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

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 alpha-bungarotoxin (alpha Bgt), indicating dependence on alpha Bgt-AChRs, most of which are alpha 7 subunit homopentamers. Slow sEPSCs were unaffected by alpha Bgt, and were blocked instead by the alpha 3/beta 2-selective alpha-conotoxin-MII (alpha CTx-MII), indicating dependence on alpha 3*-AChRs, which lack alpha 7 and contain alpha 3 subunits. Biexponential sEPSCs were mediated by both alpha Bgt- and alpha 3*-AChRs because they had fast and slow components qualitatively similar to those comprising simple events, and these were reduced by alpha Bgt and blocked by alpha CTx-MII, respectively. Fluorescence labeling experiments revealed both alpha Bgt- and alpha 3*-AChR clusters on neuron somata and neurites. Colabeling with antisynaptic vesicle protein antibody suggested that some alpha 3*-AChR clusters, and a few alpha Bgt-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.