Research tool: Validation of floxed α7 nicotinic acetylcholine receptor conditional knockout mice using in vitro and in vivo approaches

Caterina M. Hernandez, Ibdanelo Cortez, Zhenglin Gu, José O. Colón-Sáez, Patricia W. Lamb, Maki Wakamiya, Jerrel L. Yakel, Kelly Dineley

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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-Chrna7LBDEx4007Ehs) which we refer to as floxed α7 nAChR conditional knockout or α7nAChRflox. We validated the chosen approach by mating α7nAChRflox with mice expressing Cre recombinase driven by the glial acidic fibrillary protein (GFAP)-Cre promoter (GFAP-A7KO) to test whether α7nAChRflox, 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 α7nAChRflox 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 α7nAChRflox 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 α7nAChRflox 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.

Original languageEnglish (US)
Pages (from-to)3201-3214
Number of pages14
JournalJournal of Physiology
Volume592
Issue number15
DOIs
StatePublished - Aug 1 2014

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Glial Fibrillary Acidic Protein
Nicotinic Receptors
Knockout Mice
Research
Astrocytes
Exons
Synapsins
Pyramidal Cells
Central Nervous System Diseases
Locomotion
Choline
In Vitro Techniques
Neuroglia
Rodentia
Binding Sites
Learning
Gene Expression
Neurons
Health
Brain

ASJC Scopus subject areas

  • Physiology
  • Medicine(all)

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Research tool : Validation of floxed α7 nicotinic acetylcholine receptor conditional knockout mice using in vitro and in vivo approaches. / Hernandez, Caterina M.; Cortez, Ibdanelo; Gu, Zhenglin; Colón-Sáez, José O.; Lamb, Patricia W.; Wakamiya, Maki; Yakel, Jerrel L.; Dineley, Kelly.

In: Journal of Physiology, Vol. 592, No. 15, 01.08.2014, p. 3201-3214.

Research output: Contribution to journalArticle

Hernandez, Caterina M. ; Cortez, Ibdanelo ; Gu, Zhenglin ; Colón-Sáez, José O. ; Lamb, Patricia W. ; Wakamiya, Maki ; Yakel, Jerrel L. ; Dineley, Kelly. / Research tool : Validation of floxed α7 nicotinic acetylcholine receptor conditional knockout mice using in vitro and in vivo approaches. In: Journal of Physiology. 2014 ; Vol. 592, No. 15. pp. 3201-3214.
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AU - Colón-Sáez, José O.

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N2 - 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-Chrna7LBDEx4007Ehs) which we refer to as floxed α7 nAChR conditional knockout or α7nAChRflox. We validated the chosen approach by mating α7nAChRflox with mice expressing Cre recombinase driven by the glial acidic fibrillary protein (GFAP)-Cre promoter (GFAP-A7KO) to test whether α7nAChRflox, 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 α7nAChRflox 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 α7nAChRflox 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 α7nAChRflox 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.

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