Immunohistochemical localization of neuropeptides in the human ciliary ganglion

BRAIN RESEARCH ELSEVIER

Brain Research 681 (1995) 229-234

Short communication

Immunohistochemical localization of neuropeptides in the human ciliary ganglion Wolfram Kirch, Winfried Neuhuber, Ernst R. Tamm " Departr~'nt of Anatomy, University of Erlangen-Niirnberg, Universitiitsstr. 19, 91054 Erlangen, Germany Accepted 22 February 1995

Abstract

In human ciliary ganglia, 18% of neurons were in contact with substance P (SP) and 12% with calcitonin gene-related peptide (CGRP) like-imrnunoreactive(LI) varicose axons. CGRP was colocalized with SP. Numerous SP-LI and CGRP-LI non-varicose nerve fibers were found between the ganglion cells and in nerve trunks that entered the ganglia. Axons immunoreactive for neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH) or dopamine-/3-hydroxylase(DBH) never contacted neuronal cell bodies. Perikarya of ciliary neurons neither stained for any of the neuropeptides nor for DBH. 23% of ciliary perikarya were TH-immunoreactive. These observations suggest an innervation of human ciliary ganglion neurons by peptidergic primary afferent collaterals presumably of trigeminal origin.

Keywords: Ciliary ganglion; Substance P; Calcitonin gene-related peptide; Vasoactive intestinal polypeptide; Neuropeptide Y; Tyrosine hydroxylase; Dopamine-/3-hydroxylase;Human

The parasympathetic inttervation of the mammalian eye takes its origin from the ciliary and the pterygopalatine ganglia. In primates, octdar pterygopalatine axons contribute to the vascular innervation of the choroid [28,29], whereas ciliary ganglion axons mainly innervate iris sphincter and ciliary muscle [30,41]. Experimental studies indicate that in cynomolgus monkeys only 3% of the ciliary ganglion neurons supply the iris, while the rest projects to the ciliary muscle [41], a fast multi-unit smooth muscle with an extreme dense cholinergic innervation and an unusual high content of receptors for acetylcholine [5,10,16]. In various species, the ciliary neurons and their preganglionic innervation express a considerable neurochemical complexity. In the rat ciliary ganglion, virtually all ciliary neurons show immunoreactivity for choline acetyltransferase, still numerous neurons that stain also for tyrosine hydroxylase and/or dopamine-fl-hydroxylase have been observed [20]. In addition, some rat ciliary neurons express a preganglionic catecholaminergic innervation [20].

* Corresponding author. Fax: (49) (9131) 852862. Present address: National Eye Institute, Laboratory of Molecular and Developmental Biology, 6 Center Drive, MSC 2730, Building 6 / R o o m 203, Bethesda, Maryland 20892, USA. Fax: (1),(301) 402-0781. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved

SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 2 9 9 - 5

Moreover, peptidergic nerve cells as well as a peptidergic preganglionic innervation have been identified in the ciliary ganglion of rat and cat [13,15,21,22]. It is not clear, however, how findings in subprimate mammals relate to the situation in humans and higher monkeys, since the ciliary muscle, the main target of ciliary postganglionic terminals in primates, is only vestigial in rat [27]. Also in the cat eye, ciliary muscle and functional accommodative amplitude are considerable smaller than in the primate eye [27,39]. In the present study, the localization of different neuropeptides and enzymes involved in catecholaminesynthesis was investigated in human ciliary ganglia. The ciliary ganglia of 6 human donors (age range 43-93 years) were removed 3-7 h after death and fixed overnight in Zamboni's fixative [32]. As positive control, trigeminal, superior cervical and pterygopalatine ganglia, as well as the choroid of both eyes (containing an intrinsic VIP-like immunoreactive (LI) nerve cell plexus [ l i d of each of the donors were similarly processed. After washing in phosphate-buffered saline (PBS) containing 30% sucrose for 24 h, 20-~m cryostat sections were cut, mounted on slides covered with 0.1% poly-L-lysine and preincubated for 30 min in Blotto's dry milk solution. After preincubation, the sections were incubated overnight at room temperature with the primary antibodies listed in

