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

Abstract

Changes in the intracellular Ca2+ concentration ([Ca2+]i) within CA1 hippocampal pyramidal neurons were imaged using confocal laser scanning microscopy in conjunction with Ca(2+)-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 changes within highly localized regions of neurons in thick brain slices. The approach should allow further analysis of the relative contribution of voltage- and agonist-dependent influences on [Ca2+]i within neurons throughout the CNS and it raises the possibility of routinely relating subcellular [Ca2+]i changes to structural and functional modifications.