Combining Voltage and Calcium Imaging from Neuronal Dendrites

The ability to monitor membrane potential (V _m) 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 V _m and Ca²⁺ imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca²⁺ indicators. In all cases V _m 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, V _m 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 V _m 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, V _m 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.