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Table 1. Thereafter, the sections were washed in PBS, reacted for 1 h with biotinylated secondary antibodies (Amersham Buchler, Braunschweig, Germany), washed again, and covered with streptavidin-FITC (Dakopatts, Hamburg, Germany). For double-labelling experiments, sections were incubated with rabbit antibodies in combination with the non-rabbit antibodies listed in Table 1. Binding of mouse, rat, sheep and goat antibodies was visualized using biotinylated secondary antibodies specific for each species and streptavidin-FITC. The rabbit antiserum was stained with Texas red (Amersham) conjugated anti-rabbit IgG. Negative control experiments were performed using either PBS or pre-immune serum from the same host species, substituted for the primary antibody. After washing in PBS, the sections were mounted in Entellan (Merck, Darmstadt, Germany)containing 1,4-Diazabicyclo[2,2,2] octan (DABCO, Merck) and viewed with a Leitz Aristoplan microscope (Ernst Leitz GmbH, Wetzlar, Germany). A Kodak T-max 400 film was used for photography. For quantitative evaluation, a total of 15002000 cells were examined for each primary antiserum used. 23.1 + 0.9% of the ciliary neurons showed positive immunofluorescence for tyrosine hydroxylase (TH, Fig. 1A). The intensity of the staining was considerably weaker than in the similarly processed superior cervical ganglia of the same donors, where virtually all neurons were positively labelled (Fig. 1B). In contrast to the perikarya of the superior cervical neurons, ciliary neurons did not stain for dopamine-/3-hydroxylase (DBH). Ciliary neurons were not in contact with axons expressing immunoreactivity for TH or DBH. Axons immunoreactive for neuropeptide Y (NPY) were extremely rare in the ciliary ganglion and were not in obvious contact with ciliary neurons (Fig. 2A). No staining for NPY was found in perikarya of ciliary neurons, but was readily observed in the superior cervical ganglion (Fig. 2B). Staining for vasoactive intestinal polypeptide (VIP) gave similar results. Again, VIP-LI was not seen in the soma of ciliary neurons, and VIP-LI axons were rare and did not contact perikarya. In contrast, such neurones and axons were regularly found in the pterygopalatine ganglia and in the choroid of the same donors. In striking contrast to the findings for NPY and VIP, substance P

Fig. 1. Immunohistochemical localization of TH in ciliary ganglion (A) and superior cervical ganglion (B) from the same human donor. In the ciliary ganglion, some nerve cells are positively labelled (arrows), while others (asterisks) are not stained (scale bar: 15 /zm). In the superior cervical ganglion, virtually all perikarya show positive immunofluorescence for TH (scale bar: 10 /.tm).

(SP)-LI or calcitonin gene-related peptide (CGRP)-LI varicose and non-varicose nerve fibers were frequently observed in human ciliary ganglia and in nerve trunks that entered the ganglia (Fig. 3AB). SP-LI varicose axons

Table 1 Detailed description of antisera used for immunocytochemistry Primary antisera

Source

Host species

Dilution

Calcitonin gene-related peptide (CGRP)

Euro Diagnostica AB (MalmiS, Sweden) Affiniti Res. Prod. Ltd. (Nottingham, UK) Amersham Buchler (Braunschweig, Germany) Affiniti Eugene Toch Chemicon Eugene Tech (Ridgefield Park, NJ) Eugene Tech Euro-Diagnostica AB

Rabbit Sheep Rabbit Rabbit, Goat Rabbit Rat Rabbit Rabbit Rabbit

1:1000 1:800 1:80 1:200 1:200 1:200 1:200 1:200 1:1000

Neuropeptide Y (NPY) Substance P (SP) Tyrosine hydroxylase (TH) Dopamine-fl-hydroxylase (DBH) Vasoactive intestinal peptide (VIP)

W. Kirch et al. / Brain Research 681 (1995) 229-234

Fig. 2. NFY-LI in ciliary ganglion (A) and superior cervical ganglion (13) of the same human donor (scale bar: 8.75 ~m). NPY-LI axons (arrows) are rare in the human ciliary ganglion and not in obvious contact with

ciliary perikarya. The soma of the ciliary neurons (asterisk) contains lipofuscin granules that express autofluorescence,but does not show NI'Y2LI. In contrast, NTY-LI is observed in the perikarya of several neurons in the superiorcervicalganglion. closely surrounded 18.0 + 1.5% (mean + S.E.M.) of ciliary neurons, while CGRP-LI axons were in intimate contact with 12.5 + 0.7% of neurons, respectively (Fig. 3CD). In double-labelling experiments, almost all CGRP-LI axons were also SP-LI (Fig. 4AB). Ciliary neurons that were in contact with SP-LI or CGRP-LI nerve fibers showed no distinct distribution within the ganglion, nor were they restricted to any particular cell size. Perikarya of ciliary neurons did not stain for SP or CGRP. However, such neuronal cell bodies were numerous in the trigeminal ganglia of the same donors (Fig. 5AB). Similar to findings in a considerable number of mammalian species [12,15,20,38], a significant proportion of human ciliary neurons expressed immunoreactivity for TH. Also in situ hybridization experiments in rats indicate that TH is synthesized in ciliary neurons [37]. So far the role of TH in parasympathetic neurons has not been clarified.

