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

Abstract

Key pointsr Currently, no animal model exists in which selective deletion of α7 nAChRs from a specific cell type or tissue is possible through genetic manipulation.r 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 α7nAChR flox . 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.

Septal Cholinergic Neuromodulation Tunes the Astrocyte-Dependent Gating of Hippocampal NMDA Receptors to Wakefulness

“…We thus asked whether α7nAChRs required for the wakefulness-dependent regulation of D-serine availability are located on astrocytes or neurons. We used mice in which exon 4 of Chrna7 is floxed (α7nAChR flox/flox , Star Methods, Figure S7, Hernandez et al, 2014) and performed stereotaxic injections of adeno-associated viruses encoding Cre recombinase and GFP reporter. Mice were injected with an AAV5 encoding GFAP(0.7)-eGFP-T2A-iCre into area CA1 to selectively transduce astrocytes (Figure 7A), or with an AAV9 encoding eSYN-eGFP-T2A-iCre both in areas CA3 and CA1 to transduce neurons (Figure 7H).…”

“…Heterozygous α7nAChR floxed/+ mice in which exon 4 of the Chrna7 gene is flanked by loxP sites ( B6-Chrna7 LBDEx4007Ehs ) originated from Dr. K. Dineley’s lab (Department of Neurology, University of Texas, Medical Branch, Galveston, TX 77555) where they were generated as described in Hernandez et al, 2014. They were cleared fron quarantine after 2 weeks, backcrossed with C57Bl/6 mice and bred in our facility.…”

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

“…85 Additional Ab-mediated mechanisms may also contribute to propagation of AD pathology; for example, oligomer binding to membrane lipids or to a7 nicotinic cholinergic receptors might modulate the downstream signaling. 84,86,87 Our results suggest that effective therapies will need to target synaptic Ab oligomers and that antiamyloid therapies will be much less effective once synaptic p-tau pathology has developed, thus providing a potential explanation for the failure of amyloid-based trials. An additional important clinical implication is that therapies slowing Ab oligomer accumulation in the synaptic compartment will delay onset of dementia.…”

The effect of α7 nicotinic receptor activation on glutamatergic transmission in the hippocampus

“…Furthermore, the outcome of α7 nAChR activation on the hippocampal circuit is complicated by the extensive distribution of α7 nAChRs on both glutamatergic and GABAergic neurons. With the newly-generated floxed α7 nAChR transgenic mouse (Hernandez et al , 2014) combining with Cre and Cre-ER dependent systems, selective deletion of α7 nAChRs from distinct cell populations at specific developmental stages will allow us to unravel systematically the impact of α7 nAChR activation on hippocampal network both in vitro and in vivo . This will also help us to understand how the alteration of α7 nAChR function will impact cognitive function.…”