Calretinin-immunoreactivity in trigeminal neurons innervating the nasal mucosa of the rat

231

Brain Research, 629 (1993) 231-238 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 19458

Calretinin-immunoreactivity in trigeminal neurons innervating the nasal mucosa of the rat Hiroyuki Ichikawa a, Seiji Mitani b, Hiroyuki Hijiya b, Tadao Nakago b, David M. Jacobowitz c, Tornosada Sugimoto a,, a Second Department of OralAnatomy, Okayama University Dental School, Okayama, Japan, b Department of Orthodontics, Okayama University Dental School, Okayama, Japan, c Laboratory of Clinical Sciences, National Institute of Mental Health, Bethesda, MD 20892, USA (Accepted 6 July 1993)

Key words: Calretinin; Tachykinin; Nasal mucosa; Cornea; Trigeminal ganglion; Rat

Trigeminal primary neuronal cell bodies were labeled by retrograde transport of Fluoro-gold (FG) from the nasal mucosa of rats. The trigeminal ganglion containing the labeled cell bodies were processed for double stain for calretinin- and tachykinin-immunoreactivities (CR- and TK-irs). Except for a few contralateral cells, all the cells that innervated the nasal mucosa (NM cells) were confined to the ophthalmo-maxillary division of the trigeminal ganglion ipsilateral to the FG application. In the dorsal two-thirds of the ganglion, NM cells formed a cluster in the rostromedial part of ophthalmo-maxillary division (the rostromedial cluster). In the ventral third, the number of cells in the rostromediai cluster markedly decreased. Instead, numerous NM cells were found in the caudolateral part of the ophthalmo-maxillary division (the caudoventrolateral cluster). CR- and TK-irs were detected in 18% and 54% of overall population of NM cells, respectively. Virtually all of CR-immunoreactive (-ir) NM cells coexpressed TK. Although the proportion of TK-ir cells, irrespective of CR-ir, was similar for both clusters, CR-ir cells were more frequent in the caudoventrolateral cluster than in the rostromedial cluster. In the dorsal 1 / 3 of the ganglion where all the NM cells belonged to the rostromedial cluster, only 8.4% exhibited CR-ir. On the other hand, as much as 30.1% of NM cells expressed CR-ir in the ventral 1/3 where most NM cells were found in the caudoventrolateral cluster. Trigeminal cell bodies innervating the cornea and conjunctivum were located in the rostromedial part of the ophthaimo-maxillary division. About 40% and 4% of them were positive for TK and CR, respectively. As was the case for NM cells, all the CR-ir cells innervating these (peri-)ocular structures exhibited TK-ir. The nasal mucosa contained both CR- and TK-ir free nerve endings within the epithelium. The CR-ir endings almost always coexpressed TK.

INTRODUCTION

Calretinin (CR), a recently described species of the calcium-binding protein (CaBP) family 17"28'29'35'36"39'40'47'48 is widely distributed in sensory neurons of both the central and peripheral nervous systems of the rat 9A3-15'34. Lines of evidence indicate that CaBP-containing dorsal root ganglion neurons are involved in proprioception. For example, calbindin D28K and parvalbumin are localized in medium to large dorsal root ganglion neurons 4-6. These CaBPcontaining primary neurons exhibit high levels of cytochrome oxidase activity 4,5, indicative of high levels of electrophysiologic activity, and contain carbonic anhydrase 4'5 that is a marker for proprioceptive primary neurons 3°'31'37'38'41'42'49'5°. In addition, the external cuneate nucleus and the Clarke's nucleus appear to be their major termination sites 6. Like the above two CaBPs, CR has been reported to be localized in mostly * Corresponding author. Fax: (81) (86) 222-4572.

