calbindin-28k-immunoreactivity in the trigeminal ganglion neurons and molar tooth pulp of the rat

ELSEVIER

BRAIN RESEARCH

Brain Research 7 I5 (1996) 7 l-78

Research report

Calbindin-D28k-immunoreactivity in the trigeminal ganglion neurons and molar tooth pulp of the rat

Hiroyuki Ichikawa ” * , Toru Deguchi b, Yoshiaki Fujiyoshi b, Tadao Nakago b, David M. Jacobowitz ‘, Tomosada Sugimoto a

a Second Depurtment of Oral Anatomy, Okayama Unirersity Dental School, 2-5-l Shikata-Cho, Okqamu 700. Japan b Department of Orthodontics, Okayama Unixrsity Dental School, 2-5-I Shikata-Cho, Okayama 700, Japan

’ Laboratory of Clinical Sciences, National Znstitute of Mental Health, Bethesda, MD 20892, USA

Accepted 5 December 1995

Abstract

The cell body size and coexpression of carbonic anhydrase (CA), calretinin (CR) and calcitonin gene-related peptide (CGRP) of primary neurons with calbindin-D28k (CB) was examined in the trigeminal ganglion (TG) of the rat. CB-immunoreactive (-ir) cells were mostly large and preferentially distributed in the maxillary and mandibular divisions of the TG. 48% of CB-ir TG cells exhibited enzyme CA activity. 10% of CB-ir TG cells contained CR-ir. Most TG cells coexpressing CB- and CR-irs were localized to the maxillary and mandibular divisions and exhibited CA activity. 6.5% of CB-ir TG cells coexisted with CGRP-ir. 46% of TG cells coexpressing CB and CGRP exhibited CA activity. The innervation of the molar tooth pulp by CB-ir TG primary neurons was also examined. CB-ir thick and smooth nerve fibers projected from the root pulp to the pulp horn and the roof of the pulp chamber, where they became thinner and rarely entered the subodontoblastic layer. However, they could not be traced to the odontoblastic layer, predentin or dentine. The distribution pattern of CB-ir pulpal fibers was different from that of CR-ir ones. The trigeminal neuronal cells retrogradely labeled with fast blue (FB) from the maxillary molar tooth pulp contained CB- and CR-irs. 23% and 1% of the labeled cells were immunoreactive for CB and CR. respectively. The coexpression of CB- and CR-immunoreactivities (-irs) in FB-labeled cells was negligible. An immunoelectron microscopic method revealed that 21% of pulpal nerve fibers were immunoreactive for CB. and that all CB-ir nerve fibers in the root pulp were myelinated. The present study indicated that the tooth pulp primary neurons contained CB-ir but did not coexpress CB- and CR-irs and that these neurons projected their myelinated axons to the pulp.

Keywords: Calbindin-DZHk: Calretinin; Carbonic anhydrase: Trigeminal ganglion; Tooth pulp; Immunohistochemistry; Rat

1. Introduction

The use of immunohistochemistry has enabled us to classify primary afferent neurons into several subpopula- tions on the basis of their chemical markers [15,17,19,29- 3 11. Neuropeptides such as tachykinin (TK) and calcitonin gene-related peptide (CGRP) have been considered to be markers specific to small to medium-sized dorsal root ganglion (DRG) cells. TK- and CGRP-immunoreactive (-ir) primary neurons supply their peripheral receptive field with free nerve endings, project to the superficial dorsal horn, and are thus considered to participate in nociception and/or thermal sensation [5,6,16,18-20,28.30,3 11. Immu- noelectron microscopy revealed that the axons of these peptide-containing primary neurons are mostly unmyelin-

* Corresponding author. Fax: (8 1) (86) 222.4572.

0006.8993/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0006.8993(95)01550-7

ated except for a small subpopulation that are finely myelinated [ 10,181.

In the last decade, we have accumulated knowledge about chemical markers for large myelinated primary neurons subserving sensory modalities other than noci- or thermo-reception. In the DRG, a high level of enzymatic activity of carbonic anhydrase (CA) was localized to large neuronal cell bodies [2,3,9,12,22.26,35]. CA activity was detected in many large myelinated axons in peripheral nerves supplying musculature but only rarely in cutaneous nerves [22.26,35]. Therefore, the high level of CA activity is considered to be a highly specific marker of muscular proprioceptive primary neurons in the peripheral nervous system. Parvalbumin, calretinin (CR) and calbindin-D28k (CB) are calcium-binding proteins (CaBPs) that are also recognized as markers specific to muscular afferents. Their distribution in the DRG is similar to that of CA activity,

12 H. Ichikawa et al. /Brain Research 715 (19%) 71-78

and the central destination of these CaBP-containing primary neurons was demonstrated to be proprioceptive relay nuclei rather than the spinal dorsal horn [2,4,7,9,17,24,25,31]. Further, double stain studies indicated that these CaBP-containing neurons in the DRG were subclasses of the CA-positive DRG neurons [2,9].

