There have been several major accomplishments within the past fiscal year. The activation of α7 nAChRs has been shown to improve hippocampal-dependent learning and memory. However, the molecular mechanism of α7 nAChRs' action remains elusive. We previously reported that activation of α7 nAChRs induced a prolonged enhancement of glutamatergic synaptic transmission in a PKA-dependent manner. Here, we investigated any connection between the activation of the α7 nAChR and cAMP signaling in hippocampal neurons. To address this question, we employed a FRET-based biosensor to measure the intracellular cAMP levels directly via live cell imaging. We found that application of the α7 nAChR-selective agonist choline, in the presence of the α7 nAChR positive allosteric modulator PNU-120596, induced a significant change in emission ratio of F535/F470, which indicated an increase in intracellular cAMP levels. This choline-induced increase was abolished by the α7 nAChR antagonist MLA and the calcium chelator BAPTA, suggesting that the cAMP increase depends on the α7 nAChR activation and subsequent intracellular calcium rise. The selective AC1 inhibitor CB-6673567 and siRNA-mediated deletion of AC1 both blocked the choline-induced cAMP increase, suggesting that calcium-dependent AC1 is required for choline's action. Furthermore, α7 nAChR activation stimulated the phosphorylation of synapsin, which serves as a downstream effector to regulate neurotransmitter release. Our findings provide the first direct evidence to link activation of α7 nAChRs to a cAMP rise via AC1, which defines a new signaling pathway employed by α7 nAChRs. Our study sheds light into potential molecular mechanisms of the positive cognitive actions of α7 nAChR agonists and development of therapeutic treatments for cognitive impairments. Second, using mutagenesis and a combination of electrophysiology and imaging techniques, we discovered the possible involvement of an aspartate residue in the calcium permeability of the α7 nAChR. We found that the aspartate at position 44 appears to be essential since mutating it to alanine resulted in the complete disappearance of detectable calcium changes in most cells, which indicates that the extracellular domain of the α7 nAChR plays a key role in calcium permeation. Third, 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.
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