medium to large dorsal root ganglion n e u r o n s 14'34'47'48. CR-containing dorsal root ganglion neurons mostly co-express carbonic anhydrase 14 and project to the Clarke's nucleus but not to the superficial dorsal hOIT134'36. It is therefore suggested that CR-containing dorsal root ganglion neurons are also involved in proprioception. It is interesting that primary neuronal cell bodies in the trigeminal ganglion also contain CR 13'14, because trigeminal primary proprioceptors innervating the masticatory muscles and periodontal ligaments have their cell bodies in the mesencephalic trigeminal tract nucleus but not in the trigeminal ganglion. While studying the peripheral distribution of CR in the rat trigeminal system, we encountered CR-containing free nerve endings ~3. The CR-containing neurites emitted terminal branches that invaded the epithelium. Some areas of the oral, pharyngeal and laryngeal mucosa were particularly rich in CR-containing free nerve endings,

232 whereas the hairy skin was devoid of them. Furthermore, triple stain analysis indicated that CR-containing trigeminal neurons are divided into two subclasses; one coexpressing tachykinin-immunoreactivity (TK-ir) and the other exhibiting carbonic anhydrase activity 14. Because T K has never b e e n shown to localize in large myelinated axons, and because free nerve endings are considered to be derived from unmyelinated or finely myelinated axons lk12A9,Sl, it is suggested that trigeminal ganglion n e u r o n s coexpressing C R and T K have unmyelinated or finely myelinated peripheral axons and innervate the visceral but not somatic receptive field. T h e present study examines the sensory innervation of the nasal mucosa and cell bodies of origin for CR- and TK-irs. MATERIALS AND METHODS Male Sprague-Dawley rats (180-250 g) were deeply anesthetized by i.p. injection with ethyl carbamate (650 mg/kg) and pentobarbital sodium (20 mg/kg). A cotton pellet (diameter 1 mm) containing 2/,1 Fluoro-gold (FG; 4% in distilled water) was inserted into the commorn nasal meatus and retained for 2 h. Subsequently, the cotton pellet was removed from the nasal cavity. After 5 days, the animals were deeply re-anesthetized with pentobarbital sodium (40 mg/kg, i.p.) and transvascularly perfused with saline for exsanguination followed by 4% formaldehyde (prepared fresh from paraformatdehyde) in 0.1 M phosphate buffer (pH 7.3). The trigeminal ganglia were dissected, soaked in phosphate-buffered 20% sucrose overnight, frozen-sectioned horizontally at 12/,m, and then mounted on gelatin-coated glass slides. The jugular, petrosal and nodose ganglia bilaterally and the trigeminal ganglion contralateral to FG application were similarly processed in order to assess the spread of FG from the nasal cavity to surrounding tissues. The double immunofluorescence method for CR and TK was performed using rabbit anti-CR-serum (1:1000) 47,48 and rat monoclonal anti-TK antibody (l:1000; Sera Lab., UK) 16. Sections were incubated in a mixture of these primary antibodies for 24 h at 4°C. Subsequently, sections were incubated in a mixture of lissamine rhodamine B chloride (LRITC)-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch Labs) and fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG (1:500; Cappel, Cooper Biomedical). The specificities of antibodies used have been already described elsewhere 16"47'4s.The sections were viewed with an Olympus epifluorescent microscope with a U filter for FG, a G filter for LRITC and a B filter for FITC. There was generally little or no cross-over fluorescence between FG, FITC and LRITC. However, cells which were heavily labeled with LRITC showed a faint red cross-over fluorescence with a U filter. This was easily distinguished from the golden fluorescence of FG. Only FG-labeled cells detectable with a 4 x objective (lens diameter 1.0 cm) were counted. Analysis was performed on glossy prints at a final magnification of 165×. In one group of animals, the FG-moistened cotton pellet was applied to the surface of the cornea and conjunctivum instead of the nasal mucosa. The trigeminal ganglion was processed as described above. In some rats that had not received FG application, the nasal mucosa was processed for the same double immunostaining. RESULTS Because the F G tracer required a relatively long survival period for labeling the trigeminal primary neuronal cell bodies, we a v o i d e d - s u b m u c o u s injection in

