Deciphering neuronal circuitry is central to understanding brain function and dysfunction, yet it remains a daunting task. To facilitate the dissection of neuronal circuits, a process requiring functional analysis of synaptic connections and morphological identification of interconnected neurons, we present here a method for stable simultaneous octuple patch-clamp recordings. This method allows physiological analysis of synaptic interconnections among 4–8 simultaneously recorded neurons and/or 10–30 sequentially recorded neurons, and it allows anatomical identification of >85% of recorded interneurons and >99% of recorded principal neurons. We describe how to apply the method to rodent tissue slices; however, it can be used on other model organisms. We also describe the latest refinements and optimizations of mechanics, electronics, optics and software programs that are central to the realization of a combined single- and two-photon microscopy–based, optogenetics- and imaging-assisted, stable, simultaneous quadruple–viguple patch-clamp recording system. Setting up the system, from the beginning of instrument assembly and software installation to full operation, can be completed in 3–4 d.
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We thank R. Andrade, M. Beenhakker, T. Figl, V. Iyer, J. Kim, P. Neumann and K. Svoboda for technical advice and invaluable discussions, and we thank members of the Zhu laboratory for comments and technical assistance. Although we discuss here a few approaches for setting up a single- and two-photon optogenetics- and imaging-aided simultaneous multiple patch-clamp recordings system using mechanics, electronics, optics and software that we have some experience with, we anticipate alternative approaches to achieve the same goal with other instruments and software programs. We hope that this protocol will inspire more scientists to contribute their wisdom and experience to this subject. This work was supported in part by postdoctoral fellowships from the Epilepsy Foundation (G.W. and X.J.), National Institutes of Health (NIH) predoctoral training fellowships (D.R.W. and L.C.M.), a French governmental Bourse du mérite graduate fellowship and Aquimob traveling fellowship (T.L.), a Cultural and Educational Expert award from the State Administration of Foreign Experts Affairs of China (Y.S. and J.J.Z.), Chinese Ministry of Education Project 111 Program grant B13026 (Y.S. and J.J.Z.) and NIH grants R15NS081628 (Q.-Q.S. and W.Y.) and R01NS053570 (J.J.Z). This paper is the part of a dissertation in partial fulfillment of the requirements of the M.S. degree at the Bordeaux University, France (T.L.).
The authors declare no competing financial interests.
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Wang, G., Wyskiel, D., Yang, W. et al. An optogenetics- and imaging-assisted simultaneous multiple patch-clamp recording system for decoding complex neural circuits. Nat Protoc 10, 397–412 (2015). https://doi.org/10.1038/nprot.2015.019
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