exhibited parvalbumin-ir (Fig. 2A). We suspect this profile to be a terminal specialization of otherwise myelinated axons. On the other hand, CGRP-ir profiles included both myelinated (15.4% or 4/26, 2-3 fzm in diameter, including the myelin sheath thickness) and unmyelin-

H. Ichikawa et al. / Brain Research 679 (1995) 205-211 209

ated fibers (84.6% or 22/26, axon diameters of 0.1-1 /xm). Most CGRP-ir fibers had a structure typical of unmyelinated fibers; i.e., multiple axons shared a single sheath of Schwann cell cytoplasm. These Schwann units usually contained both CGRP-ir and immunonegative axons.

4. Discussion

The present study demonstrated that the tooth pulp primary neurons exhibited parvalbumin- and CR-ir. This is consistent with our previous observation that nerve fibers in the rat molar tooth pulp contained these CaBPs [9]. A small but substantial population of TG neurons coexpresses parvalbumin and CGRP though they are extremely rare in the DRG [10]. Our double-immunofluorescence method revealed that trigeminal neurons coexpressing parvalbumin and CGRP innervated the molar tooth pulp. Because parvalbumin-ir nerve fibers in the root pulp were considered to be all myelinated, peripheral axons of the tooth pulp primary neurons coexpressing parvalbumin and CGRP were also all myelinated. On the other hand, the present observation that 80% of CR-ir tooth pulp primary neurons contained TK-ir is consistent with our previous findings that most CR-ir pulpal nerve fibers contained TK-ir [9]. Because the tooth pulp contains only a few CR- and TK-ir nerve fibers [9], these nerve fibers were not examined by an immuno-EM method in this study. However, it is suggested that pulpai fibers coexpressing CR and TK are unmyelinated for TK-ir myelinated fibers have never been demonstrated in peripheral organs.

The tooth pulp primaries have larger cell bodies than cutaneous TG neurons [30,33]. Our previous study demonstrated that ~ 30% of trigeminal neuronal cell bodies retrogradely labeled from the rat tooth pulp showed CA activity [30-32]. Because CA activity was not examined in this study, we did not get any evidence for the coexistence of CaBPs and CA activity. Never- theless, we believe that parvalbumin-ir tooth pulp primary neurons contain CA activity because 90% of parvalbumin-containing trigeminal neurons exhibit CA activity [10]. This suggestion is supported by our EM findings that both parvalbumin-ir and CA-containing pulpal fibers are large and myelinated [32].

We demonstrated that the rat root pulp contained two types of CR-ir nerve fibers, smooth (TK-ir) and varicose (TK-immunonegative) fibers [9]. These fibers are considered to be derived from TK-ir and TK-immunonegative tooth pulp primary neurons, respectively. Because CR-ir trigeminal neurons are divided into two subtypes, TK-ir and CA-containing ones [12], the TK-immunonegative tooth pulp primary neurons and their peripheral axons may contain CA activity. If

this was the case, it would also be predicted that the TK-negative subtypes of CR-ir tooth pulp primary neurons have large myelinated axons.

The tooth pulp primary neurons project mainly to the rostral subdivisions of the brain stem trigeminal sensory nuclear complex [2,21,27,30,35,39]. In addition, smaller populations of tooth pulp primary projections are directed to laminae I and II of the medullary dorsal horn (trigeminal subnucleus caudalis) as well as the solitary tract nucleus [30,35]. Because the solitary tract nucleus and the superficial laminae of the dorsal horn do not appear to receive dense innervation of parvalbumin-ir primary neurons [4], parvalbumin-ir tooth pulp primary neurons probably project to the rostral subdivisions of the trigeminal nuclear complex. This may be supported by the previous findings that parvalbumin was localized to large primary neurons [3,4,10] and that large trigeminal primary neurons pro- jected to the rostral subdivisions of the trigeminal sensory complex [36]. On the other hand, CGRP-ir fibers have been demonstrated in both caudal and rostral subdivisions of the trigeminal nuclear complex [19,20,28]. Because the projection sites of parvalbumin- ir tooth pulp primary neurons are thought to be the rostral subdivisions, the tooth pulp primaries coexpressing parvalbumin and CGRP probably project to the rostral subdivisions.

