Brain Research, 547 (1991) 82-88 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391165258

82

BRES 16525

Peripheral territory and neuropeptides of the trigeminal ganglion neurons centrally projecting through the oculomotor nerve demonstrated by fluorescent retrograde double-labeling combined with immunocytochemistry R. Bortolami 1, L. Calz~ 2, M.L. Lucchi 1, L. Giardino 2, E. Callegari 1, E. Manni 3, V.E. Pettorossi 4, A.M. B a r a z z o n i I and G. L a l a t t a C o s t e r b o s a ~ Ilnstitute of Veterinary Anatomy, University of Bologna, Bologna (Italy), 21nstitute of Human Physiology, University of Cagliari, Cagliari (Italy), 31nstitute of Human Physiology, Universitd Cattolica S. Cuore, Rome (Italy) and ~Institute of Human Physiology, University of Perugia, Perugia (Italy) (Accepted 13 November 1990)

Key words: Trigeminal ganglion neuron; Trigeminal-oculomotor nerve fiber; Peripheral ophthalmic territory; Fluorescent retrograde labeling; Immunocytochemistry; Calcitonin gene-related peptide; Substance P; Cholecystokinin-8

The peripheral territories of sheep trigeminal neurons which send their central process to the brainstem through the oculomotor nerve were investigated by the use of fluorescent tracers in double-labeling experiments. For this purpose Diamidino yellow (DY) injection into the oculomotor nerve was combined with Fast blue (FB) injection either into the extraocular muscles (EOMs), or the cornea, or the superior eyelid. Double-labeled DY+FB cells were found in the ophthalmic region of the trigeminal ganglion in addition to single-labeled DY or FB cells. The DY and DY+FB-labeled trigeminal cells were analysed immunocytochemically for their content of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, and cholecystokinin-8 (CCK-8)-like. All single-labeled DY cells showed SP-, CGRP- or CCK-8-1ike immunoreactivity. Double-labeled DY+FB neurons innervating the EOMs were immunoreactive for each of the three peptides, whereas double-labeled neurons supplying the cornea were only CGRP-Iike positive. The findings suggest that, in the sheep, trigeminal neurons which send their process centrally through the oculomotor nerve supply the EOMs, the cornea, and the superior eyelid and contain neuropeptides which are usually associated with pain sensation.

INTRODUCTION In previous investigations we demonstrated that in the sheep and some other mammals 4'11-14 the oculomotor nerve contains sensory fibers which are the central processes of trigeminal ganglion cells located in the ophthalmic region of the ganglion. Just caudally to the orbital fissure, the ophthalmic branch of the trigeminal nerve forms, in fact, an anastomosis with the neighboring oculomotor nerve 11. Through this anastomosis many processes, approximately 400, of trigeminal ganglion cells leave the ophthalmic nerve and, making U-turns, enter the oculomotor nerve and run towards the brainstem within this nerve. These primary trigeminai afferent fibers running in the oculomotor nerve terminate mostly in the subnucleus gelatinosus of the nucleus caudalis trigemini, similarly to the thermal and nociceptive classical primary trigeminal afferents entering the brainstem through the trigeminal root. Most of the terminals of the

trigeminal fibers running in the oculomotor nerve are presynaptic to the classical trigeminal afferents, thus making axo-axonic contacts 4. Since the axo-axonic synapses are considered to be the morphological basis of presynaptic inhibition 8'9, the trigeminal fibers travelling in the oculomotor nerve can modulate other trigeminal primary fibers. Electrophysiological observations supported such data TM. Our recent electrophysiological investigations 15, utilizing a selective stimulation for activating the nociceptive afferents, strongly suggest that these trigeminal fibers running into the oculomotor nerve are involved in the transport of pain signals arising from the superior eyelid and conjunctiva. In the present investigation, by the use of fluorescent tracers in double-labeling experiments, we have attempted to anatomically define in the sheep other peripheral ophthalmic territories supplied by trigeminal ganglion neurons centrally projecting through the oculo-

Correspondence: R. Bortolami, Institute of Veterinary Anatomy, University of Bologna, Via Belmeloro 12, 40126 Bologna, Italy.

83 m o t o r n e r v e . M o r e o v e r , by c o m b i n i n g the f l u o r e s c e n t r e t r o g r a d e tracing t e c h n i q u e w i t h i m m u n o c y t o c h e m i s t r y , we have

searched

neuropeptides

in t h e l a b e l e d n e u r o n s

for s o m e

w h i c h a r e usually a s s o c i a t e d with p a i n

s e n s a t i o n , n a m e l y s u b s t a n c e P (SP)-, calcitonin g e n e related peptide (CGRP)-, and cholecystokinin-8 (CCK8)-like 6'7 n e u r o p e p t i d e s .

