Calibration and Quantification of 2-Photon Voltage-Sensitive Dye Recordings in Single Dendritic Spines

Abstract

Quantitative measurements of membrane potential in dendritic spines are necessary to investigate the biophysical properties of these structures, which are initial sites of cortical excitatory synaptic information processing. Voltage-sensitive dyes (VSDs), which are developed in our lab, are capable of recording membrane potential from dendritic spines. However, the ability of VSDs to resolve subthreshold membrane potential fluctuations depends on their sensitivity to membrane potential along with the attainable signal-to-noise ratio of optical membrane potential recordings. We analyze the sensitivity of new generation VSDs by calibrating membrane potential fluctuations in single dendritic spines. Using 2-photon excitation techniques, we find that the calibration of signals is highly consistent among different dendritic spines, even spines that differ significantly in size. Spines can be targeted at their centers using a “single-voxel” recording approach for consistent, reproducible recordings. Dendrites must be targeted along the lateral edge, which is less consistent and calibration shows reduced sensitivity to voltage. Since signal-to-noise levels depend on dye loading and internal concentration, we use a controlled loading technique in order to reproducibly load dyes being tested. By quantifying signal-to-noise we are able to follow molecular changes to experimental VSDs that lead to improvements in this crucial metric. With continued advances in optical membrane potential recording techniques, we come closer to the ultimate goal: single sweep recordings of synaptic potentials in dendritic spines.