Combining Voltage and Calcium Imaging from Neuronal Dendrites

The ability to monitor membrane potential (Vm) and calcium (Ca2+) transients at multiple locations on the same neuron can facilitate further progress in our understanding of neuronal function. Here we describe a method to combine Vm and Ca2+ imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca2+ indicators. In all cases Vm optical signals are linear with membrane potential changes, but the calibration of optical signals on an absolute scale is presently possible only in some neurons. The interpretation of Ca2+ optical signals depends on the indicator Ca2+ buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300 μM Bis-Fura-2, Vm optical signals cannot be calibrated and the physiological Ca2+ dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in Vm and Ca2+ fluorescence signals under different conditions can provide meaningful new information on local dendritic integration. In cerebellar Purkinje neurons, loaded with JPW-1114 and 1 mM Fura-FF, Vm optical signals can be calibrated in terms of mV and Ca2+ optical signals quantitatively reveal the physiological changes in free Ca2+. Using these two examples, the method is explained in detail.