MATERIALS AND METHODS Ten sheep (weighing 20-25 kg) were used in this study and were anesthetized with Ketamine (4 mg/kg, i.v.) + diazepam (2 mg/kg, i.m.). The fluorescent tracers Diamidino yellow dihydrochloride (DY) and Fast blue (FB) were used as 2% aqueous suspensions and were injected by means of Hamilton microsyringes as follows: (1) in 5 subjects DY was injected into the oculomotor nerve and FB into EOMs; (2) in 3 subjects DY was injected into the oculomotor nerve and FB into the cornea; (3) in 2 subjects DY was injected into the oculomotor nerve and FB into the superior eyelid, its conjunctiva and the adjacent skin of the frontal region.

Procedures for fluorescent tracer injections (a) D Y injection into the oculomotor nerve. An appropriate craniotomy was performed and the intracranial portion of the right oculomotor nerve was exposed so as to avoid damage of the ipsilateral hemisphere. A strip of plastic film was carefully pulled underneath the nerve to prevent leakage of the tracer to subjacent structures. 1-5/A of DY suspension was slowly injected into the nerve half-way between its emergence from the interpeduncular fossa and its passage through the dura mater. The injection site was then swabbed and covered by Tissucol (Immuno AG, Vienna). (b) FB injection into the EOMs. All operations were performed ipsilaterally to the DY-injected oculomotor nerve. The two palpebral commissures were incised and the eyelids retracted; an incision was performed along the conjunctival fornix and the eyeball was exposed. Each muscle selected for the injection was carefully isolated and a plastic film was i n t e n d between it, the eyeball and the other muscles. 40-50/A of FB suspension was slowly injected in each muscle at different sites along its longitudinal axis. In each subject 4 muscles were simultaneously injected. It was avoided to inject in the same animal the EOMs which have been shown to project intermingled in the same area of the trigeminal ganglionTM. After injections a cotton swab was carefully pressed on the site of the injections to prevent leakage of the dye as well as to facilitate its infiltration. Then the muscle was wrapped in the abovementioned plastic film and the palpebral commissures were sutured. (c) FB injection into the cornea. 20-30/~l of FB suspension was slowly injected through multiple penetrations into the thickness of the cornea, ipsilaterally to the DY-injected oculomotor nerve. A cotton swab was then pressed on the site of the injections in order to prevent leakage of the tracer. (d) FB injection into the superior eyelid, its conjunctiva and the adjacent skin of the frontal region. 100/~1 of FB suspension was injected at different sites into the thickness of these structures, ipsilaterally to the DY-injected oculomotor nerve. Tissue preparation All the animals recovered very well and after a survival time of 5 days were deeply anesthetized and perfused through the common carotid arteries. After a brief rince with heparinized saline solution at room temperature followed by the same ice-cold solution, the perfusion was continued with ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The right trigeminal ganglion was then removed, immersed in the same f'utative for 90 min and rinsed for at least 72 h in an ice-cold 0.1 M phosphate buffer, pH 7.4, containing 10% sucrose. The trigeminal ganglion was then quickly

frozen with CO 2. Consecutive longitudinal cryostat sections (12/~m thickness) of the whole ganglion were thaw-mounted on gelatinecoated slides. From groups of 4 serial sections, each first section was exposed to a stream of dry air immediately after removal from the cryostat, mounted with Eukitt and examined to identify the DY and FB retrograde labelings. 20 series of the three other sections were processed for SP-, CGRP- and CCK-8-like immunoreactivity according to the indirect immunofluorescence method 5. In order to compare the SP-, CGRP- and CCK-8-like immunostaining in the trigeminal ganglion of the sheep with that already described in the same ganglion of other mammals, some series of each ganglion were submitted to immunoperoxidase procedure (PAP) according to the technique of Sternberger TM. All the remaining sections were utilized for retrograde labeling control.

