Analysis of calcium channels in single spines using optical fluctuation analysis

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Abstract
Most synapses form on small, specialized postsynaptic structures known as dendritic spines1. The influx of Ca2+ ions into such spines—through synaptic receptors and voltage-sensitive Ca2+ channels (VSCCs)—triggers diverse processes that underlie synaptic plasticity2. Using two-photon laser scanning microscopy3, we imaged action-potential-induced transient changes in Ca2+ concentration in spines and dendrites of CA1 pyramidal neurons in rat hippocampal slices4. Through analysis of the large trial-to-trial fluctuations in these transients, we have determined the number and properties of VSCCs in single spines. Here we report that each spine contains 1–20 VSCCs, and that this number increases with spine volume. We are able to detect the opening of a single VSCC on a spine. In spines located on the proximal dendritic tree, VSCCs normally open with high probability (∼0.5) following dendritic action potentials. Activation of GABAB receptors reduced this probability in apical spines to ∼0.3 but had no effect on VSCCs in dendrites or basal spines. Our studies show that the spatial distribution of VSCC subtypes and their modulatory potential is regulated with submicrometre precision.