this study. Such an injection would have caused retention of the tracer at a high concentration at the injection site and gradual spread to the surrounding structures over a long period of time, resulting in extensive labeling of neurons innervating the surrounding tissues. Instead, we maintained a high concentration of the tracer in contact with the target neurons only transiently (2 h). This m e t h o d p r o d u c e d intense labeling of the target neurons and avoided tracer spread to the surrounding tissues. Following nasal F G application, very weak fluorescence could be seen in many neurons in the jugular, nodose and petrosal ganglia as well as the mandibular division (lateral part) of the trigeminal ganglia bilaterally. We consider these neurons to be labeled by spread of F G to the u p p e r alimentary tract. This weak fluorescence could be barely seen with a 10 × but not 4 × objective lens. O n the other hand, strong fluorescence, that could be clearly observed with a 4 x objective, was found in only a limited area of the trigeminal ganglion where primary neuronal cell bodies innervating the nasal mucosa have been reported to be located (see below). We, therefore, regard these strongly fluorescent neurons to be retrogradely labeled from the nasal mucosa that had b e e n in direct contact with the cotton pellet holding the tracer solution. T h e y will be refered to as the N M (nasal mucosa) cells, hereafter. N M cells were mostly seen in the trigeminal ganglion ipsilateral to the F G application. Except for the dorsalmost and ventralmost parts of the ganglion, about 63 N M cells (range: 3 2 - 1 7 6 cells) were found in each section. N M ceils contralateral to the application were rare (at most 1 cell per section). N M cells could not be seen in the jugular, petrosal or nodose ganglia. N M cells were distributed in the rostromedial half (ophthalmo-maxillary division) o f the ganglion. T h e caudolateral half corresponding to the mandibular division was essentially devoid of N M cells. In the dorsal 2 / 3 , N M cells were located only at the medial edge of the ganglion (rostromedial N M cell cluster). In the ventral 1 / 3 , n u m e r o u s N M cells were found in the caudolateral part of the ophthalmo-maxillary division (caudoventrolateral N M cell cluster). A l t h o u g h the rostromedial cluster extended t h r o u g h o u t the entire dorsoventral dimension of the ganglion, the n u m b e r of cells in this cluster markedly decreased in the ventral 1 / 3 of the ganglion. 1254 N M cells labeled in 3 trigeminal ganglia were examined for TK-ir and CR-ir (Table I). TK-ir and CR-ir (Figs. 1 A - C ) were detected in 54% (674 cells) and 18% (227 cells) of these cells, respectively. 96.9% of C R - i m m u n o r e a c t i v e (-ir) N M cells coexpressed TK. Therefore, 32.7% of TK-ir N M cells coexpressed CR.

233 TABLE I The number of TK- and CR-ir cells innervating the nasal mucosa in the dorsal, middle and ventral parts The data were obtained from 3 TGs. Five sections in each area were calculated for the analysis. The numbers in parentheses indicate the percentage within each part.

Dorsal Middle Ventral Sum

TK

TK + CR

CR

Neither

Total

228 (50.33) 130 (35.23) 96 (22.22)

38 (8.39) 52 (14.09) 130 (30.09)

0 (0) 2 (0.54) 5 (1.16)

187 (41.28) 185 (50.14) 201 (46.53)

453

454 (36.20)

220 (17.54)

7 (0.56)

573 (45.69)

369 432 1254

The CR-ir was relatively rare among NM cells in the rostromedial cluster. On the other hand, the caudoventrolateral NM cell cluster contained many CR-ir cells

(compare Fig. 2A-E with F and G). Because the rostromedial and caudoventrolateral clusters of NM cells were not clearly separated, we could not establish an objective border between them. For convenience of analysis, the series of sections were devided into 3 classes of dorsoventral hierarchy. The proportion of total TK-ir cells (both with and without CR-ir, about 53%) remained unchanged dorsoventrally. However, that of CR-ir cells was greatest in the ventral 1/3 (30.1%) and smallest in the dorsal 1/3 (8.4%). Consequently, the proportion of TK-ir, CR-negative cells decreased from dorsal to ventral (Fig. 3 and Table I). Following FG application to the cornea and conjunctivum, cell bodies were labeled only in the rostromedial part of the ophthalmo-maxillary division. About 40% of the labeled cells (208 out of 535 cells recovered from 2 ganglia ipsilateral to application) exhibited TK-ir but only 4% (22 cells) expressed CR. As it was the case for NM ceils, almost all CR-ir cells (except for 1 cell) coexpressed TK.

