There have been several major accomplishments within the past fiscal year. The activation of &#945;7 nAChRs has been shown to improve hippocampal-dependent learning and memory. However, the molecular mechanism of &#945;7 nAChRs' action remains elusive. We previously reported that activation of &#945;7 nAChRs induced a prolonged enhancement of glutamatergic synaptic transmission in a PKA-dependent manner. Here, we investigated any connection between the activation of the &#945;7 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 &#945;7 nAChR-selective agonist choline, in the presence of the &#945;7 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 &#945;7 nAChR antagonist MLA and the calcium chelator BAPTA, suggesting that the cAMP increase depends on the &#945;7 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, &#945;7 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 &#945;7 nAChRs to a cAMP rise via AC1, which defines a new signaling pathway employed by &#945;7 nAChRs. Our study sheds light into potential molecular mechanisms of the positive cognitive actions of &#945;7 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 &#945;7 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 &#945;7 nAChR plays a key role in calcium permeation. Third, we have generated mice in which the fourth exon of the &#945;7 nAChR gene (Chrna7) is flanked by loxP sites (B6-Chrna7(LBDEx4007Ehs)) which we refer to as floxed &#945;7 nAChR conditional knockout or &#945;7nAChR(flox). We validated the chosen approach by mating &#945;7nAChR(flox) with mice expressing Cre recombinase driven by the glial acidic fibrillary protein (GFAP)-Cre promoter (GFAP-A7KO) to test whether &#945;7nAChR(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 &#945;7nAChR(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 &#945;7 nAChR binding sites were absent on GFAP-positive astrocytes in hippocampal slices obtained from GFAP-A7KO offspring from &#945;7nAChR(flox) and GFAP-Cre crosses. Finally, we validated that Cre recombinase (Cre)-mediated excision led to functional, cell- and tissue-specific loss of &#945;7 nAChRs by demonstrating that choline-induced &#945;7 nAChR currents were present in Cre-negative, but not synapsin promoter-driven Cre-positive, CA1 pyramidal neurons. Additionally, electrophysiological characterization of &#945;7 nAChR-mediated current traces was similar in terms of amplitude and time constants of decay (during desensitization) for the &#945;7nAChR(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 &#945;7 nAChR expression in a cell-specific manner.
NIH ExPorter
NIH ExPorter