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.
J Neurophysiol. 2011 Nov;106(5):2216-31
[PMID:
21795621]
Nat Methods. 2007 Feb;4(2):139-41
[PMID:
17195846]
J Neurosci. 2009 Oct 7;29(40):12440-8
[PMID:
19812320]
Nat Methods. 2007 Jan;4(1):47-9
[PMID:
17179932]
Hum Gene Ther. 2011 Jun;22(6):669-77
[PMID:
21319997]
Cold Spring Harb Protoc. 2012 Sep 01;2012(9):998-1004
[PMID:
22949715]
J Neurosci. 2015 Feb 4;35(5):2293-307
[PMID:
25653383]
J Neurosci. 2014 May 28;34(22):7704-14
[PMID:
24872574]
Neuron. 2011 Aug 25;71(4):617-31
[PMID:
21867879]
Exp Physiol. 2011 Jan;96(1):34-9
[PMID:
20562296]
Front Neuroanat. 2015 Jul 01;9:80
[PMID:
26190977]
Nat Neurosci. 2007 May;10(5):663-8
[PMID:
17435752]
Neuron. 2016 Feb 17;89(4):711-24
[PMID:
26804990]
Neuron. 2013 Dec 18;80(6):1368-83
[PMID:
24360541]
J Neurosci. 2012 Apr 4;32(14):4992-5001
[PMID:
22492054]
J Neurosci. 2013 Sep 25;33(39):15333-42
[PMID:
24068800]
Nat Neurosci. 2010 Jun;13(6):739-44
[PMID:
20436481]
Nat Neurosci. 2011 Apr;14(4):513-8
[PMID:
21399632]
J Neurosci. 2015 Feb 18;35(7):2959-74
[PMID:
25698734]
Proc Natl Acad Sci U S A. 2011 May 3;108(18):7595-600
[PMID:
21504945]
Nature. 2009 Feb 26;457(7233):1142-5
[PMID:
19151697]
J Neurosci. 2013 Jan 9;33(2):748-60
[PMID:
23303952]
Cold Spring Harb Protoc. 2015 Apr 01;2015(4):368-74
[PMID:
25834255]
Nature. 1984 Aug 9-15;310(5977):498-500
[PMID:
6205278]
Neuron. 2011 Oct 6;72(1):111-23
[PMID:
21982373]
Nat Neurosci. 2008 Mar;11(3):360-6
[PMID:
18246064]
Nat Protoc. 2006;1(6):3166-73
[PMID:
17406580]
Cold Spring Harb Protoc. 2015 Apr 01;2015(4):375-85
[PMID:
25834254]
Nat Methods. 2014 Mar;11(3):338-46
[PMID:
24509633]
Nat Neurosci. 2010 Mar;13(3):387-92
[PMID:
20081849]
