Combining voltage and calcium imaging from neuronal dendrites.

The ability to monitor membrane potential (V(m)) and calcium (Ca(2+)) 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 Ca(2+) imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca(2+) 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 Ca(2+) optical signals depends on the indicator Ca(2+) buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300 microM Bis-Fura-2, V(m) optical signals cannot be calibrated and the physiological Ca(2+) dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in V (m) and Ca(2+) 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 Ca(2+) optical signals quantitatively reveal the physiological changes in free Ca(2+). Using these two examples, the method is explained in detail.