High-throughput multiplex voltage-clamp/current-clamp evaluation of acutely isolated neurons.

Abstract

The patch-clamp technique remains the gold-standard for the investigation of excitable cells. However, the manual implementation of this technique is slow and low throughput. While recently developed high-throughput robotic patch-clamp methods have proven valuable for drug screening, they have predominantly focused on investigating receptors and channels overexpressed in heterologous cell lines. We recently developed an automated high-throughput patch-clamp approach that enables the simultaneous and unbiased analysis of acutely dissociated neurons in their native state. To analyze and manage the large and complex datasets resulting from this methodology, we have also developed open-source software with an easy-to-use graphical user interface to fit data from each neuron with appropriate biophysical equations to functionally characterize each individual neuron. Here we describe a protocol that provides a streamlined set of procedures, including (1) the dissociation and isolation of neurons from intact tissue; (2) the designing and performing of patch-clamp experiments on the robotic system; and (3) the analysis of data using predetermined, unbiased filtration criteria. This methodology can be used for diverse applications ranging from the assessment of neuronal biophysics to drug development. The protocol requires 6-18 h including cell preparation, experimental execution and analysis of the generated data. Graduate-student-level expertise in animal dissection, electrophysiology and biophysics is required.