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TH-IR neurons in human ciliary ganglia might synthesize dopamine but not noradrenaline, since they do not stain for DBH. On the other hand, ciliary neurons in rats do not take up exogenous DOPA, indicating lack of aromatic-Lamino-acid decarboxylase (AADC) which catalyzes the conversion of L-DOPA to dopamine [20]. Moreover, a substantial number of TH-IR neurons in sensory ganglia of rat and guinea pig do not express AADC-IR [17,18]. In contrast to the rat [20], but alike to ciliary ganglia in other species (monkey, dog, cat, mouse, guinea pig) [12,20,38], we did not observe an innervation by TH or DBH immunoreactive axons in humans. In addition, perikarya of human ciliary neurons did not stain with antibodies against NPY, VIP, CGRP or SP, which is contrary to findings in other species. In the rat ciliary ganglion, all of these neuropeptides have been identified in neuronal perikarya [15,22,34]. In cats, approximately one third of ciliary perikarya have been reported to be immunoreactive for NPY [13] or SP [42], while staining for CGRP was absent [13]. In cynomolgus monkeys, faint to moderate staining for SP has been demonstrated in 60% of ciliary perikarya by Zhang et al. [42], a finding which was not confh'med by other investigators [12,40]. Similar to our findings in humans, perikarya of monkey ciliary neurons did not stain for CGRP or NPY [12,40]. Immunoreactivity of somata for VIP was shown in a small population of ciliary perikarya in the cynomolgus monkey [40], but not in the rhesus monkey [12]. Presumably, lack of immunostaining in human material might be due to post mortem loss of antigen. On the other hand, in each of the human donors we were able to visualize these peptides in characteristic localizations outside the ciliary ganglion. In accordance with other studies on human material [1,11,26], immunoreactivity for SP and CGRP was present in trigeminal ganglion neurons, NPY in neurons of the superior cervical neurons and VIP in pterygopalatine as well as in intrinsic neuronal cell bodies of the choroid. We therefore assume that these neuropeptides are either not present in perikarya of human ciliary neurons or only expressed to very small amounts. Differences between human ciliary neurons and those of other mammalian species, seem also to exist regarding their peptiderglc preganglionic innervation. In marked contrast to findings in the rhesus monkey [12], NPY-immunoreactive axons were rare and did not contact ciliary neuronal perikarya, which is in accordance with studies in cynomolgus monkey, rat and cat [13,34,40]. Similar to cynomolgus monkey and cat [40,42], but in contrast to rhesus monkey and rat [12,34], a distinct population of human ciliary neuronal cell bodies appears to be endowed by SP-LI and CGRP-LI axons. Compared to our findings in humans, SP-LI axon terminals were rare in the cynomolgus monkey ciliary ganglion and surrounded only 0.5-3% of neuronal perikarya [42]. Electron microscopy demonstrated that these SP-labelled axon terminals in the cynomolgus monkey formed synapses with dendritic profiles of ciliary neurons [42]. In the ciliary ganglion of

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birds, where SP-LI preganglionic terminals are similarly observed [9], also physiological studies indicate a role for SP as neurotransmitter [6,7]. The nature and origin of SP-LI and CGRP-LI axons entering the human ciliary ganglion and contacting cell bodies there remains to be clarified. SP-LI nerve cells have been demonstrated in the Edinger-Westphal nucleus of cats and cynomolgus monkeys [24,42], and may also be present in humans. On the other hand, the finding of colocalization of CGRP-LI and SP-LI in terminals around human ciliary neurons suggests an origin from sensory neurones in the trigeminal ganglion, where colocalization of SP/CGRP has been demonstrated in many species including man [26]. In lower mammals such as rat and guinea pig, CGRP-LI trigeminal neurons are more numerous than SP-LI neurons, and essentially all SP-LI neurons are also CGRP-LI [19,23,31,36]. In primates the situation is different, since in the trigeminal ganglion of man and cynomolgus monkeys, a considerable number of SP-LI