lmmunofluorescence procedure (IF) The sections were first incubated in 0.1 M phosphate buffered saline (PBS) at room temperature for 30 rain which was followed by incubation at 4 °(2 for 12 h in a humid atmosphere with the primary antiserum diluted in PBS containing 0.3% Triton X-100 (vol/vol). After the sections had been rinsed in PBS for 30 rain (3 x 10 rain) they were incubated at 37 °C for 30 rain in humid atmosphere with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulins diluted in PBS containing 0.3% Triton X-100 and rinsed in PBS (3:1 vol/vol). PAP The sections were incubated for 48 h at 4 °C with the primary antibody diluted with PBS containing 0.3% (vol/vol) Triton X-100 and normal sheep serum 1% (vol/vol). The excess of the primary antibody was washed out as follows: PBS + 0.2% Triton X-100, 10 min; PBS + 0.1% Triton X-100, 10 min; PBS 10 min; PBS + bovine serum albumin 0.1% (wt/vol) (Sigma, The Netherlands), 10 min; PBS + normal sheep serum 0.1%, 30 min; PBS 10 rain. They were then incubated for 2 h at room temperature with the sheep anti-rabbit serum diluted in PBS + 0.2% Triton X-100 + normal sheep serum 1% and washed twice in PBS for 10 min each. The PAP complex was finally applied. After 1 h of incubation the sections were washed twice for 10 min, each one with PBS. They were then treated with a solution containing 3,3"-diaminobenzidine-HCl (0.5 mg/ml) (DAB, Sigma, The Netherlands) in 50 mM Tds (= tris(hydroxymethyl)aminomethano)-HCl (Sigma, The Netherlands), pH 7.4; after 10 rain of incubation hydrogen peroxide was added to get a final concentration of 0.003%, and the reaction was carried out for an additional time that differs for the different antisera, but that is strictly the same in all the staining performed with each antiserum (4-20, mostly 8 win). After treatment with DAB/hydrogen peroxide/Tris buffer, the sections were rinsed in PBS. As a result of this, the slides were washed in distilled water and dried for 1 h, warming in an oven at 37 °C, dehydrated and coverslipped in Eukitt. These sections have been examined in a Microphot-FXA Nikon light microscope using bright- and dark-field optics. Antlsera Commercially available antisera were used in this study. Anti SPand anti CCK-antisera were purchased from Incstar (Stillwater, Minnesota, U.S.A.), anti-CGRP from Peninsula Laboratories Europe Ltd. (Merseyside, U.K.), and used at the following dilutions both for PAP and IF staining: SP 1/500, CGRP 1/600, CCK 1/300.

TABLE I

D Y and DY+FB labeled cells (% of the total number, mean + $.E.M.) DY DY + FB EOMs DY + FB cornea

0.49 + 0.41 0.26 + 0.41 0.10 + 0.17

85 Bovine serum albumin (BSA) was added to the diluted antisera. The primary polyclonal antisera were raised in rabbit and prepared as follows. Polydonai anti-SP: antigen SP coupled to Keyhole Limpet Hemoeyanin (KLH) with earbodiimide (CDI). Polyclonal anti-CGRP: antigen synthetic, rat CGRP conjugated to BSA with glutaraldehyde. CCK: antigen CCK-8 conjugated to KLH with CDI. FITC conjugate goat anti-rabbit serum was purchased from DAKO and used at 1:10 dilution. Bridge antiserum: sheep antirabbit (BIOYEDA), 1:50; peroxidase conjugate-antiserum: rabbit anti-sheep (BIOYEDA), 1:100. All stainings have been abolished using the primary antiserum preadsorbed with 100/zg of antigen/ml diluted antiserum. Tissue analysis

For both fluorescent labeling and immunocytochemistry the sections were examined in an epifluoreseent Leitz microscope fitted with appropriate filter setting for alternative observation of FB/DY labelings (Leitz filter system A) and FITC-indueed immunofluorescence (Leitz filter system 12). Alternating the filter combination, areas of the ophthalmic region of the trigeminal ganglion were photographed (obj. 25x, opt. 2:1) in each selected section. Ektachrome P 800/1600, or T-Max 400 films (Kodak) were used for photography. On the slides about 700-800 cells for each ganglion were counted in order to determine the % of labeled cells and the % of immunoreactive (IR)-labeled cells. RESULTS Retrograde tracing (Table I)

A n u m b e r of trigeminal ganglion cells showed a DY-labeled nucleus, indicating tracer uptake from their central process running in the DY-injected ipsilateral oculomotor nerve. They were located in the dorsal area of the ophthalmic region. They were intermingled with a large n u m b e r of perikarya retrogradely labeled with FB transported from the ophthalmic-injected territories (Fig. 1). Clearly double-labeled D Y + F B neurons were also present in all the experiments (Fig. 2); however, they were numerous when E O M s and cornea were injected, while rare after FB injection into the superior eyelid, its conjunctiva and the adjacent skin of the frontal region. Both small (approx. 20--30/~m in diameter), medium (approx. 30--40/~m in diameter) and large (approx. 50-60 /zm in diameter) sized D Y + F B - l a b e l e d cells were found, although the majority of them were medium-sized. Immunocytochemistry and retrograde tracing