Fig. 1. Photomicrographs illustrating labeled cell bodies in the ventral part of trigeminal ganglion following FG application to the nasal mucosa (A-C); nasal mucosa (D and E). B and D demonstrate fluorescence for CR-ir, while C and E for TK-ir. Five cells were unequivocally judged to he NM cells (FG-positive) in A. Among these 4 exhibited fluorescence for both CR- and TK-irs (arrows in A-C). An NM cell (arrowhead) is negative for both CR- and TK-irs (A-C), Double immunostain reveals nerve fibers coexpressing CR-ir (D) and TK-ir (E) within the epithelium and along the basal lamina of the nasal mucasa, Scale bar (for all micrographs) = 50 ram.

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Fig. 2. Camera lucida drawings of serial horizontal sections at a 200 mm interval of the trigeminal ganglion ipsilateral to the FG application to the nasal mucosa (A to H; from dorsal to ventral). The lower power picture on the left illustrates topographic distribution of NM cells (dots) irrespective of immunoreactivities, whereas the higher power picture on the right shows NM cells with both CR- and TK-irs (filled circles), with only TK-ir (open circle), and without CR- or TK-ir (crosses). In the dorsal 2/3 of the ganglion (B-E), only the rostromedial cluster can be seen where most NM cells are devoid of CR-ir. In the ventral 1/3 (F and G), many NM cells exhibiting both CR- and TK-irs can be seen in the caudoventrolateral but not the rostromedial cluster. MAN, mandibular branch of the trigeminal nerve; O-M, ophthalmo-maxillary division of the trigeminal nerve. Scale bar (for the lower power pictures); 2 ram.

236 number of cells

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against the dorso-ventral axis of the trigeminal ganglion ipsilateral to the FG application. The distribution of NM cells with both CR- and TK-irs is greatest in the ventral and smallest in the dorsal 1/3 of the ganglion. The dorso-ventral relation is inverted for the NM cells with only TK-ir. Double immunostaining of the nasal mucosa revealed numerous nerve fibers with the fluorescence indicative of CR-ir (Figs. 1D, E). In the lamina propria, several CR-ir fibers formed a bundle that traveled parallel to the basal lamina. Solitary fibers could be seen only along the basal lamina and within the epithelium. The intraepithelial fiber trajectory was parallel to the basal lamina, and located at the basal 1 / 3 of the full thickness of the epithelium. Both basal and intraepithelial CR-ir fibers had varicosities. Unspecific staining was slight in the ganglion but was relatively intense in the mucous connective tissue. This was caused by the well-known unspecific sticking of the anti-rat IgG secondary antibodies to the rat connective tissue. By lowering the concentration of this second antiserum to 1:1000, we could clearly visualize the basal and intraepithelial TK-ir nerve fibers though the fluorescence intensity was somewhat compromized. Both the basal and intraepithelial CR-ir fibers usually coexpressed TK-ir. DISCUSSION Earlier studies with H R P application to the anterior ethmoidal nerve revealed cell bodies of origin in the ophthalmic division of the trigeminal ganglion in the cat 22'e3. A previous study as well as this study, that

