Ca2+ imaging of CNS axons in culture indicates reliable coupling between single action potentials and distal functional release sites

A combination of Ca2+ imaging and current-clamp recording in cultured cortical neurons was used to evaluate the reliability of coupling between the action potential and rises in Ca2+ at distal release sites as a possible source of variability in CNS synaptic transmission. Local domains of enhanced Ca2+ influx were observed at varicosities on axon collaterals. Functional assay of vesicle turnover using FM1-43 and parallel electron microscopy confirmed that these varicosities were release sites. Single action potentials reliably (>95% of the time) resulted in a presynaptic Ca2+ transient at all presumed release sites including those on distal collaterals. Variability in the amplitude of presynaptic Ca2+ transients at individual boutons was estimated to be on average less than 20%. We conclude that the coupling of somatic action potentials to distal release sites is generally a reliable process, although nonlinearity in the relationship between Ca2+ influx and neurotransmitter release may amplify the effects of relatively small fluctuations in Ca2+ influx.