TK-ir neurons are a subset of CGRP-ir ones in the primary sensory ganglia [18,29]. TK-ir projection has been repeatedly shown in laminae I and II of the medullary dorsal horn and the solitary tract nucleus [5,6,41]. Because CR-ir fibers in the superficial dorsal horn are insensitive to dorsal root ganglionectomy [22], this area is not the major projection site of CR-ir primary neurons. Lines of evidence suggest that tooth pulp primaries coexpressing CR and TK project to the solitary tract nucleus and that those exhibiting TK alone project to the superficial laminae of the medullary dorsal horn [1,5,6,15,22,41].

In conclusion, we have demonstrated that tooth pulp primary neurons contain CaBPs. The coexistence and immuno-EM analysis indicated that the parvalbumin-ir (CGRP-ir or -immunonegative) and CR-ir (TK- Jr) neurons send their myelinated and unmyelinated axons, respectively, to the molar tooth pulp. The projection site of parvalbumin-containing tooth pulp primary neurons is probably the rostral subdivisions of the trigeminal nucleus whereas CR-containing ones may project mainly to the solitary tract nucleus.

References

[1] Arai, R., Winsky, L., Arai, M. and Jacobowitz, D.M., Immuno- histochemical localization of calretinin in the rat hindbrain, J. Comp. Neurol., 310 (1991) 21-44.

210 11. Ichikawa et al. /Brain Research 679 (1995) 205-211

[2] Arvidsson, J. and Gobel, S., An HRP study of the central projections of primary trigeminal neurons that innervate tooth pulp in the cat, Brain Res., 210 (1981) 1-16.

[3] Carr, P.A., Yamamoto, T., Karmy, G., Baimbridge, K.G. and Nagy, J.I., Analysis of parvalbumin and calbindin D28K- immunoreactive neurons in dorsal root ganglia of rat in relation to their cytochrome oxidase and carbonic anhydrase content, Neuroscience, 33 (1989) 363-371.

[4] Celio, M.R., Calbindin D-28k and parvalbumin in the rat nervous system, Neuroscience, 35 (1990) 375-475.

[5] Cuello, A.C. and Kanazawa, I., The distribution of substance P immunoreactive fibers in the rat central nervous system, J. Comp. Neurol., 178 (1978) 129-156.

[6] Cuello, A.C., Fiacco, M.D. and Paxinos, G., The central and peripheral ends of the substance P-containing sensory neurons in the rat trigeminal system, Brain Res., 152 (1978) 499-509.

[7] Duc, C., Barakat-Walter, I. and Proz, B., Peripheral projections of calretinin-immunoreactive primary sensory neurons in chicken hindlimbs, Brain Res., 622 (1993) 321-324.,

[8] Fried, K., Arvidsson, J., Robertson, B., Brodin, E. and Theodor- sson, E., Combined retrograde tracing and enzyme/immuno- histochemistry of trigeminal ganglion cell bodies innervating tooth pulps in the rat, Neuroscience, 33 (1989) 101-109.

[9] Ichikawa, H., Deguchi, T., H., Mitani, S., H., Nakago, T., Jacobowitz, D.M., Yamaai, T. and Sugimoto, T., Neural parvalbumin and calretinin in the tooth pulp, Brain Res., 647 (1994) 12~4-130.

[10] Ichikawa, H., Deguchi, T., H., Nakago, T., Jacobowitz, D.M. and Sugimoto, T., Parvalbumin, calretinin and carbonic anhydrase in the trigeminal and spinal primary neurons of the rat, Brain Res., 655 (1994) 241-245.

[11] Ichikawa, H., Jacobowitz, D.M. and Sugimoto, T., Calretinin- immunoreactivity in the oro-facial and pharyngeal regions of the rat, Neurosci. Lett., 146 (1992) 155-158.

