title: Recording, labeling, and transfection of single neurons in deep brain structures.
abstract: Genetic tools that permit functional or connectomic analysis of neuronal circuits are rapidly transforming neuroscience. The key to deployment of such tools is selective transfection of target neurons, but to date this has largely been achieved using transgenic animals or viral vectors that transduce subpopulations of cells chosen according to anatomical rather than functional criteria. Here, we combine single-cell transfection with conventional electrophysiological recording techniques, resulting in three novel protocols that can be used for reliable delivery of conventional dyes or genetic material in vitro and in vivo. We report that techniques based on single cell electroporation yield reproducible transfection in vitro, and offer a simple, rapid and reliable alternative to established dye-labeling techniques in vivo, but are incompatible with targeted transfection in deep brain structures. In contrast, we show that intracellular electrophoresis of plasmid DNA transfects brainstem neurons recorded up to 9 mm deep in the anesthetized rat. The protocols presented here require minimal, if any, modification to recording hardware, take seconds to deploy, and yield high recovery rates in vitro (dye labeling: 89%, plasmid transfection: 49%) and in vivo (dye labeling: 66%, plasmid transfection: 27%). They offer improved simplicity compared to the juxtacellular labeling technique and for the first time offer genetic manipulation of functionally characterized neurons in previously inaccessible brain regions.
authors.affiliation: Australian School of Advanced Medicine, Macquarie University, Sydney, 2109, NSW, Australia.
authors.forename: Bowen
authors.initials: B
authors.lastname: Dempsey
authors.affiliation: Australian School of Advanced Medicine, Macquarie University, Sydney, 2109, NSW, Australia.
authors.forename: Anita J
authors.initials: AJ
authors.lastname: Turner
authors.affiliation: Australian School of Advanced Medicine, Macquarie University, Sydney, 2109, NSW, Australia.
authors.forename: Sheng
authors.initials: S
authors.lastname: Le
authors.affiliation: Australian School of Advanced Medicine, Macquarie University, Sydney, 2109, NSW, Australia.
authors.forename: Qi-Jian
authors.initials: QJ
authors.lastname: Sun
authors.affiliation: Australian School of Advanced Medicine, Macquarie University, Sydney, 2109, NSW, Australia.
authors.forename: Lama
authors.initials: L
authors.lastname: Bou Farah
authors.affiliation: Department of Physiology, The University of Melbourne, Parkville, 3010, VIC, Australia.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Allen
authors.affiliation: Australian School of Advanced Medicine, Macquarie University, Sydney, 2109, NSW, Australia.
authors.forename: Ann K
authors.initials: AK
authors.lastname: Goodchild
authors.affiliation: Australian School of Advanced Medicine, Macquarie University, Sydney, 2109, NSW, Australia.
authors.forename: Simon
authors.initials: S
authors.lastname: McMullan
keywords: Electroporation; gene delivery; juxtacellular; labeling