The Journal of Neuroscience, November 1987, 7(11): 35743587 Catecholaminergic Properties of Cholinergic Neurons and Synapses in Adult Rat Ciliary Ganglion Story C. Landis,’ Patrick C. Jackson,l,a John R. Fredieu,l,b and Jean ThibauW ‘Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, and 2CoIlege de France, Paris, France Parasympathetic neurons of the ciliary ganglion are inner- The developmental mechanisms responsible for these mixed vated by preganglionic cholinergic neurons whose cell bod- transmitter phenotypes and the functional consequences re- ies lie in the brain stem; the ganglion cells in turn provide main to be elucidated. cholinergic innervation to the intrinsic muscles of the eye. Noradrenergic innervation of the iris is supplied by sympa- thetic neurons of the superior cervical ganglion. Using im- The ciliary ganglion is classified as a parasympathetic ganglion munocytochemical and histochemical techniques, we have based on anatomical, biochemical, and pharmacological crite- examined the ciliary ganglion of adult rats for the expression ria. The ganglion lies close to its target tissues, the iris and ciliary of cholinergic and noradrenergic properties. As expected, body; the preganglionic neurons lie in the brain stem (Warwick, the postganglionic ciliary neurons possessed detectable 1954; Loewy et al., 1978; Johnson and Purves, 198 1). In the levels of choline acetyltransferase immunoreactivity (ChAT- cat, the mammal studied most extensively, the ganglion contains IR). Unexpectedly, many ciliary neurons also exhibited im- high levels of ChAT, reflecting enzyme present in both pregan- munoreactivity for tyrosine hydroxylase (TH-IR). Some had glionic terminals and postganglionic perikarya (Buckley et al., dopamine&hydroxylase-like (DBH-IR) immunoreactivity, but 1967). The postganglionic terminals in the iris contain high none contained detectable catecholamines, even after treat- levels of ChAT (Ehinger et al., 1966) and ACh (Ehinger et al., ment with nialamide and L-DOPA. A sparse plexus of fibers 1970a). The pharmacology of transmission at both ganglionic exhibiting faint TH-IR was present in the irises of acutely (Langley and Anderson, 1892; Perry and Talesnik, 1953) and sympathectomized rats. The terminals of preganglionic ax- postganglionic synapses is cholinergic (Schaeppi and Koella, ons in the ciliary ganglion exhibited not only immunoreac- 1964; Ehinger et al., 1968). tivity for ChAT, but also for TH and contained stores of en- According to the traditional view of the autonomic nervous dogenous catecholamine. Neither ciliary neurons nor their system, postganglionic sympathetic neurons are noradrenergic, preganglionic innervation accumulated detectable stores of parasympathetic ones are cholinergic, and the preganglionic exogenous catecholamines. Rats sympathectomized as neurons of both divisions are uniformly cholinergic. Thus, the neonates by treatment with 6-hydroxydopamine subse- parasympathetic and functionally cholinergic neurons of the quently had a greater proportion of neurons possessing de- ciliary ganglion and their preganglionic inputs would not be tectable TH-IR in the ciliary ganglion; both the TH-IR peri- expected to express catecholaminergic properties. A number of karya and their axons in the iris were more intensely observations, however, make it clear that in the autonomic ner- immunofluorescent. TH-IR was present in the ciliary neuron vous system, neurotransmitter-related properties cannot be at- cell bodies of mouse, guinea pig, and ferret. These species, tributed singly or strictly on the basis of cell class. For example, however, lacked detectable TH-IR or catecholamine stores in the sympathetic division, a minority population of postgan- in preganglionic terminals. These observations indicate that glionic neurons are functionally choline@ (Langley, 189 1, 1922; mature, functionally cholinergic neurons from 2 different em- Sjoqvist, 1963; Leblanc and Landis, 1986) and developing ad- bryonic origins, postganglionic ciliary neurons derived from renergic sympathetic neurons can become cholinergic both in the neural crest and preganglionic neurons derived from the vitro (e.g., Johnson et al., 1976, 1980; Patterson and Chun, 1977; neural tube, display several catecholaminergic properties. Landis, 1980; Potter et al., 1986) and in viva (Landis and Keefe, 1983; Leblanc and Landis, 1986). Under certain conditions in vitro (Furshpan et al., 1976; Landis, 1976; Reichardt and Pat- Received Oct. 10, 1986; revised Mar. 2, 1987; accepted Apr. 30, 1987. terson, 1977; Higgins et al., 1981; Iacovitti et al., 1981; Potter This work was supported by USPHS Grants NS 02253 and NS 15549 and et al., 1986) and in vivo (Siegel et al., 1982; Landis and Keefe, Training Grant NS07009 