Nicotinic synapses formed between chick ciliary ganglion neurons in culture resemble those present on the neurons in vivo

Publisher Website
Google Scholar
Abstract
We studied nicotinic synapses between chick ciliary ganglion neurons in culture to learn more about factors influencing their formation and receptor subtype dependence. After 4–8 days in culture, nearly all neurons displayed spontaneous excitatory postsynaptic currents (sEPSCs), which occurred at about 1 Hz. Neurons treated with tetrodotoxin displayed miniature EPSCs (mEPSCs), but these occurred at low frequency (0.1 Hz), indicating that most sEPSCs are actually impulse driven. The sEPSCs could be classified by decay kinetics as fast, slow, or biexponential and, reminiscent of the situation in vivo, were mediated by two major nicotinic acetylcholine receptor (AChR) subtypes. Fast sEPSCs were blocked by α‐bungarotoxin (αBgt), indicating dependence on αBgt‐AChRs, most of which are α7 subunit homopentamers. Slow sEPSCs were unaffected by αBgt, and were blocked instead by the α3/β2‐selective α‐conotoxin‐MII (αCTx‐MII), indicating dependence on α3*‐AChRs, which lack α7 and contain α3 subunits. Biexponential sEPSCs were mediated by both αBgt‐ and α3*‐AChRs because they had fast and slow components qualitatively similar to those comprising simple events, and these were reduced by αBgt and blocked by αCTx‐MII, respectively. Fluorescence labeling experiments revealed both αBgt‐ and α3*‐AChR clusters on neuron somata and neurites. Colabeling with antisynaptic vesicle protein antibody suggested that some α3*‐AChR clusters, and a few αBgt‐AChR clusters are associated with synaptic sites, as is the case in vivo. These findings demonstrate the utility of ciliary ganglion neuron cultures for studying the regulation of nicotinic synapses, and suggest that mixed AChR subtype synapses characteristic of the neurons in vivo can form in the absence of normal inputs or targets. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 265–279, 2001