title: Long-lasting single-neuron labeling by in vivo electroporation without microscopic guidance.
abstract: In order to make a direct link between the morphological and functional study of the nervous system, we established an experimental protocol for labeling individual neurons persistently without microscopic guidance by injecting a plasmid encoding fluorescent protein electroporatively after recording their activity extracellularly. Using a glass pipette filled with electrolyte solution containing a plasmid encoding green fluorescent protein (GFP), single-neuron recording and electroporation were performed on anesthetized rats. When performing the electroporation at the completion of recording, the degree of contact between the target neuron and the electrode tip was adjusted by monitoring the change of the trace of recorded action potentials and the increase of electrode resistance. The expression of GFP and its immunostaining with a polyclonal antibody enabled us to clearly see the basic structural components such as cell bodies, axons, dendrites, and even smaller components such as spines. Identification of the morphological subtypes of neurons was possible with every labeled neuron. The optimum condition for labeling was a 30% increase of the electrode resistance, and the labeling success rate evaluated 3 days after labeling was 40%. The rate evaluated one month after labeling was only slightly lower (33%). We also confirmed experimentally that this recording and labeling procedure can be similarly successful in head-fixed behaving rats. This new experimental protocol will be a breakthrough in systems neuroscience because it makes a direct link between the morphology and behavior-related activity of single neurons.
authors.affiliation: Division of Systems Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai 980-8577, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Oyama
authors.forename: Shinya
authors.initials: S
authors.lastname: Ohara
authors.forename: Sho
authors.initials: S
authors.lastname: Sato
authors.forename: Fuyuki
authors.initials: F
authors.lastname: Karube
authors.forename: Fumino
authors.initials: F
authors.lastname: Fujiyama
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Isomura
authors.forename: Hajime
authors.initials: H
authors.lastname: Mushiake
authors.forename: Toshio
authors.initials: T
authors.lastname: Iijima
authors.forename: Ken-Ichiro
authors.initials: K
authors.lastname: Tsutsui
mesh_terms: D000818:Animals; D018274:Electroporation / Q000379:methods*; D049452:Green Fluorescent Proteins; D008297:Male; D009474:Neurons / Q000166:cytology* / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D049452:Green Fluorescent Proteins
keywords: Behavioral neurophysiology; Electroporation; Fluorescent protein; Plasmid; Single-unit recording