In the trigeminal ganglion of the sheep, processed for P A P without eolchicine treatment, SP-, C G R P - or

TABLE II lmmunoreactive cells (% of the total number, mean ± S. E. M. )

The count has been made on 700-800 cells. Only the positive cells in which the nucleus was evident were counted. SP CGRP CCK-8

43.7 ± 2.9 55.5 + 3.8 19.9 ± 4.2

TABLE III DY and DY+FB labeled cells and immunoreactivity (% of the immunoreactive ceils, mean ± S. E. M. )

In the estimations of the % of the DY + FB-labeled immunoreactive cells at least 20 and 10 double-labeled neurons were considered in each ganglion after FB injection into the EOMs and into the cornea respectively.

DY DY+FBEOMs DY+FBcornea

SP

CGRP

CCK~

2.6±0.9 2.0±0.3 -

3.6±3.0 2.1±0.8 0.41±0.35

3.9±2.5 0.3±0.3 -

CCK-8-1ike I R neurons were present, although in different percentages (Table II). No somatotopic organization of the trigeminal ganglion cells containing SP-, C G R P - or CCK-8-1ike immunoreactivity, either for cells within the ophthalmic region or for the ganglion as a whole, was evident. The immunostaining concerned small, medium and large sized neurons. Two staining patterns were evident for SP- and CGRP-Iike: a very strong labeling or a dotted labeling throughout the cytoplasm. Processing for immunofluorescence in some cases caused diffusion of the D Y into neighboring satellite cells and a decrease in fluorescence intensity of the FB tracer; however, by controlling the untreated serial sections no diffusion of the tracers into other surrounding neurons was observed. Therefore, SP-, C G R P - and CCK-8-1ike immunoreactivity was considered in neurons to be unequivocally identified by the retrograde transport of the dyes. Alternating the filter combinations, a large proportion of DY-labeled cells appeared immunostained for SP-, C G R P - or CCK-8-1ike (Table III, Fig. 3). On the consecutive sections most of the DY-labeled cells were found to contain more than one peptide: SP-like coexisted with CGRP-Iike peptides, or with CCK-8-1ike, and CGRP-Iike with CCK-8-1ike; occasionally in a single DY-labeled cell all three peptides (Fig. 4) were found. After FB injection into the E O M s , the D Y + F B labeled neurons repeated the I R pattern of DY-labeled cells, whereas after FB injection into the cornea only CGRP-Iike I R double-labeled cells were found (Table III, Fig. 5). Following FB injection into the superior eyelid and the adjacent skin of the frontal region the double-labeled D Y + F B cells were in such a small number that a statistical evaluation of their immunoreactivity was not possible. H o w e v e r , the rare D Y + F B cells were found to demonstrate SP-like IR. The retrogradely single-labeled FB cells, after injection of the tracer into the ophthalmic territories, were SP-, C G R P or CCK-8-1ike IR.

