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.

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Figure 1: Action-potential-evoked Ca2+ influx in spines and dendrites.
Figure 2: Variance analysis.
Figure 3: Failures of action-potential-evoked Ca2+ influx in spines.
Figure 4: GABAB receptor activation reduces Ca2+ influx in apical spines.
Figure 5: Number of VSCCs opened by an action potential in apical (squares) and basal (circles) spines plotted as a function of spine volume.

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Acknowledgements

We thank B. J. Burbach and P. O'Brien for help with experiments, and Z. Mainen, R. Malinow, M. Maravall, W. Regehr, R. Weinberg and R. Yasuda for comments on the manuscript. This work was supported by the Pew, Klingenstein and Mathers Foundations, the Howard Hughes Medical Institute, NIH and a Helen Hay Whitney Fellowship.

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Correspondence to Karel Svoboda.

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