and was carried out during the tenure of an Established 1983) functionally cholinergic sympathetic neurons exhibit ad- Investigatorship of the American Heart Association (S.C.L.) and with funds con- tributed in part by the Massachusetts Affiliate. We wish to thank Dr. Nancy Ip renergic properties. In the parasympathetic division of the auto- for performing tyrosine hydroxylase assays and Drs. David Potter, Bill Matthew, nomic nervous system, mature submandibular ganglion neurons and Tom Jesse11 for critically reading the manuscript. contain dopamine-P-hydroxylase (DBH) enzyme activity and Correspondence should be addressed to Story C. Landis, Department of Phar- macology, Case Western Reserve Medical School, Cleveland, OH 44106. immunoreactivity (Coyle et al, 1974; Grzanna and Coyle, 1978). = Present address: Department of Anatomy, Dalhousie University, Halifax, Nova A small proportion of ciliary neurons in the chick embryo nor- Scotia, Canada. h Present address: Department of Anatomy, Case Western University, Cleve- mally express tyrosine hydroxylase-immunoreactivity (TH-IR) land, OH 44106. and the proportion of these TH-IR neurons is significantly in- Copyright 0 1987 Society for Neuroscience 0270-6474/87/l 13574-14$02.00/O creased in cell culture (Iacovitti et al., 1985; Teitelman et al., The Journal of Neuroscience, November 1987, 7(11) 3575 1985). Finally, Bjorklund and colleagues (1985) have shown that antiserum has been described previously (Grzanna et al., 1976). The ciliary terminals in the irises of adult rats acquire detectable phenylethanolamine N-methyl transferase (PNMT) antiserum was raised in rabbit against PNMT purified from bovine adrenal medulla and was levels ofTH-IR following sympathectomy. In the course ofother obtained commercially (Eugene Tech International). Synaptic terminals studies, we observed identical TH-IR fibers in the irises of rats were identified using a mouse monoclonal antibody that recognizes a sympathectomized at birth by treatment with 6-hydroxydopa- synaptic vesicle antigen (Matthew et al., 198 1). mine. In tracing the origin of these fibers, we found that the Immunocytochemistry. Binding of the primary ChAT antiserum was visualized with a double-cycle peroxidase-antiperoxidase procedure ciliary ganglia of normal as well as chronically sympathecto- (PAP; Stemberger et al., 1970; Ordronneau et al., 198 1). Six-week-old mized adult rats contained significant numbers of TH-IR neu- rats were deeply anesthetized with ether and perfused through the heart rons. In addition, preganglionic terminals in the ciliary ganglion for 10 min with 4% paraformaldehyde in 0.1 M phosphate buffer, pH contained not only TH-IR, but also significant stores of cate- 7.3, containing 15% by volume of a saturated aqueous solution of picric cholamine, most likely dopamine. A preliminary report of these acid. The ciliary ganglia were dissected along with a segment of the oculomotor nerve and immersed for an additional 50 min in the same results has appeared (Landis et al., 1983). fixative solution. The ganglia were rinsed several times with 0.1 M phosphate buffer and then equilibrated with 30% sucrose in phosphate buffer. In some cases, 20 pm cryostat sections were cut, while in others, Materials and Methods the ganglia were frozen and thawed and then processed as whole mounts. Animals. Adult albino rats were used in most experiments. In addition, The sections or ganglia were incubated overnight at room temperature ciliary ganglia from adult Long Evans hooded rats, albino and pigmented in mouse anti-ChAT (the kind gift of F. Eckenstein, Harvard Medical mice, albino guinea pigs, and pigmented ferrets were examined for both School, Boston), diluted 1:lOOO in buffer containina 0.1 M Tris buffer. TH-IR and catecholamine histofluorescence. Some rats were acutely pH 7.3, 150 rnh sodium chloride, 1% BSA, and 5%-Triton X-100. The sympathectomized by surgical removal of the superior cervical ganglion tissue was rinsed several times with incubation buffer, then incubated and examined 2, 4, or 7 d after surgery. In addition, other rats were in affinity-purified goat anti-mouse immunoglobulins diluted I:40 in chemically sympathectomized as neonates or as adults by treatment incubation buffer containing 10% rat serum. After rinsing in incubation with 6-hydroxydopamine; rat pups were injected with 100 mg/kg in- buffer, the sections were incubated in a mouse monoclonal antibody to traperitoneally (i.p.) daily for the first postnatal week and adults were a PAP complex diluted 1:80 in incubation buffer containina rat serum. injected with 100 mg/kg i.p. for 3 d and examined on the fourth (Clark The sections were then cycled a second time through the goat anti- et al., 1972; Finch et al., 1973). To increase