86 DISCUSSION In double-labeling experiments we have found that the DY injected into the intracranial portion of the oculomotor nerve labels cells in the ipsilateral trigeminal ganglion, in agreement with previous studies utilizing horseradish peroxidase 3,14. The DY-labeled neurons are located exclusively within the ophthalmic region of the ganglion, intermingled to a large number of perikarya labeled with FB injected respectively into EOMs, cornea, superior eyelid and its conjunctiva, and the adjacent skin of the frontal region. In each section examined for the retrograde labeling at least one or two DY-fluorescent nuclei are present, corresponding to about 5%0 of the neurons within the ophthalmic region. Moreover in all the experiments some of the DY-labeled cells have been shown to contain the FB tracer too. The double labeling clearly indicates that the peripheral processes of some trigeminal ganglion cells centrally projecting through the oculomotor nerve provide sensory innervation to the territories injected with the FB, particularly to the EOMs (2.5%~) and the cornea (1%~). The DY+FB-labeled cells innervating the eyelid, its conjunctiva and the adjacent skin of the frontal region are rare in spite of the massive injection of FB in these peripheral territories. The electrophysiological experiments 15 showed evident activation in the oculomotor nerve following stimulation of the superior eyelid. Therefore the small number of double-labeled cells does not fit well with those physiological findings; however, it could be explained with the hypothesis that each of these neurons could have peripheral processes richly branched and thus supplying a wide area. DY-labeled neurons, which should correspond to the perikarya of trigeminal A delta and C group fibers, previously identified electrophysiologically in the oculomotor nerve 14'15, have been found immunoreactive to SP-, CGRP-, or CCK-8-1ike. Two of the peptides were often co-localized in the same neuron, whereas only occasionally all three peptides were found in the same neuron. According to the literature, SP, CGRP, and CCK-8 are neuropeptides involved in conveying nociceptive information (SP ~°) and in the processing of nociceptive stimuli (CGRP1; CCK-8~8). CGRP, in addition, would potentiate, according to Oku et al. 17, the release of SP from the primary afferent terminal and promote the transmission of nociceptive information induced by mechanical noxious stimuli. Trigeminal neurons supplying the EOMs are immunoreactive for SP-, CGRP- or CCK-8-1ike whether they send central processes either through the trigeminal root or through the oculomotor nerve. Regarding the corneal territory, on the contrary, differences exist if the immu-

Fig. 4. Consecutivesections of a trigeminai ganglion cell (arrowed) showing DY-labeled nucleus (visible in b) after DY injection into the ocuiomotor nerve and displaying: a: SP, b: CGRP and c: CCK-8 immunostaining. DY-negative cells display the coexistence of two (square) or three (open circle) peptides (filter system combination A + I2). Bar = 23 #m.

87

01

Ca

6 v

Fig. 6. Schematic drawing illustrating the peripheral and central processes of a double-labeled neuron (single arrow) observed in the trigeminal ganglion after injection of Diamidino yellow dihydrochloride (DY) into the oculomotor nerve (OM) and Fast blue (FB) into the extraocular muscles. The same result (not illustrated here) was also obtained after injection of Fast blue into the cornea or the superior eyelid, its conjunctiva and the adjacent skin of the frontal region. A trigeminal ganglion neuron (double arrow) which innervates muscle spindles in the extraocular muscles and projects centrally via the trigeminal root according to our previous observationsn is also represented. C1C2, first and second segments of the cervical spinal cord; MAN, mandibular division; MAX, maxillary division; OMN, oculomotor nucleus; OPH, ophthalmic division; PC, pars caudalis of spinal trigeminal complex; PI, parts interpolaris of spinal trigeminal complex; PO, pars oralis of spinal trigeminal complex; TG, trigeminal ganglion. noreactivity of FB only or D Y + F B trigeminal neurons is considered. The FB neurons, projecting the corneal sensitivity through the trigeminal root, are positive for SP-, C G R P - or CCK-8-1ike, whereas the D Y + F B neurons sending the corneal information through the oculom o t o r nerve have CGRP-like immunoreactivity only. Concerning the E O M s in particular, it could be suggested that the fibers of D Y + F B trigeminal neurons could be group III and IV afferent fibers originating in free nerve endings and possessing nociceptive properties. This hypothesis can be supported by the fact that all the trigeminal fibers present in the oculomotor nerve belong to A delta and C groups, according to their conduction velocity 14"15, and also by the fact that in the sheep, species in which the E O M s are endowed with spindles, the proprioceptive impulses from these muscles reach the brainstem through the trigeminal root only 11. The present anatomical and immunocytochemical findings provide evidence that SP-, C G R P - , or CCK-8-1ike I R trigeminal afferents from ophthalmic territories REFERENCES 1 Bates, R.EL., Buckley, G.A. and McArdle, C.A., Comparison of the antinociceptive effects of centrally administered calcitonins and ealcitonin gene-related peptide, Br. J. Pharm., 82 (1984) 295 (Abstract).

(EOMs, cornea, eyelid) enter the brainstem not only via trigeminal root but also via oculomotor nerve (Fig. 6). Such primary trigeminal afferents running within the oculomotor nerve may have a functional implication on the trigeminal information. In fact, in the subnucleus gelatinosus of the nucleus caudalis trigemini they are for the most part presynaptic to the classical trigeminal root afferents 4. The mechanisms by which peptidergic primary afferents interact synaptically with one another remain to be worked out. However, our electrophysiological data 14 demonstrated that the trigeminal afferent fibers running within the oculomotor nerve enhance the excitability of the trigeminal fibers entering the brainstem via trigeminal root and that the increased excitability possibly results in a presynaptic inhibition. Acknowledgements. This study was supported by grants from the MURST of Italy and from the CNR (Medicina Preventiva e Riabilitativa, Sottoprogetto: Controllo del Dolore). The authors thank mr E. Ferrari for his expert technical assistance, and Ms M.L. Polsoni for secretarial assistance.

