Confocal imaging of dendritic Ca2+ transients in hippocampal brain slices during simultaneous current‐ and voltage‐clamp recording

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Abstract
Changes in the intracellular Ca2+ concentration ([Ca2+]i) within CA1 hippocampal pyramidal neurons were imaged using confocal laser scanning microscopy in conjunction with Ca2+ ‐sensitive fluorescent indicators. The imaging was performed in thick hippocampal brain slices while simultaneously measuring or controlling electrical activity with sharp microelectrodes or whole‐cell patch‐clamp electrodes. The combination of imaging and electrophysiology was essential for interpreting the changes in [Ca2+]i. We compared the increases in [Ca2+]i produced by either of two methods–direct depolarization of the cell via the somatic electrode or high‐frequency stimulations of synaptic inputs. The increases in [Ca2+]i in the soma and proximal dendrites caused by both methods were of comparable magnitude and they always decayed within seconds in healthy cells. However, the spatial patterns of distal Ca2+ increases were different. Separate sets of synaptic inputs to the same cell resulted in different spatial patterns of [Ca2+]i transients. We isolated and observed what appeared to be a voltage‐independent component of the synaptically mediated [Ca2+]i transients. This work demonstrates that the combination of neurophysiology and simultaneous confocal microscopy is well suited for visualizing and analyzing [Ca2+]i within neurons throughout the CNS and it raises the possibility of routinely relating subcellular [Ca2+]i changes to structural and functional modifications.