applied tracers to the cornea in the rats, revealed primary neuronal cell bodies in the rostromedial part of the ophthalmo-maxillary division of the trigeminal ganglion 2°. It is likely, therefore, that the rostromedial NM cell cluster in this study represents the location of NM neurons whose peripheral axons travel through the ophthalmic nerve branch(es), most probably through the anterior ethmoidal nerve. On the other hand, the caudoventrolateral cluster appears to consist of those with peripheral axons traveling through the maxillary nerve branch(es). When H R P - W G A (HRP-labeled wheat germ agglutinin) was applied to the nasal mucosa of the cat, cell bodies were labeled in the maxillary division in addition to the ophthalmic division 4~ Similar tracer application in rats by Anton and Pappel disclosed caudoventrolateral NM ceils 2. A few examples were illustrated in the ventralmost section in their Fig. 2, though they were not discussed in the text 2. Because all the TK-containing primary neurons in the trigeminal and dorsal root ganglia expressed substance P (SP) 1°'45, TK-ir cells in this study are considered to be equivalent to SP-ir cells though they might have contained tachykinins other than SP. Therefore, this study confirms the previously reported observation that approximately 50% of the primary neurons innervating the nasal mucosa expressed SP 22. The proportion of TK-ir cells to those innervating the cornea and the conjunctivum appeared to be higher than that of SP-ir cells innervating the cornea 2°. This suggests that a large population of neurons innervating the conjunctivum contain TK. CR had been considered to be a specific marker for large primary neurons innervating the muscular proprioceptors and exteroceptive corpuscular receptors for its c o l o c a l i z a t i o n with carbonic anhydrase14'30'31"37'38'41'42'49"50. O u r recent study, however, demonstrated subsets of CR-ir primary neurons to contain T K and form free nerve endings 13'~4. Cell bodies of primary neurons coexpressing C R and T K were smaller than those coexpressing C R and carbonic anhydrase with some size range of overlap t4. As predicted from the fact that trigeminal primary neurons innervating the nasal mucosa are smaller than the overall trigeminal ganglion cell population 22'23, virtually all CR-ir NM cells belonged to the TK-containing subclass. This appears reasonable for there is no musculature equiped with the muscle spindle or corpuscular endings in the vicinity of F G application site. In addition, the nasal mucosa examined in this study actually contained neurites exhibiting CR-ir and TK-ir simultaneously. It was previously demonstrated that the topographic distribution of cell bodies coexpressing C R and T K was

237 uneven in the trigeminal ganglion t4. They were abundant in the mandibular and maxillary divisions, and rare in the rostromedial part (presumed ophthalmic division) of the trigeminal ganglion in rats. Coincidently, CR-ir NM cells in the present study were abundant in the caudoventrolateral cluster but not in the rostromedial cluster. Further, the cells labeled after FG application to the cornea and the conjunctivum were found only in the rostromedial part and mostly devoid of CR-ir. We suspect this difference in CR content between cells of the ophthalmic and maxillary divisions may be related to the difference in central projection. Transganglionic transport of HRP revealed that primary neurons comprising the anterior ethmoidal nerve of the cat projected to the brain stem trigeminal sensory nuclear complex but not the solitary tract nucleus 22. Similar results were obtained following HRP application to the cornea and periocular structures 27. On the other hand, HRP application to the maxillary nerve branches revealed a substantial primary projection to the solitary tract nucleus 1'18'24-26'32'44. Central projection of the anterior ethmoidal nerve has not been studied in rats but, to our knowledge, ophthalmic primary neurons have never been shown to project to the solitary tract nucleus. On the other hand, a considerable amount of primary projections have been reported from the maxillary and mandibular primary neurons in rats, particularly those innervating the intraoral structures 1,42,43,. More directly, the HRP-WGA injection into the nasal mucosa resulted in labeling of axonal endings in the solitary tract nucleus 2. Because the superficial laminae of the spinal dorsal horn and the trigeminal subnucleus caudalis are major termination sites of primary neurons containing SP 7'8'21'33 but not C R 34'36, CR-ir NM cells appear to project outside the subnucleus caudalis. Presently, the solitary tract nucleus is the only nucleus that appears to receive both CR-ir and SP-ir primary projection3,S,21,36. We have described the innervation of the nasal mucosa by a recently discovered subset of TK (SP)-containing trigeminal primary neurons. This group of primary neurons is characterized by its content of CR TM. They emit fine peripheral axons that form free nerve endings in the epithelium of nasal mucosa. Bibliographical analysis indicates that they project to the solitary tract nucleus but not to the superficial laminae of the trigeminal subnucleus caudalis. They are abundant in the maxillary but not ophthalmic component of primary neurons innervating the nasal mucosa of rats. Acknowledgments.

This work was supported by a Grant-In-Aid for General Scientific Research from the Ministry of Education, Science and Culture of Japan, No. 04771409 to H.I.

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