We have examined the specificity and the mechanism of acetylcholine receptor (AChR) accumulation at embryonic chick nerve-muscle contacts that form in culture. Spinal cord motoneurons were identified in vitro after labeling them in vivo with Lucifer Yellow-wheat germ agglutinin conjugates. All of their processes induced receptor clusters on contacted myotubes; after 24 to 48 hr of co-culture, the incidence of neurite-associated receptor patches (NARPs) was approximately 1.2/100 microns of contact. In contrast, NARPs were rarely associated with spinal cord interneurons (approximately 0.1/100 microns of contact). Neurons dissociated from ciliary ganglia induce NARPS to the same extent as motoneurons. The relative contribution to NARPs of AChRs present in the membrane prior to plating ciliary ganglion neurons and of "new" AChRs inserted 8, 11, or 17 hr after addition of neurons was assessed with two fluorescent receptor probes. Rhodamine-conjugated alpha-bungarotoxin was used to label either old or new receptors; a monoclonal, anti-receptor antibody visualized with fluorescein-second antibody was used to label all (new and old) receptors. Analysis of digitized fluorescence images showed that NARPs contained both new and old receptors but that within the first 24 hr of co-culture the majority (60 to 80%) were new. We estimate that cholinergic neurites increase the rate of receptor insertion 4- to 5-fold during the first 8 hr of NARP formation. The contribution of new receptors to NARPs declines with time. After 3 days of co-culture, receptors inserted over an 8-hr interval comprised only 20% of the total NARP complement.(ABSTRACT TRUNCATED AT 250 WORDS)

Although presynaptic input can influence the number and distribution of ACh receptors (AChRs) on muscle, the role of cellular interactions in the development of transmitter sensitivity in neurons is less clear. To determine whether presynaptic input modifies neuronal AChR channel function and distribution, we must first ascertain the profile of changes in receptor properties relative to the timing of synapse formation. We have examined the temporal aspects of synaptogenesis in the lumbar sympathetic ganglia of the embryonic chick in anatomical experiments with anterograde 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate labeling of presynaptic inputs and cytochrome oxidase histochemistry. Biophysical studies of sympathetic neurons, within hours of removal from animals at different stages relative to synapse formation, show that both the properties and distribution of AChR channels are modified concurrent with a significant increase in presynaptic input to the neurons. The most striking change in AChR channel distribution is revealed by patching multiple sites on the surface of individual neurons. Following innervation in vivo, many neurons express only one of the four AChR channel subtypes and the AChRs are clustered in discrete, high-activity patches. Furthermore, when neurons at this stage express more than one AChR channel subtype, the different classes are often spatially segregated from one another on the cell surface. This contrasts with patches from neurons removed earlier on, which have lower overall activity, often comprised of multiple channel subtypes. Comparison of the AChR properties of acutely dispersed neurons to those of neurons maintained in vitro indicates that most features of AChR channels are conserved despite their removal from presynaptic and other in vivo influences. These findings are consistent with inductive interactions between pre- and postsynaptic neurons playing an important regulatory role in transmitter receptor expression.

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