Studying Neuronal Function Ex Vivo Using Optogenetic Stimulation and Patch Clamp.

Abstract

Optogenetics has become a key method to interrogate the function of neural populations and circuits in the brain. This technique combines the targeted expression of light-activated proteins with subsequent manipulation of neural activity by light. Opsins such as Channelrhodopsin-2 (ChR2), which is a light-gated cation-channel, can be fused to or coexpressed with fluorescent proteins to allow for visualization and concurrent activation of neurons and their axonal projections. Via stereotaxic delivery of viral vectors, ChR2 can be constitutively or conditionally expressed in specific neurons in defined brain regions. Subsequently, identified axonal projections can be studied functionally ex vivo in combination with patch-clamp recordings in brain slices. This optogenetic mapping of neural circuitry has enabled the identification and characterization of novel synaptic connections and the detailed investigation of known anatomical connections previously not amenable with electrical stimulation techniques. Here, we describe a protocol for investigating functional properties of local and long-range connectivity in the brain using blue-light activated ChR2 variants and whole-cell patch-clamp recordings in acute brain slices.