title: Laser-scanning photostimulation of optogenetically targeted forebrain circuits.
abstract: The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.
authors.affiliation: Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Lee
authors.forename: Ying-Wan
authors.initials: YW
authors.lastname: Lam
authors.forename: Kazuo
authors.initials: K
authors.lastname: Imaizumi
authors.forename: S Murray
authors.initials: SM
authors.lastname: Sherman
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D009415:Nerve Net / Q000502:physiology*; D009474:Neurons / Q000502:physiology; D010775:Photic Stimulation / Q000379:methods*; D016548:Prosencephalon / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012243:Rhodopsin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013569:Synapses / Q000502:physiology; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D012243:Rhodopsin