Instead of measuring synaptic responses using electrophysiological methods, Oertner et al. opted for visualizing postsynaptic NMDA (N-methyl-D-aspartate)-receptor-dependent calcium transients. Glutamate release increased intracellular calcium at single spines, allowing the authors to distinguish trials in which release occurred (when there was a calcium transient) — successes — from those in which there was no vesicle fusion (no calcium signal) — failures. They measured how the transients changed under conditions that affect release probability, such as paired-pulse facilitation (PPF). In PPF, two action potentials that arrive in quick succession lead to a larger synaptic response after the second stimulus. If release were monovesicular, then PPF should not lead to further increases in the amplitude of a calcium transient, because the amount of glutamate that is released by a single vesicle is relatively homogeneous. However, Oertner et al. found that calcium increases in response to the second stimulus were significantly larger than those evoked by single action potentials, implying that PPF causes more glutamate to be released at single synapses. The simplest explanation for this finding is that transmitter is released from more than one vesicle. Indeed, the data agree with the predictions of the classical binomial model for multiple release sites, and the authors propose the existence of five independent release sites per active zone.

Measuring postsynaptic responses with this optical method arguably provides the most direct evidence for multivesicular release at a single central synapse. Clearly, the ability of a single active zone to release more than one vesicle at a time markedly increases the dynamic range of the synapse. Whether long-term plastic changes in transmission make use of this capacity is an intriguing question.

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