We have demonstrated that the trigeminal ganglion (TG) contained numerous neuronal cell bodies with CA activity or immunoreactivities for parvalbumin and CR [9,10,12,14]. This is contradictory to the previous notion that primary neurons with these markers are proprioceptive because the trigeminal primary proprioceptors have their cell bodies located in the mesencephalic trigeminal tract nucleus. A small number of neurons innervating the extraocular muscles are the only exception for the rule [23]. We also demonstrated in the TG that virtually all parvalbumin-ir cells also exhibited CA activity, while a substantial subpopulation of CR-ir cells lacked histochemically demonstrable CA activity but contained tachykinin-ir [9,12]. Further, CA-containing and parvalbumin-ir TG pri- maries supplied the tooth pulp with myelinated axons. while CR-ir ones supplying the tooth pulp were rare [8,10,32-341. In addition, a substantial population of parvalbumin-ir tooth pulp primary neurons coexpressed CGRP-ir [lo].

In this study, therefore, we have examined the cell body size and coexpression of CA, CR and CGRP by the trigeminal primary neurons with CB. We also demonstrate that the tooth pulp receives innervation of the CB- and CR-ir trigeminal primary neurons and that CB-ir pulpal fibers are myelinated.

2. Materials and methods

Eight male Sprague-Dawley rats (180-250 g) were used for the cell size and coexpression study in the TG or molar tooth pulp. They 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). TGs, maxillae and mandibles containing molar teeth were dissected and immersed in the same fixative. The maxillae and mandibles were decalcified with 4.13% ethylene diaminetetraacetic acid disodium salt (EDTA) in 0.1 M phosphate buffer (pH 7.4) for 3 weeks at 4°C. TGs and decalcified materials were immersed in a phosphate- buffered saline containing 20% sucrose overnight, frozen sectioned at 12 pm, and thaw-mounted on gelatin-coated glass slides.

For cell size analysis of CB-ir TG cells, an ABC (avidin-biotin-horseradish peroxidase complex) method was performed using mouse monoclonal anti-CB antibody (1:20000; Sigma). Sections of ganglia were incubated with the primary antibody for 24 h at 4°C followed by biotinyl- ated horse anti-mouse IgG and ABC-complex (Vector

Laboratories). Following nickel ammonium sulfate-in- tensified diaminobenzidine reaction, the sections were dehydrated in a graded series of alcohols, cleared in xylene and cover-slipped with Entellan (Merck). The microscopic image (X 215) of the cell bodies was projected over a digitizer tablet using a drawing tube. The cross-sectional area of those cell bodies that contained the nuclear profile was recorded.

For the coexpression study in the TG and molar tooth pulp, a double immunofluorescence method was used. Sections of ganglia and molar tooth pulps were incubated with a mixture of the mouse monoclonal anti-CB antibody (1:500) and rabbit anti-CR serum [39] (l:lOOO), or of the mouse anti-CB antibody and rabbit anti-CGRP serum (1:lOOO; Cambridge Research Biochemicals) for 24 h at 4°C. Subsequently, sections were incubated in a mixture of fluorescein isothiocyanate (FIT0conjugated donkey anti- mouse IgG (1: 100; Jackson ImmunoResearch Labs) and lissamine rhodamine B chloride (LRITC)-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch Labs). After taking pictures with a fluorescent microscope, some sections of ganglia were further processed for CA enzyme histochemistry using a modification of Hansson’s method [32].

