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Cold Spring Harb Protoc. 2016 Oct 3;2016(10). doi: 10.1101/pdb.prot090084.

Combining Optogenetics and Electrophysiology to Analyze Projection Neuron Circuits.

Author information

1
Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611.
2
McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.

Abstract

A set of methods is described for channelrhodopsin-2 (ChR2)-based synaptic circuit analysis that combines photostimulation of virally transfected presynaptic neurons' axons with whole-cell electrophysiological recordings from retrogradely labeled postsynaptic neurons. The approach exploits the preserved photoexcitability of ChR2-expressing axons in brain slices and can be used to assess either local or long-range functional connections. Stereotaxic injections are used both to express ChR2 selectively in presynaptic axons of interest (using rabies virus [RV] or adeno-associated virus [AAV]) and to label two types of postsynaptic projection neurons of interest with fluorescent retrograde tracers. In brain slices, tracer-labeled postsynaptic neurons are targeted for whole-cell electrophysiological recordings, and synaptic connections are assessed by sampling voltage or current responses to light-emitting diode (LED) photostimulation of ChR2-expressing axons. The data are analyzed to estimate the relative amplitude of synaptic input and other connectivity parameters. Pharmacological and electrophysiological manipulations extend the versatility of the basic approach, allowing the dissection of monosynaptic versus disynaptic responses, excitatory versus inhibitory responses, and more. The method enables rapid, quantitative characterization of synaptic connectivity between defined pre- and postsynaptic classes of neurons.

PMID:
27698240
PMCID:
PMC5476926
DOI:
10.1101/pdb.prot090084
[Indexed for MEDLINE]
Free PMC Article

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