title: Research tool: Validation of floxed α7 nicotinic acetylcholine receptor conditional knockout mice using in vitro and in vivo approaches.
abstract: There is much interest in α7 nicotinic acetylcholine receptors (nAChRs) in CNS function since they are found throughout peripheral tissues as well as being highly expressed in brain regions implicated in attention, learning and memory. As such, the role of these receptors in many aspects of CNS function and disease is being actively investigated. To date, only one null mouse model (A7KO) is available which is non-conditional and constitutive. Since α7 nAChRs are present on neurons and glia (including astrocytes), as well as being developmentally regulated, there is an unmet need for the technical capability to control α7 nAChR gene expression. Therefore we have generated mice in which the fourth exon of the α7 nAChR gene (Chrna7) is flanked by loxP sites (B6-Chrna7(LBDEx4007Ehs)) which we refer to as floxed α7 nAChR conditional knockout or α7nAChR(flox). We validated the chosen approach by mating α7nAChR(flox) with mice expressing Cre recombinase driven by the glial acidic fibrillary protein (GFAP)-Cre promoter (GFAP-A7KO) to test whether α7nAChR(flox), GFAP-A7KO and appropriate littermate controls performed equally in our standard Rodent In Vivo Assessment Core battery to assess general health, locomotion, emotional and cognitive behaviours. Neither α7nAChR(flox) nor GFAP-A7KO exhibited significant differences from littermate controls in any of the baseline behavioural assessments we conducted, similar to the 'first generation' non-conditional A7KO mice. We also determined that α7 nAChR binding sites were absent on GFAP-positive astrocytes in hippocampal slices obtained from GFAP-A7KO offspring from α7nAChR(flox) and GFAP-Cre crosses. Finally, we validated that Cre recombinase (Cre)-mediated excision led to functional, cell- and tissue-specific loss of α7 nAChRs by demonstrating that choline-induced α7 nAChR currents were present in Cre-negative, but not synapsin promoter-driven Cre-positive, CA1 pyramidal neurons. Additionally, electrophysiological characterization of α7 nAChR-mediated current traces was similar in terms of amplitude and time constants of decay (during desensitization) for the α7nAChR(flox) and wild-type (WT) mice. Thus, we have in vivo and in vitro evidence that the Chrna7 exon 4 targeting strategy does not alter behavioural, cognitive, or electrophysiological properties compared to WT and that Cre-mediated excision is an effective approach to delete α7 nAChR expression in a cell-specific manner.
authors.affiliation: Mitchell Center for Neurodegenerative Diseases, University of Texas Medical Branch at Galveston (UTMB), Galveston, TX, USA Department of Neurology, UTMB, Galveston, TX, USA.
authors.forename: Caterina M
authors.initials: CM
authors.lastname: Hernandez
authors.affiliation: Mitchell Center for Neurodegenerative Diseases, University of Texas Medical Branch at Galveston (UTMB), Galveston, TX, USA Department of Neurology, UTMB, Galveston, TX, USA.
authors.forename: Ibdanelo
authors.initials: I
authors.lastname: Cortez
authors.affiliation: Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health (NIEHS/NIH), Department of Health and Human Services, Research Triangle Park, NC, USA.
authors.forename: Zhenglin
authors.initials: Z
authors.lastname: Gu
authors.affiliation: Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health (NIEHS/NIH), Department of Health and Human Services, Research Triangle Park, NC, USA.
authors.forename: José O
authors.initials: JO
authors.lastname: Colón-Sáez
authors.affiliation: Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health (NIEHS/NIH), Department of Health and Human Services, Research Triangle Park, NC, USA.
authors.forename: Patricia W
authors.initials: PW
authors.lastname: Lamb
authors.affiliation: Animal Resource Center, UTMB, Galveston, TX, USA Institute for Translational Sciences, UTMB, Galveston, TX, USA.
authors.forename: Maki
authors.initials: M
authors.lastname: Wakamiya
authors.affiliation: Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health (NIEHS/NIH), Department of Health and Human Services, Research Triangle Park, NC, USA.
authors.forename: Jerrel L
authors.initials: JL
authors.lastname: Yakel
authors.affiliation: Mitchell Center for Neurodegenerative Diseases, University of Texas Medical Branch at Galveston (UTMB), Galveston, TX, USA Department of Neurology, UTMB, Galveston, TX, USA Rodent In Vivo Assessment Core, UTMB, Galveston, TX, USA Center for Addiction Research, UTMB, Galveston, TX, USA ktdinele@utmb.edu.
authors.forename: Kelly T
authors.initials: KT
authors.lastname: Dineley
mesh_terms: D000200:Action Potentials; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D056547:CA1 Region, Hippocampal / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D005091:Exons; D018390:Gene Targeting / Q000379:methods*; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D018782:Maze Learning; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000235:genetics* / Q000378:metabolism
chemical_list: C579166:Chrna7 protein, mouse; D005904:Glial Fibrillary Acidic Protein; D064569:alpha7 Nicotinic Acetylcholine Receptor; C045073:Cre recombinase; D019426:Integrases