For demonstration of the origin of CB and CR in the tooth pulp, four male rats (200-300 g) were used. Under deep anesthesia by i.p. injection with ethyl carbamate (650 mg/kg) and pentobarbital sodium (20 mg/kg), 2% fast blue (FB, Sigma, USA) in distilled water was applied to the left first and second maxillary molar pulps. The method for the retrograde tracer application was described in detail elsewhere [33]. After 3 days, the animals were reanes- thetized with ether, and transvascularly perfused with 4% formaldehyde. The left trigeminal ganglion and the part of the brainstem including the mesencephalic trigeminal tract nucleus were frozen, sectioned and mounted on gelatin- coated glass slides. The brainstem of the rats that had received tracer application, was cryosectioned at 50 pm and examined with fluorescent illumination for possible FB labeling. A double immunofluorescence method for CB and CR was performed as described above. The sections were viewed with an Olympus epifluorescent 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 electron microscopic analysis of CB-ir pulpal fibers, male rats (180-250 g) were deeply anesthetized with ether and transvascularly perfused with 0.05% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). Maxillae containing molar teeth were dissected and decalcified with EDTA. Then. unfrozen 50 pm-thick horizontal sections through the molar radices were cut with a Mi-

H. Ichikawa et al. / Brain Research 715 11996) 71-78 13

croslicer (Dosaka EM, Japan) and stained for CB-ir with an ABC method as described above. The sections were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4), dehydrated through a graded series of alcohols, and embedded in Polybed 812. Some sections were examined without further stain, while others after staining with lead citrate for lo-40 s.

The specificities of the antibodies used have been al- ready described elsewhere [ 11,13,39].

3. Results

3.1. Cell size analysis of CB-ir TG neurons

CB-ir cells were distributed throughout the TG, though those in the maxillary and mandibular divisions were predominant. CB-ir TG cells measured 157.1 l-2052.07 w2 (mean 5 S.D. = 795.06 + 375.78 pm*, n = 363). 97.23% of CB-ir cells were distributed in a range of 200-1800 pm*. Although most of them were distributed in the large class of the TG cells (> 400 pm*, 83.18%), medium CB-ir TG cells were also observed (200-400 pm*, 14.05%). Small CB-ir cells were very rare (< 200 pm2, 0.83%).

3.2. Coexpression of CB with CA, CR and CGRP in the TG

A half of CB-ir TG cells exhibited CA activity (48.1% or 305/634 in 6 sections from 4 ganglia). 9.7% (26/267)

of CB-ir TG cells exhibited CR-ir, while 8.6% (26/304 in 3 sections from 2 ganglia) of CR-ir cells showed CB-ir (Fig. lA-C). Most of TG cells coexpressing CB- and CR-irs were located in the caudolateral half (maxillary and mandibular divisions) of the TG (88.5% or 23/26) and exhibited CA activity (80.8% or 21/26). 15.2% of cells coexpressing CB-ir and CA activity also exhibited CR-ir in the TG.

6.5% (24/367 in 3 sections from 2 ganglia) of CB-ir TG cells contained CGRP-ir. A half (45.8% or 11/24) of TG cells coexpressing CB and CGRP showed CA activity, while only 6.6% (1 l/167) of TG cells coexpressing CB-ir and CA activity exhibited CGRP-ir.

3.3. CB- and CR-ir nerve fibers in the tooth pulp

The molar tooth pulp in the maxilla and mandible contained CB-ir smooth but not varicose fibers (Fig. 2A). In the root pulp, they are thick and projected straight until they reached the coronal pulp. No CB-ir fibers were detected in the vicinity of the odontoblast in the root pulp. On entering the coronal pulp, they sprayed out but the smooth morphology of the individual fibers remained unchanged. Accompanied by blood vessels, CB-ir fibers as- cended toward the pulp horn and the roof of the pulp chamber. A few of them became thinner and entered the subodontoblastic layer (Fig. 2B). However, their smooth morphology remained unchanged. They could not be traced to the odontoblastic layer, predentin or dentine.

As described previously [8], CR-ir varicose and smooth fibers were observed in the root pulp. They projected

Fig. 1 Microphotographs of triple stain for CB (A). CR (B) and CA (12) in the mandibular division of the TG. Some CB-ir TG cells exhibit CR-ir and activit .y (arrows in A. B, C), whereas one CB-ir TG cell lacks CR-ir and CA activity (arrowheads in A, B, C). Bar (in A) = 100 pm, Figs. A, B and C at the same magnification.

CA are

H. Ichikawa et al. / Brain Research 715 (1996) 71-78

Fig. 2. A-D: immunofluorescent microphotographs of CB (A, B, C) and CR (D) in the molar tooth pulp. The root pulp contains thick and smooth CB-ir nerve fibers (A). CB-ir nerve fibers are observed in the subodontoblastic layer of the pulp horn, but not in the odontoblastic layer (B). The double immunofluorescent method reveals that no pulpal nerve fibers coexpress CB- and CR-ir. Arrows in C and D indicate CB-ir CR-immunonegative fibers in the subodontoblastic layer. Bar (in A) = 100 urn. Figs, A, B, C and D are at the same magnification. d = dentine; od = odontoblastic layer. E-F: double immunofluorescent microphotographs of CB (F) and CR (G) in trigeminal neurons retrogradely labeled with FB (E) from the first and second molar teeth. Three FB-labeled neurons show CB-ir but not CR-ir (arrows in E, F and G). Bar (in A) = 100 km. Figs. A. B and C are at the same magnification.

