Using Multiphoton Imaging for Targeted Electrophysiological Recording and Live Cell Imaging of Fluorescently Labeled Neurons from Isolated Retinas

Abstract

A central goal of neuroscience is to understand how neural computations are implemented by neural circuits. An excellent model system is the mammalian retina. Besides its important role in visual processing, the retina offers technical advantages for circuit interrogation at the cellular and synaptic levels due to its experimental accessibility and well-defined cell types. Recent development of genetic and molecular tools in mice has made the mouse retina a preferred choice for studying retinal circuitry, since an increasing repertoire of cell types can be specifically labeled by fluorescent proteins. However, measuring the light response of fluorescently tagged retinal neurons is challenging because excitation of fluorophores at visible wavelengths often leads to rapid photopigment bleaching that prevents subsequent recording of light responses from retinal neurons. One way to circumvent this problem is to use multiphoton excitation in the infrared range to visualize fluorescent protein-expressing cells. In this chapter, we describe a detailed protocol for multiphoton-targeted electrophysiological recording from fluorescently labeled retinal neurons while preserving their sensitivity to visual stimulation. This technique also enables live imaging of the three-dimensional morphology of the recorded neurons. With the continued development of cell-specific markers in the mouse retina, this method is expected to be widely used for harnessing the power of genetic and molecular tools in retinal circuit analysis.