Yamawaki Naoki, Suter Benjamin A, Wickersham Ian R, Shepherd Gordon M G
Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611.
McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Cold Spring Harb Protoc. 2016 Oct 3;2016(10):pdb.prot090084. doi: 10.1101/pdb.prot090084.
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.
本文描述了一组基于视紫红质-2（ChR2）的突触回路分析方法，该方法将病毒转染的突触前神经元轴突的光刺激与逆行标记的突触后神经元的全细胞电生理记录相结合。该方法利用了脑片中表达ChR2的轴突保留的光兴奋性，可用于评估局部或远程功能连接。立体定位注射既用于在感兴趣的突触前轴突中选择性表达ChR2（使用狂犬病病毒[RV]或腺相关病毒[AAV]），也用于用荧光逆行示踪剂标记两种感兴趣的突触后投射神经元。在脑片中，对示踪剂标记的突触后神经元进行全细胞电生理记录，并通过对表达ChR2的轴突进行发光二极管（LED）光刺激的电压或电流响应采样来评估突触连接。对数据进行分析以估计突触输入的相对幅度和其他连接参数。药理学和电生理操作扩展了基本方法的通用性，允许剖析单突触与双突触反应、兴奋性与抑制性反应等。该方法能够快速、定量地表征定义的突触前和突触后神经元类别之间的突触连接。