H. Ichikawa et d/Brain Reseurch 715 (1996) 71-78

Fig. 3. Electron micrographs of CB-ir in horizontal sections of the molar root pulp. CB-ir products are all localized in the axoplasm of myelinated that were mostly medium to large fibers (arrows in A). At higher magnification. strong reaction can be observed in a thick myelinated fiber (a big an B). whereas unmyelinated and finely myelinated fibers are always devoid of it (small arrows in B). Bars (in A and B) = 5 and 1 pm, respectively

fibers row in

76 H. lchikawa et al. /Brain Research 715 119961 71-78

toward the roof of the pulp chamber and pulp horn and extended their terminals into the subodontoblastic and odontoblastic layers.

The double immunofluorescence method revealed that the distribution patterns of CB-ir and CR-ir fibers were different in the molar tooth pulp (Fig. 2C and D). In the root pulp, the number of CB-ir nerve fibers was more numerous than that of CR-ir ones. Both CB- and CR-ir nerve fibers were rare in the coronal pulp. The subodontoblastic layer contained abundant CR-ir nerve terminals, while CB-ir ones were rare (Fig. 2C and D). CB-ir smooth fibers were thicker than CR-ir smooth or varicose ones throughout the molar tooth pulp.

3.4. CB- and CR-ir in tooth pulp primary neurons

At 3 days after FB application to the upper molar tooth pulps. many cell bodies were labeled in the TG (up to 26 cells/sections). They had various intensities of FB fluorescence in the cytoplasm and nucleus, and were located in the maxillary division of the ganglion. All these FB-labeled cells are 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 neuron was not observed in the mesencephalic trigeminal tract nucleus.

The retrograde and immunohistochemical labeling methods revealed that FB-labeled trigeminal primary neurons contained CB- and CR-irs (Fig. 2E, F and G). 22.6% (62/274) of the labeled cells were immunoreactive for CB. About 1% (3/274) of FB-labeled cells contained CR-ir. Only 1 FB-labeled cell coexpressed CB and CR, i.e. two FB-labeled CR-ir cells lacked CB-ir.

3.5. Ultrastructure of CB-ir nerue,fibers in the root pulp

CB-ir nerve fibers were easily identified with an electron microscope at a low magnification by electron dense granular precipitates in their axoplasm (Fig. 3A). At a higher magnification, the immunoreaction products were extensively distributed over the neurofilaments, micro- tubles and the membrane (Fig. 3B). Among the mem- branes, 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. A total of 90 axons were examined in 3 nerve bundles. Among these, 53 axons were unmyelinated, while 37 were myelinated. CB-ir axons were all myelinated and constituted 54.1% (20/37) of myelinated axons or 21.2% (20/90) of all the examined population irrespective of myelination. The CB-ir axons mostly belonged to the largest class of the pulpal fibers observed in this study (mostly 3-5 pm in short diameter including the myelin sheath thickness).

4. Discussion

The previous studies of the DRG have demonstrated that CB-ir primary neurons are predominantly large and lack CGRP-ir [2,3]. Present observations extended this rule to the TG. However, there appeared to be difference between the TG and DRG on the content of nerve cells coexpressing CB-ir and CA activity. About 50% of CB-ir neurons in the TG exhibited CA activity. This percentage is relatively low compared with the previous findings in the DRG that exhibited 76% of CB-ir cells with CA activity. The coexpression of CaBPs and CA has been considered to be a specific marker for proprioceptive primary neurons in the DRG [9,12]. A major difference between the DRG and the TG appears to be their content of proprioceptive primary neurons. It is needless to say that primary neuronal cell bodies of the spinal nervous system, including the muscular afferents, are located in the DRG. However, the trigeminal proprioceptors innervating the masticatory muscles and the periodontal ligament are located in the mesencephalic trigeminal tract nucleus. The smaller population of primary neurons coexpressing CB and CA in the TG may reflect a smaller proportion of proprioceptors in the TG compared to the DRG.