2 Bortolami, R., Lucchi, M.L., Pettorossi, V.E., Callegari, E. and Manni, E., Localization and somatotopy of sensory cells innervating the extraocular muscles of lamb, pig and cat. Histochemicai and electrophysiological investigation, Arch. ltal. Biol., 125 (1987) 1-15. 3 Bortolami, R. and Manni, E., Aspetti morfo-funzionali del

88 nucleo mesencefalico del trigemino e del ganglio di Gasser, Atti Soc. Ital. Anat., XXXVII (1981) 63-121. 4 Bortolami, R., Veggetti, A., Callegari, E., Lucchi, M.L. and Paimieri, G., Afferent fibers and sensory ganglion cells within the oculomotor nerve in some mammals and man. I. Anatomical investigations, Arch. Ital. Biol., 115 (1977) 355-385. 5 Coons, A.H., Fluorescent antibody methods. In J.E Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399-422. 6 Cuello, A.C. and Matthews, M.R., Peptides in peripheral sensory nerve fibres. In ED. Wall and R. Melzack (Eds.), Textbook of Pain, Churchill Livingstone, Edinburgh, 1984, pp. 65-79. 7 Dalsgaard, C.-J., The sensory system. In A. Bj6rklund, T. H6kfelt and C. Owman (Eds.), Handbook of Chemical Neuroanatomy, vol. 6, The Peripheral Nervous ,System, Elsevier, Amsterdam, 1988, pp. 599-636. 8 Eccles, J.C., Kostyuk, EG. and Schmidt, R.E, Presynaptic inhibition of the central actions of flexor reflex afferents, J. Physiol., 161 (1962) 258-281. 9 Gray, E.G., A morphological basis for presynaptic inhibition?, Nature, 193 (1962) 82-83. 10 Henry, J.L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 439-451. 11 Manni, E., Bortolami, R. and Desole, C., Peripheral pathway of eye muscle proprioception, Exp. Neurol., 22 (1968) 1-12. 12 Manni, E., Bortolami, R., Pettorossi, V.E. and Callegari, E.,

13

14

15

16 17

18 19

Trigeminal afferent fibers in the trunk of the oculomotor nerve of lambs, Exp. Neurol., 50 (1976) 465-476. Manni, E., Bortolami, R., Pettorossi, V.E., Lucchi, M.L. and Callegari, E., Afferent fibers and sensory ganglion cells within the oculomotor nerve in some mammals and man. II. Electrophysiological investigations, Arch. Ital. Biol., 116 (1978) 16--24. Manni, E., Bortolami, R., Pettorossi, V.E., Lucchi, M.L., Cailegari, E. and Draicchio, F., Influence of oculomotor nerve afferents on central endings of primary trigeminal fibers, Arch. ltal. Biol., 126 (1987) 29-39. Manni, E., Draicchio, E, Pettorossi, V.E., Carobi, C., Grassi, S., Bortolami, R. and Lucchi, M.L., On the nature of the afferent fibers of oculomotor nerve, Arch. ltal. Biol., 127 (1989) 99-108. Manni, E. and Pettorossi, V.E., Somatotopic localization of the eye muscle afferents in the semilunar ganglion, Arch. ltal. Biol., 114 (1976) 178-187. Oku, R., Satoh, M., Fujii, N., Otaka, A., Yajima, H. and Takagi, H., Calcitonin gene-related peptide promotes mechanical nociception by potentiating release of substance P from the spinal dorsal horn in rats, Brain Research, 403 (1987) 350-354. Stacher, G., Effects of cholecystokinin and caerulein on human eating behavior and pain sensation: a review, Psyehoneuroendocrinology, 11 (1986) 39-48. Sternberger, L.A., lmmunocytochemistry, Wiley, New York, 1979.

Peripheral territory and neuropeptides of the trigeminal ganglion neurons centrally projecting through the oculomotor nerve demonstrated by fluorescent retrograde double-labeling combined with immunocytochemistry.

The peripheral territories of sheep trigeminal neurons which send their central process to the brainstem through the oculomotor nerve were investigate...
6MB Sizes 0 Downloads 0 Views

Recommend Documents