title: High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses.
abstract: In recent years, optogenetic approaches have significantly advanced the experimental repertoire of cellular and functional neuroscience. Yet, precise and reliable methods for specific expression of optogenetic tools remain challenging. In this work, we studied the transduction efficiency of seven different adeno-associated virus (AAV) serotypes in primary cortical neurons and revealed recombinant (r) AAV6 to be the most efficient for constructs under control of the cytomegalovirus (CMV) promoter. To further specify expression of the transgene, we exchanged the CMV promoter for the human synapsin (hSyn) promoter. In primary cortical-glial mixed cultures transduced with hSyn promoter-containing rAAVs, expression of ChR2opt (a Channelrhodopsin-2 variant) was limited to neurons. In these neurons action potentials could be reliably elicited upon laser stimulation (473nm). The use of rAAV serotype alone to restrict expression to neurons results in a lower transduction efficiency than the use of a broader transducing serotype with specificity conferred via a restrictive promoter. Cells transduced with the hSyn driven gene expression were able to elicit action potentials with more spatially and temporally accurate illumination than neurons electrofected with the CMV driven construct. The hSyn promoter is particularly suited to use in AAVs due to its small size. These results demonstrate that rAAVs are versatile tools to mediate specific and efficient transduction as well as functional and stable expression of transgenes in primary cortical neurons.
authors.affiliation: Institute of Complex Systems ICS-8, Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Electronic address: lei.jin@fz-juelich.de.
authors.forename: Lei
authors.initials: L
authors.lastname: Jin
authors.affiliation: Institute of Complex Systems ICS-8, Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Electronic address: wienke.lange@web.de.
authors.forename: Wienke
authors.initials: W
authors.lastname: Lange
authors.affiliation: Institute of Complex Systems ICS-4, Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Electronic address: Annika.meisenberg@gmail.com.
authors.forename: Annika
authors.initials: A
authors.lastname: Kempmann
authors.affiliation: Institute of Complex Systems ICS-8, Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Electronic address: v.maybeck@fz-juelich.de.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Maybeck
authors.affiliation: Institute of Complex Systems ICS-4, Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Electronic address: guenther_a@gmx.net.
authors.forename: Anne
authors.initials: A
authors.lastname: Günther
authors.affiliation: Institute of Complex Systems ICS-4, Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Electronic address: nadine.gruteser@t-online.de.
authors.forename: Nadine
authors.initials: N
authors.lastname: Gruteser
authors.affiliation: Institute of Complex Systems ICS-4, Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Electronic address: a.baumann@fz-juelich.de.
authors.forename: Arnd
authors.initials: A
authors.lastname: Baumann
authors.affiliation: Institute of Complex Systems ICS-8, Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Electronic address: a.offenhaeusser@fz-juelich.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Offenhäusser
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D006801:Humans; D009474:Neurons / Q000166:cytology*; D062308:Optogenetics / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D012243:Rhodopsin / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D016704:Synapsins / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D016704:Synapsins; D012243:Rhodopsin
keywords: Channelrhodopsin; Electrophysiology; Neuronal networks