[12] Ichikawa, H., Jacobowitz, D.M. and Sugimoto, T., Calretinin- immunoreactive neurons in the trigeminal and dorsal root ganglia of the rat, Brain Res., 617 (1993) 96-102.

[13] Ichikawa, H., Jacobowitz, D.M., Winsky, L. and Helke, C.J., Calretinin-immunoreactivity in vagal and glossopharyngeal sensory neurons of the rat; distribution and coexistence with puta- tive transmitter agents, Brain Res., 557 (1991) 316-321.

[14] Ichikawa, H., Matsuo, S., Wakisaka, S. and Akai, M., Fine structure of calcitonin gene related-peptide-immunoreactive nerve fibers in the rat temporomandibular joint, Arch. Oral BioL, 35 (1990) 727-730.

[15] Ichikawa, H., Mitani, S., Hijiya, H., Nakago, T., Jacobowitz, D.M. and Sugimoto, T,, Calretinin-immunoreactivity in the trigeminal neurons innervating the nasal mucosa of the rat, Brain Res., 629 (1993) 231-238.

[16] Ichikawa, H., Nishikawa, S., Wakisaka, S., Matsuo, S., Takano, Y. and Akai, M., Coexistence of vasoactive intestinal polypeptide- and substance P-like immunoreactivities in the tongue of the guinea pig, Neurosci. Lett., 89 (1988) 283-285.

[17] Jacobowitz, D.M. and Winsky, L., Immunohistochemical localization of calretinin in the forebrain of the rat, J. Comp. Neurol., 304 (1991) 198-218.

[18] Ju, G., H6kfelt, T., Brodin, E., Fahrenkrug, J., Fischer, J.A., Frey, P., Elde, R.P. and Brown, J.C., Primary sensory neurons of the rat showing calcitonin gene related-peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecyctokinin- immunoreactive ganglion cells, Cell Tissue Res., 247 (1987) 417- 431.

[19] Kawai, Y., Takami, K., Shiosaka, S., Emson, P.C., Hillyard, C.J., Girgis, S., Maclntyre, I. and Tohyama, M., Topographic localization of calcitonin gene-related peptide in the rat brain: an immunohistochemical analysis, Neuroscience, 15 (1985) 747-763.

[20] Kruger, L., Sternini, C., Brecha, N.C. and Mantha, P.W., Distri- bution of calcitonin gene-related peptide immunoreactivity in relation to the rat central somatosensory projection, J. Comp. NeuroL, 273 (1988) 149-162.

[21] Marfurt, C.F. and Turner, D.F., The central projections of tooth pulp afferent neurons in the rat as determined by the transganglionic transport of horseradish peroxidase, J. Comp. Neurol., 223 (1984) 535-547.

[22] Olgart, L., H6kfelt, T., Nilsson, G. and Pernow, B., Localization of substance P-like immunoreactivity in nerves in the tooth pulp, Pain, 4 (1977) 153-159.

[23] Peyronnard, J.M., Charron, L., Lavoie, J., Messier, J.P. and Dubreuil, M., Carbonic anhydrase and horseradish peroxidase: double labelling of rat dorsal root ganglion neurons innervating motor and sensory peripheral nerves, Anat. Embryol., 177 (1988) 353-359.

[24] Poter, J.D., Guthrie, B.L. and Sparks, D.L., Innervation of monkey extraocular muscles: localization of sensory and motor neurons by retrograde transport of horseradish peroxidase, J. Comp. Neurol., 218 (1983) 208-219.

[25] Ren, K., Ruda, M.A. and Jacobowitz, D.M., Immunohistochem- ical localization of calretinin in the dorsal root ganglion and spinal cord of the rat, Brain Res. Bull., 31 (1993) 13-22.

[26] Riley, D.A., Ellis, S. and Bain, J.L.W., Ultrastructural cyto- chemical localization of carbonic anhydrase activity in rat peripheral sensory and motor nerves, dorsal root ganglia and dorsal column nuclei, Neuroscience, 13 (1984) 189-206.