neurons does not show colocalization with CGRP-LI [25,26]. SP/CGRP-LI terminals have also been shown in the rat sphenopalatine ganglion [35], where anterograde tracing studies demonstrated a trigeminal innervation [2]. Such an innervation might also be present in the human ciliary ganglion. Indeed, it is well documented that axons from the nasociliary nerve enter the human ciliary ganglion [14]. Another possibility might be an origin from the pterygopalatine ganglion, since SP/CGRP-LI perikarya have been demonstrated in the sphenopalatine ganglion of rats [15] and anterograde tracing studies in the same species indicate that some axons from the sphenopalatine ganglion traverse the ciliary ganglion [3]. On the other hand, these axons do not contact ciliary neurons [3], and ocular or orbital nerve fibers deriving from the human pterygopalatine ganglion do not seem to enter the ciliary ganglion [28]. An origin of ciliary ganglion CGRP/SP-LI axons from the superior cervical ganglion seems unlikely, since only some CGRP-LI neurons, but no SP-LI neurons

::ii Fig. 3. SP-LI and CGRP-LI in human ciliary ganglion. CGRP-LI nerve fibers (arrows) are stained in nerve trunks that enter the ciliary ganglion (A) and within the ganglion between ciliary nerve cells (B). Some nerve cells (asterisk) are in close contact with positively stained varicose axons (arrowhead). In addition, SP-LI (C) and CGRP-LI (D) varicose nerve endings may form pericellular basket-like formations around ciliary ganglion perikarya (scale bar: 8.75 ~m).

W. Kirch et al. /Brain Research 681 (1995) 229-234

have been found in the htmaan superior cervical ganglion [11. Numerous SP-LI and CGRP-LI varicose axons have been observed in the human ciliary muscle [33]. It is assumed that these peptides are locally released by means of an axon reflex in course of the irritative response of the eye [4]. In humans, intraocular inflammations or trigeminal neuralgia may be accompanied by severe spasms of the ciliary muscle [8]. It is tempting to speculate that in such conditions a connection of trigeminal S P / C G R P axon collaterals with ciliary ganglion cells might function as an alternative pathway to a reflex arc through the brainstem which enables trigeminal neurons to modulate ciliary muscle tone. Clearly, more experimental studies are needed to verify this hypothesis. The excellent photograp]hic help of Marco G/iBwein is gratefully acknowledged. This research was supported by a grant from the Deutsche Forschungsgemeinschaft (Ta

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(

Fig. 5. SP-LI (A) and CGRP-LI (B) in human trigeminal ganglion (same donor as in Fig. 3, scale bar: 5 /xm). The perikarya of many trigeminal neurons are positively labelled for SP-LI or CGRP-LI.

115/5-1). The authors dedicate this paper to Prof. W. Zenker on the occasion of his 70th birthday.

References

Fig. 4. Double labelling for SP-LI (A) and CGRP-LI (B) in human ciliary ganglion (scale bar: 8.75 /zm). In such experiments, almost all CGRP-LI varicose nerve endings in pericellular baskets are also SP-LI, indicating axonal coexistence.

[1] Baffi, J., Gores, T., Slowik, F., Horvath, M., Leldca, N., Pasztor, E. and Palkovits, M., Neuropeptides in the human superior cervical ganglion, Brain Res., 570 (1992) 272-278. [2] Beckers, H.J.M., Klooster, J., Vrensen, G.F.J.M. and Lamers, W.P.M.A., Ultrastructural identification of trigeminal nerve terminals in the pterygopalatinc ganglion of rats: an anterograde tracing and immunohistochemical study, Brain Res., 557 (1991) 22-30. [3] Beckers, H.J.M., Klooster, J., Vrensen, G.F.J.M. and Lamers, W.P.M.A., Facial parasympathetic innervation of the rat choroid, lacrimal glands and ciliary ganglion: an ultrastructural pterygopalatine tracing and immunohistochemical study, Ophthalmic Res., 25 (1993) 319-330. [4] Bill, A., The 1990 Endre Balazs Lecture. Effects of some neuropeptides on the urea, Exp. Eye Res., 53 (1991) 3-11. [5] Bozler, E., Conduction, automaticity and tonus of visceral muscle, Experientia, 4 (1948) 213-218.