A small but substantial population of TG neurons coexpressed CB and CR. We consider that these neurons are large because they also exhibited CA activity and because all neurons exhibiting CA activity are large in the TG and DRG [12]. The neurons coexpressing CB, CR and CA were localized to the maxillary and mandibular divisions but not the ophthalmic division in the TG. While peripheral targets of these neurons were unclear, our retrograde tracing and immunohistochemical methods indicated that such neurons innervating the molar tooth pulp were ex- tremely rare. This is also supported by the present finding that the distribution patterns of CB- and CR-ir nerve fibers were different in the tooth pulp.

The tooth pulp primary neurons exhibited CB- and CR-irs but did not coexpress these CaBPs. Our electron microscopic study revealed that all CB-ir fibers were large and myelinated. Because the root pulp contains only a few CR-ir nerve fibers, the ultrastructure of these nerve fibers was not examined in this study. However, it is suggested that most CR-ir pulpal fibers are unmyelinated because they contain TK-ir and because TK-ir myelinated fibers have never been demonstrated in peripheral organs. Thus, CB- and CR-ir trigeminal neurons probably supply the molar tooth pulp with their myelinated and unmyelinated axons, respectively. The present study also demonstrated that CR-ir tooth pulp primary neurons were very rare but that CR-ir nerve fibers were abundant in the subodontoblastic layer. This would indicate that CR-ir unmyelinated nerve fibers arborize repeatedly and terminate in the subodontoblastic layer.

Our previous studies have demonstrated that about 30% of trigeminal neuronal cell bodies retrogradely labeled

H. lchikawa et al. /Brain Reseurch 715 f 19961 7/&7X 77

from the rat tooth pulp show the enzyme activity for CA or the immunoreactivity for parvalbumin [ 10,32-341. Because CA activity or parvalbumin-ir was not examined in this study, we did not obtain any evidence for the coexistence of CB with CA and parvalbumin in the tooth pulp primary neurons. However, we believe that CB-ir tooth pulp primary neurons also contained CA activity and parvalbumin- ir, because CB-ir primary neurons in the DRG coexpress CA and parvalbumin [2] and because pulpal fibers containing these calcium binding proteins and CA were large and myelinated [10,34]. If this is the case, it might be deduced that the coexpression of CaBPs and CA is not a specific marker for proprioceptors in the TG.

The tooth pulp primary neurons project mainly to the rostra1 subdivisions of the brainstem trigeminal sensory nuclear complex [ 1,21,27,36,38]. In addition, smaller popu- lations 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 [36]. However, the superficial laminae of the dorsal horn do not appear to receive dense innervation of CB-ir primary neurons [4,24]. The solitary tract nucleus is also unlikely to be a projection site of CB-ir trigeminal neurons, because the trigeminal neurons projecting to the solitary tract nucleus are mainly small to medium [40] and because CB-ir trigeminal neurons were mostly large. Thus, CB-ir tooth pulp primary neurons probably project to the rostra1 subdivisions of the trigeminal nuclear complex. This may be supported by the previous findings that large trigeminal primary neurons project to the rostra1 subdivisions of the trigeminal sensory nuclear complex [37].

In conclusion, the present study indicated that the TG contain neuronal cell bodies coexpressing CB and CA and that they are unlikely to be muscular afferents. A CR-immunonegative subset of CB-ir neurons send their myelinated axons to the molar tooth pulp and are most probably nociceptors. Their central projection site may be the rostra1 subdivisions of the trigeminal sensory nuclear complex.

References

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

[2] 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, Nruroscirnce, 33 (1989) 363-371.

[3] Carr, P.A., Yamamoto, T., Karmy, G.. Baimbridge. K.G. and Nagy. J.I., Parvzalbumin is highly colocalized with calbindin D28K and rarely with calcitonin gene-related peptide in dorsal root ganglia neurons of rat, Brain Res., 497 (1989) 163-170.

[4] Celio, M.R., Calbindin-D28k and parvalbumin in the rat nervous system, Neuroscience, 35 (1990) 3755475.

[5] Cuello. A.C. and Kanazawa, I., The distribution of substance P

immunoreactive fibers in the rat central nervous system. J. Conzp. Neurd.. I78 (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. Bruin Res., 152 (1978) 499-509.

[7] Due. C., Barakat-Walter, 1. and Proz. B., Peripheral projections of calretinin-immunoreactive primary sensory neurons in chicken hindlimbs, Bruin Rrs.. 622 (1993) 321-324.