[27] Shigenaga, Y., Suemune, S., Nishimura, M., Nishimori, T., Sato, H., Ishidori, H., Yoshida, A., Tsuru, K., Tsuiki, Y., Dateoka, Y., Nasution, I.D. and Hosoi, M., Topographic representation of lower and upper teeth within the trigeminal sensory nuclei of adult cat as demonstrated by the transganglionic transport of horseradish peroxidase, J. Comp. Neurol., 251 (1986) 299-316.

[28] Skofitsch, G. and Jacobowitz, D.M., Calcitonin gene-related peptide: detailed immunohistochemical distribution in the rat central nervous system, Peptides, 6 (1985a) 721-746.

[29] Skofitsch, G. and Jacobowitz, D.M., Calcitonin gene-related peptide: coexists with substance P in capsaicin sensitive ganglia of the rat, Peptides, 6 (1985b) 747-754.

[30] Sugimoto, T. and Takemura, M., Tooth pulp primary neurons: cell size analysis, central connection, and carbonic anhydrase activity, Brain Res. Bull., 30 (1993) 221-226.

[31] Sugimoto, T., Takemura, M., Ichikawa, H. and Akai, M., Car- bonic anhydrase activity in the trigeminal primary afferent neuronal cell bodies with peripheral axons innervating the mandibular molar tooth pulps of the rat, Brain Res., 505 (1989) 354-357.

[32] Sugimoto, T., Takemura, M. and Mukai, N., Histochemical demonstration of carbonic anhydrase activity within the mandibular molar and incisor tooth pulps of the rat, Brain Res., 529 (1990) 245-254.

[33] Sugimoto, T., Takemura., M. and Wakisaka, S., Cell size analysis of primary neurons innervating the cornea and tooth pulp of the rat, Pain, 32 (1988) 375-381.

[34] Szabolcs, M.J., Kopp, M. and Schaden, G.E., Carbonic anhydrase activity in the peripheral nervous system of rat: the enzyme as a marker for muscle afferents, Brain Res., 492 (1989) 129-138.

[35] Takemura, M., Sugimoto, T. and Shigenaga, Y., Difference in central projection of primary afferents innervating facial and intraoral structures in the rat, Exp. Neurol., 111 (1991) 324-331.

[36] Tashiro, T., Higo, S. and Matsuyama, T., Soma size comparison of the trigeminal ganglion cells giving rise to the ascending and descending tracts: a horseradish peroxidase study in the cat, Exp. Neurol., 84 (1984) 37-46.

[37] Uddman, R., Grunditz, T. and Sundler, F., Calcitonin gene-related peptide: a sensory transmitter in dental pulps? Scand. J. Dent. Res., 94 (1986) 219-226.

H. Ichikawa et al. /Brain Research 679 (1995) 205-211 211

[38] Wakisaka, S., Nishikawa, S., Ichikawa, H., Matsuo, S., Takano, Y. and Akai, M., The distribution and origin of substance P-like immunoreactivity in the rat molar pulp and periodontal tissue, Arch. Oral Biol., 30 (1985) 813-818.

[39] Westrum, L.E., Canfield, R.C. and O'Connor, T.A., Each ca- nine tooth projects to all brain stem trigeminal nuclei in cat, Exp. Neurol., 74 (1981) 787-799.

[40] Winsky, L., Nakata, H., Martin, B.M. and Jacobowitz, D.M., Isolation, partial amino acid sequence, and immunohistochemical localization of a brain-specific calcium-binding protein, Proc. Natl. Acad. Sci. USA, 86 (1989) 10139-10143.

[41] Zhang, W.-B., Li, J.S. and Li, H.M., SP-like immunoreactivity in the primary trigeminal neurons projecting to the nucleus tractus solitarii, Brain Res., 558 (1991) 87-89.

parvalbumin- and calretinin-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

Documents