Membrane Potential Imaging in Neurons using Fluorinated Voltage-Sensitive Dyes and a Custom Multiphoton Brain Slice Microscope

Abstract

In order to fully understand the physiology of fundamental neurophysiological processes such as synaptic integration and synaptic plasticity, direct recording of changes in membrane potential neuronal dendrites and spines is essential. In an effort to improve voltage-sensitive dye measurements of synaptic potentials and backpropagating action potentials, our group has developed new fluorinated dyes with enhanced photostability. We have also made performance improvements on our custom, non-linear optical microscope for greater sensitivity. By modifying a commercial Zeiss microscope we have added two “up front” epifluorescence detection channels and one transfluorescence detection channel. Optics for these new light paths were optimized using numerical ray tracing. Here we show that we are able to fill individual neurons with these dyes via somatic patch pipettes and record membrane potential changes in the soma and dendrites of Purkinje neurons in cerebellar brain slices. Using voltage clamp protocols, membrane potential was changed in a stepwise fashion, resulting in changes in membrane fluorescence. When excited with 1060 nm light, the new dyes typically produced changes in fluorescence (dF/F) between 3 and 7 % for 50 mV changes in membrane potential. Feasibility of using second harmonic generation to record membrane potential with these dyes was investigated in a cultured cell line by measuring dSHG/SHG, kinetics, and intensity as a function of dye concentration. Work funded by the following NIH grants: R01-EB001963 and P41-RR013186.