[X] Ichikawa, H., Deguchi. T., H.. Mitani, S.. H.. Nakago, T.. Ja- cobowitz, D.M., Yamaai, T. and Sugimoto. T., Neural parvalbumin and calretinin in the tooth pulp, Bruin Rrs.. 647 (1994) I24- 130.

[9] 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, Bruin Res., 655 (1994) 241-245.

[IO] Ichikawa, H., Deguchi, T., H., Nakago, T., Jacobowitz. D.M., and Sugimoto. T., Parvalbumin- and calretinin-immunoreactive trigeminal neurons innervating the rat molar tooth pulp. Bruin Res.. 679 (1995) 205-211.

I Ichikawa. H. and Helke, C.J., Parvalbumin and calbindmD28k in vagal and glossopharyngeal neurons of the rat, Brain RL’s., 675 (1995) 337-341.

I 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.

[ 131 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. Ord/ Bid., 35 (1990) 727- 730.

[14] 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.

[15] Ju, G.. Hiikfelt. 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 Tiss. Res.. 247 (1987) 417-431.

[ 161 Kawai, Y.. Takami, K.. Shiosaka. S., Emson. P.C., Hillyard. C.J.. Girgis, S., MacIntyre, I. and Tohyama, M.. Topographic localization of calcitonin gene-related peptide in the rat brain: an immunohistochemical analysis, Neurosciencr, I5 (1985) 747-763.

[ I71 Kazimierczak. J., Sommer, E.W.. Philippe. E. and Droz, B.. Car- bonic anhydrase activity in primary sensory neurons, I. Require- ments for the cytochemical localization in the dorsal root ganglion of chicken and mouse by light and electron microscopy, Cc/l Tim. Res.. 245 (1986) 487-495.

[18] Kruger. L.. Sampogna. S.L., Rodin, B.E., Claugue, J.. Brecha, N. and Yeh, Y.. Thin-fiber cutaneus innervation and its intraepidermal contribution studied by labeling methods and neurotoxin treatment in rats, Sonzatosens. Res., 2 (1985) 335-356.

[IY] Kruger, L., Silverman, J.D., Mantha. P.W., Sternini, C. and Brecha. N.C., Peripheral patterns of calcitonin gene-related peptide general somatic sensory innervation: Cutaneus and deep terminations. .I. Camp. Neurnl., 280 (1989) 29 I-302.

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

[2l] 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. Camp. Neural., 223 (1984) 535-547.

]22] Peyronnard. J.M., Charron, L.. Lavoie. J., Messier, J.P. and Dubreuil,

H. Ichikaw et al/Brain Research 715 (19961 71-78

M., Carbonic anhydrase and horseradish peroxidase: double la- belling of rat dorsal root ganglion neurons innervating motor and sensory peripheral nerves. Anat. Embryl., 177 (1988) 353-359.

[23] 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. Camp. Neural., 218 (1983) 208-219.

[24] Ren, K. and Ruda, M.A., A comparative study of the calcium-binding proteins calbindin-D28k, calretinin. calmodulin and parvalbumin in the rat spinal cord, Brain Res. Rw., 19 (1994) 163-179.

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

[26] Riley. D.A., Ellis, S. and Bain, J.L.W.. Ultrastructural cytochemical 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. Camp. Neural., 251 (1986) 299-316.

[28] Silverman, J.D. and Kruger, L., Calcitonin gene-related peptide-immunoreactive innervation of the rat head with emphasis on special- ized sensory structures, J. &nip. Neural., 280 (1989) 303-330.

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

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

[3l] Sommer. E.W., Kazimierczak. J. and Droz, B., Neuronal subpopula-

tions in the dorsal root ganglion of the mouse as characterized by combination of ultrastructural and cytochemical features. Brain Rex, 346 (1985) 310-326.

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

[33] Sugimoto, T.. Takemura, M., Ichikawa, H. and Akai, M., Carbonic 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.

[34] 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 Rex, 529 (1990) 245-254.

[35] 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.

[36] Takemura, M., Sugimoto. T. and Shigenaga, Y., Difference in central projection of primary afferents innervating facial and intrao- ral structures in the rat, Exp. Neuro/.. I1 1 (1991) 324-331.

[37] 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. Neural., 84 (1984) 37-46.

[38] Westrum, L.E.. Canfield, R.C. and O’Connor, T.A., Each canine tooth projects to all brainstem trigeminal nuclei in cat. I!?.x~. Neural., 74 (1981) 787-799.

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

[40] 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 Rex, 558 (1991) 87-89.

calbindin-28k-immunoreactivity in the trigeminal ganglion neurons and molar tooth pulp of the rat

Documents