The distribution of alpha-bungarotoxin binding sites on chick ciliary ganglion neurons was examined at the ultrastructural level by incubating ganglia with horseradish peroxidase-conjugated toxin and examining the peroxidase-stained and thin-sectioned ganglia with the electron microscope. Both in embryonic and in adult ganglia heavy labeling was restricted to the surface membrane of short processes emerging from the ciliary and choroid cell somata in the region of preganglionic innervation. Less dense labeling occasionally was present on the smooth surface membrane of the soma in the same region. In contrast, the pre- and postsynaptic membranes of most synapses were clearly not labeled even in the immediate vicinity of heavily labeled processes. The labeling represented specific binding of the toxin conjugate since it could be prevented by d-tubocurarine and hexamethonium or by unconjugated toxin. The conjugated toxin was not excluded from the synaptic cleft on the basis of size because a substantially larger protein conjugate, a horseradish peroxidase-labeled monoclonal antibody, was able to enter the cleft and heavily label synaptic membranes as well as soma membranes. Even neurons in adult ganglia had very little synaptic labeling after exposure to the conjugated toxin. These results strongly suggest that the high affinity alpha-bungarotoxin binding sites on chick ciliary ganglion neurons are different from the synaptic ACh receptors which would be expected to be concentrated in the postsynaptic membrane. Clustering of the alpha-bungarotoxin binding sites in the vicinity of synapses, however, may reflect a related synaptic function.

Survival and development of chick ciliary ganglion neurons in vivo appear to depend on information from the embryonic eye structure that contains the postsynaptic targets of the neurons. We have tested embryonic eye extracts on ciliary ganglion neurons in dissociated cell culture for stimulation of growth and development. Control conditions were chosen that permitted the long term maintenance of the neurons in the absence of tissue extracts of conditioned medium. The conditions included coating the culture substratum with fibroblast material and increasing the K+ concentration in the culture medium to 25 mM. Neurons survived for at least 3 weeks in control conditions. Two major components were resolved in eye extracts that stimulated growth and development of the neurons above the basal levels obtained with control conditions. One component, with an apparent molecular weight of about 2 X 10(4) by gel filtration analysis, stimulated neuronal growth without increasing the levels of choline acetyltransferase activity per neuron. The second component, with an apparent molecular weight of about 5 X 10(4), increased development of choline acetyltransferase levels per neuron but had no effect on neuronal growth. Both components were effective in normal K+ as well as 25 mM K+. These components may represent mechanisms by which the postsynaptic target tissue acts in vivo to direct the growth and development of ciliary ganglion neurons.

Chick ciliary ganglion neurons have previously been shown to contain a component that shares an antigenic determinant with the "main immunogenic region" of the alpha-subunit in nicotinic acetylcholine receptor from skeletal muscle and electric organ. Ultrastructural studies of antibody binding in the ganglion have shown that the cross-reacting antigen exposed on the surface of the neurons is located predominantly in synaptic membrane. Here we show that the neuronal antigen can be identified in detergent extracts of ciliary and sympathetic ganglia, but not in extracts of heart, liver, spinal cord, retina, or dorsal root ganglia. In the ciliary ganglion the component is present as an integral membrane constituent, and, when detergent solubilized, it sediments as a 10 S species and binds to concanavalin A. The component is distinct from the alpha-bungarotoxin-binding site on the neurons since toxin-binding sites and antibody-binding sites can be precipitated separately in ganglion extracts. The component reaches peak levels per ganglionic protein between embryonic days 8 and 12. These are some of the properties expected for the nicotinic acetylcholine receptor on ciliary ganglion neurons.

A large family of genes encoding subunits of nicotinic ACh receptors (AChRs) has been identified in vertebrates and shown to be expressed in the nervous system. The multiplicity of genes raises questions about which gene products coassemble to produce native receptor subtypes and how the expression of receptor genes is regulated in neurons. We report here that five neuronal AChR genes are expressed in the chick ciliary ganglion at both early and late times in development. Quantitative RNase protection experiments demonstrated that at embryonic day 18 (E18) the ganglion contains about 1800 copies of alpha 7 transcript per neuron, 900 copies of alpha 3 transcript per neuron, and 200-300 copies each of alpha 5, beta 2, and beta 4 transcripts per neuron. The same five genes are expressed at significantly lower levels at E8 but show the same rank order of abundance in transcripts per neuron. Few, if any, transcripts were found for the alpha 2, alpha 4, alpha 8, and beta 3 AChR genes in ciliary ganglion RNA at either E8 or E18. The 6- and 13-fold increases previously reported for two classes of AChRs on the neurons between E8 and E18 approximate the 4-14-fold increases observed here in AChR gene mRNA levels per neuron over the same time period. The alpha 3, alpha 5, alpha 7, and beta 4 genes have previously been correlated with subunits of ciliary ganglion AChRs, but the beta 2 gene has not. The abundance of beta 2 transcripts raises the possibility either that the known AChRs in the ganglion have a more complex subunit composition than previously described or that additional receptor subtypes remain to be discovered. Northern blot analysis revealed no changes in transcript pattern for the alpha 3, alpha 5, and beta 4 genes between E8 and E18; a small change may occur in the transcript pattern for the alpha 7 gene. In situ hybridizations demonstrated that alpha 5 and beta 4 transcripts are expressed in essentially all ciliary ganglion neurons as has been shown previously for the more abundant alpha 3 transcript and inferred for the alpha 7 transcript. The results indicate that neurons can stably coexpress multiple AChR genes, including three of the alpha type, and that transcript levels may be rate limiting for accumulation of AChRs during development.

An alpha-neurotoxin, Bgt 3.1, that reversibly blocks the ACh response of chick ciliary ganglion neurons has been used to identify 2 classes of high-affinity binding sites on the cells in culture. The first class appears to be the alpha-bungarotoxin binding site on the neurons. The second class of Bgt 3.1 sites is distinct from the alpha-bungarotoxin binding sites and has the properties expected for the functional nicotinic ACh receptor on the cells. Equilibrium binding and kinetic studies indicate a Kd value of 5-6 nM for Bgt 3.1 at the second class of sites. The kinetics and affinity of binding are consistent with those inferred from previous physiological studies for Bgt 3.1 inhibition of receptor function. Bgt 3.1 binding to the sites is completely inhibited by each of the cholinergic ligands ACh, carbachol, nicotine, d-tubocurarine, and trimethaphan, but not by alpha-bungarotoxin. Highest site densities are found in cultures of ciliary and sympathetic ganglion neurons, cell types known to have ganglionic nicotinic ACh receptors. Low levels of sites may be present in cultures of spinal cord and dorsal root ganglion neurons; no binding is found in cultures of skeletal myotubes or cardiac cells when alpha-bungarotoxin is used to block Bgt 3.1 binding to alpha-bungarotoxin sites. These results demonstrate that Bgt 3.1 can be used as a specific probe for the nicotinic ACh receptor on chick autonomic neurons.