Direct recording of nicotinic responses in presynaptic nerve terminals

Nicotinic acetylcholine receptors are widely expressed in the nervous system, but their functions remain poorly understood. One attractive hypothesis is that the receptors act presynaptically to modulate synaptic transmission. We provide a direct demonstration of presynaptic nicotinic receptors in situ by using whole-cell patch-clamp techniques to record currents in large presynaptic calyces that midbrain neurons form on ciliary neurons. Bath application of nicotine induced inward currents in the calyces capable of generating action potentials that overrode the limited space clamp achievable. The inward currents reversed near 0 mV and showed inward rectification common for neuronal nicotinic receptors. Tetrodotoxin (TTX) blocked the action potentials but not the inward currents. alpha-Bungarotoxin blocked both, consistent with the presynaptic receptors containing alpha7 subunits. Recording from the postsynaptic ciliary neurons during nicotine exposure revealed EPSCs that TTX blocked, presumably by blocking presynaptic action potentials. The postsynaptic cells also displayed bimodal inward currents caused by their own nicotinic receptors; the bimodal currents were not blocked by TTX but were blocked partially by alpha-bungarotoxin and completely by D-tubocurarine. Dye-filling with Lucifer yellow from the recording pipette confirmed the identity of patched structures and showed no dye transfer between calyx and ciliary neuron. When calyces or ciliary neurons were labeled en mass with neurobiotin and biocytin through nerve roots, dye transfer was rarely observed. Thus, electrical synapses were infrequent and unlikely to influence calyx responses. Immunochemical analysis of preganglionic nerve extracts identified receptors that bind alpha-bungarotoxin and contain alpha7 subunits. The results unambiguously document the existence of functional presynaptic nicotinic receptors.