CONFOCAL IMAGING OF DENDRITIC CA2-SLICES DURING SIMULTANEOUS CURRENT-CLAMP AND VOLTAGE-CLAMP RECORDING( TRANSIENTS IN HIPPOCAMPAL BRAIN)

Changes in the intracellular Ca2+ concentration ([Ca2+](i)) within CA1 hippocampal pyramidal neurons were imaged using confocal laser scanni ng microscopy in conjunction with Ca2+-sensitive fluorescent indicator s. The imaging was performed in thick hippocampal brain slices while s imultaneously 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 ch anges in [Ca2+](i). We compared the increases in [Ca2+](i) produced by either of two methods-direct depolarization of the cell via the somat ic electrode or high-frequency stimulations of synaptic inputs. The in creases in [Ca2+](i) in the soma and proximal dendrites caused by both methods were of comparable magnitude and they always decayed within s econds in healthy cells. However, the spatial patterns of distal Ca2increases were different. Separate sets of synaptic inputs to the same cell resulted in different spatial patterns of[Ca2+](i) transients. W e isolated and observed what appeared to be a voltage-independent comp onent of the synaptically mediated [Ca2+](i) transients. This work dem onstrates that the combination of neurophysiology and simultaneous con focal microscopy is well suited for visualizing and analyzing [Ca2+](i ) changes within highly localized regions of neurons in thick brain sl ices. The approach should allow further analysis of the relative contr ibution of voltage- and agonist-dependent influences on [Ca2+](i) with in neurons throughout the CNS and it raises the possibility of routine ly relating subcellular [Ca2+](i) changes to structural and functional modifications. (C) 1994 Wiley-Liss, Inc.