234

W. Kirch et al. / Brain Research 681 (1995) 229-234

[6] Dryer, S.E. and Chiapinelli, V.A., Properties of choroid and ciliary neurons in the avian ciliary ganglion and evidence for substance P as a neurotransmitter, J. Neurosci., 5 (1985) 2654-2661. [7] Dryer, S.E. and Chiapinelli, V.A., Substance P depolarizes nerve terminals in an autonomic ganglion, Brain Res., 336 (1985) 190-194. [8] Dnke-Elder, S. and Scott, G.I., Accommodation spasm. In S. DukeElder (Ed.), System of Ophthalmology. Vol. XII. Neuroophthalmology, Henry Kimpton, London, 1971, pp. 706-709. [9] Erichsen, J.T., Karten, H.J., Eldred, W.D. and Brecha, N.C., Localization of substance P-like and enkephalin-like immunoreactivities within preganglionic terminals of the avian ciliary ganglion: light and electron microscopy, J. Neurosci., 2 (1982) 994-1003. [10] Erickson-Lamy, K.A., Polansky, J.R., Kaufman, P.L. and Zlock, D.M., Cholinergic drugs alter ciliary muscle response and receptor content, Invest. Ophthalmol. Vis. Sci., 28 (1987) 375-383. [11] Fliigel, C., Tamm, E.R., Mayer, B. and Liitjen-Drecoll, E., Species differences in choroidal vasodilative innervation: evidence for specific intrinsic nitrerglc and VIP-positive neurons in the human eye, Invest. Ophthalmol. Vis. Sci., 35 (1994) 592-599. [12] Grimes, P.A., Koeberlein, B., Tigges, M. and Stone, R.A., Neuropoptide Y localizes to preganglionic axon terminals in monkey ciliary ganglion, Invest. Ophthalmol. Iris. Sci. (ARVO abstracts), 35 (1994) 2050 [13] Grimes, P.A., McGlinn, A.M. and Stone, R.A., An immunohistochemically distinct population of cat ciliary ganglion cells, Brain Res., 535 (1990) 323-326. [14] Grimes, P.A. and yon Sallmann, L., Comparative anatomy of the ciliary nerves, Arch. Ophthalmol., 64 (1960) 111-121. [15] Hardebo, J.E., Suzuki, N., Ekblad, E. and Owman, C., Vasoactive intestinal polypeptide and acetylcholine coexist with neuropeptide Y, dopamine-b-hydmxylase, substance P or calcitonin gene-related peptide in neuronal subpopulations in cranial parasympathetic ganglia of rat, Cell Tissue Res., 267 (1992) 291-300. [16] Ishikawa, T., Fine structure of the human ciliary muscle, Invest. Ophthalmol. Vis. Sci., 1 (1962) 587-608. [17] Kummer, W., Bachmann, S., Neuhuber, W.L., H~inze, J. and Lang, R.E., Tyrosine-hydroxylase-containing vagal afferent neurons in the rat nodose ganglion are independent from neuropeptide-Y-containing populations and project to esophagus and stomach, Cell Tissue Res., 271 (1993) 135-144. [18] Kummer, W., Gibbins, I.L., Stefan, P. and Kapoor, V., Catecholamines and catecholamine-synthesizing enzymes in guinea-pig sensory ganglia, Cell Tissue Res., 261 (1990) 595-606. [19] Kuwayama, Y., Terenghi, G., Polak, J.M., Trojanowski, J.Q. and Stone, R.A., A quantitative correlation of substance P-, calcitonin gene-related peptide- and cholecystokinin-like immunoreactivity with retrogradely labeled trigeminal ganglion cells innervating the eye, Brain Res., 405 (1987) 220-226. [20] Landis, S.C., Jackson, P.C., Fredieu, J.R. and Thibault, J., Catecholaminergic properties of cholinergic neurons and synapses in adult rat ciliary ganglion, J. Neurosci., 7 (1987) 3574-3587. [21] Leblanc, G.G. and Landis, S.C., Target speciflty of neuropetide Y-immunoreactive cranial parasympathetic neurons, J. Neurosci., 8 (1988) 146-155. [22] Leblanc, G.G., Trimmer, B.A. and Landis, S.C., Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: coexistence with vasoactive intestinal peptide and choline acetyltransferase, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 3511-3515. [23] Lee, Y., Kawai, Y., Shiosaka, S., Takami, K., Hillyard, C.J., Girgis, S., MacIntyre, I., Emson, P.C. and Tohyama, M., Coexistence of calcitonin gene-related peptide and substance P-like peptide in single cells of the trigeminal ganglion of the rat: immunohistochemical analysis, Brain Res., 330 (1985) 194-196. [24] Maciewicz, R., Phipps, B.S., Foote, W.E., Aronin, N. and DiFiglia, M., The distribution of substance P-containing neurons in the cat Edinger-Westphal nucleus: relationship to efferent projection systems, Brain Res., 270 (1983) 217-230.

[25] Prins, M., Van der Weft, F., Baljct, B. and Otto, J.A., Calcitonin gene-related peptide and substance P immunoreactivity in the monkey trigeminal ganglion, an electron microscopic study, Brain Res., 629 (1993) 315-318. [26] Quartu, M., Diaz, G., Floris, A., Lai, M.L., Priestley, J.V. and Del Fiaceo, M., Calcitonin gene-related peptide in the human trigeminal sensory system at developmental and adult life stages: immunohistochemistry, neuronal morphometry and coexistence with substance P, J. Chem. Neuroanat., 5 (1992) 143-157. [27] Rohen, J.W., Ciliarki~rper (Corpus ciliate). In W. von M/Jllendoft and W. Bargmann (Eds.) Handbuch der mikroskopischen Anatoraie des Menschen. Vol. 3, pt. 4. Haut und Sinnesorgane. Das Auge und seine Hilfsorgane, Springer Veflag, Heidelberg, New York, 1964, pp. 189-237. [28] Ruskell, G.L., An ocular parasympathetic nerve pathway of facial nerve origin and its influence on intraocular pressure, Exp. Eye Res., 10 (1970) 319-330. [29] Ruskell, G.L., Facial parasympathetic innervation of the choroidal blood-vessels in monkeys, Exp. Eye Res., 12 (1971) 166-172. [30] Ruskell, G.L. and Griffiths, T., Peripheral nerve pathway to the ciliary muscle, Exp. Eye Res., 28 (1979) 277-284. [31] Skofitsch, G. and Jacobowitz, D.M., Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat, Peptides, 6 (1985) 747-754. [32] Stefanini, M., de Martino, C. and Zamboni, C., Fixation of ejaculated spermatozoa for electron microscopy, Nature, 216 (1967) 173-174. [33] Stone, R.A., Kuwayama, Y. and Laties, A.M., Regulatory peptides in the eye, Experientia, 43 (1987) 791-800. [34] Stone, R.A., McGlinn, A.M., Kuwayama, Y. and Grimes, P.A., Peptide immunoreactivity of the ciliary ganglion and its accessory cells in the rat, Brain Res., 475 (1988) 389-392. [35] Suzuki, N., Hardebo, J.E. and Owman, C., Trigeminal fibre collaterals storing substance P and calcRonin gene-related peptide associate within ganglion cells containing choline acetyRransferase and vasoactive intestinal polypeptide in the sphenopalatine ganglion of the rat. An axon reflex modulating parasympathetic ganglionic activity?, Neuroscienee, 30 (1989) 595-604. [36] Terenghi, G., Polak, J.M., Ghatei, M.A., Mulderry, P.K., Butler, J.M., Unger, W.G. and Bloom, S.R., Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye, J. Comp. Neurol., 233 (1985) 506-516. [37] Tyrrell, S., Siegel, R.E. and Landis, S.C., Tyrosine hydroxylase and neuropeptide Y are increased in ciliary ganglia of sympatheetomized rats, Neuroscience, 47 (1992) 985-998. [38] Uemura, Y., Suglmoto, T., Nomura, S., Nagatsu, I. and Mizuno, N., Tyrosine hydroxylase-like immunurcactivity and catecholamine fluorescence in ciliary ganglion neurones, Brain Res., 416 (1987) 200203. [39] Vakkur, G.J., Bishop, P.O. and Kozak, W., Visual optics in the cat, including posterior nodal distance and retinal landmarks, Vision Res., 3 (1963) 289-314. [40] Van der Weft, F., Innervation of the lacrimal gland in the cynomolgus monkey. A retrograde tracing and immunohistochemical study. In F. van der Weft (Ed.), Autonomic and Sensory lnnervation of Some Orbital Structures in the Primate, Thesis, Universiteit van Amsterdam, Amsterdam, 1993, pp. 51-70. [41] Warwick, R., The ocular parasympathetic nerve supply and its mesencephalic sources, J. Anat., 88 (1954) 195-203. [42] Zhang, Y.L., Tan, C.K. and Wong, W.C., Localisation of substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca faseicularis) and cat: a fight- and electron-microscopic study, Cell Tissue Res., 276 (1994) 163-171.