Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABAA receptors in cortical pyramidal cells

Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABA(A)R gamma 2(77I) lox) in which the gamma 2 subunit of the GABA(A) receptor (GABA(A)R) was mutated to become zolpidem insensitive (gamma 2(77I)) and used viral vectors to swap gamma 2(77I) with wild-type, zolpidem-sensitive gamma 2 subunits (gamma 2(77F)). The verification of unaltered density and subcellular distribution of the virally introduced gamma 2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged gamma 2 subunits, with which cortical cultures of GABA(A)R gamma 2(-/-) mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged gamma 2 ((AU1)gamma 2(77F)) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the (AU1)gamma 2(77F) subunit (Cre-2A-(AU1)gamma 2(77F)) into the neocortex of GABA(A)R gamma 2(77I) lox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the (AU1 gamma)2(77F) subunits in perisomatic GABAergic synapses. In line with this, miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of gamma 2(77I) and (AU1)gamma 2(77F) GABA(A)Rs in cortical pyramidal cells. This transgenic-viral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that might endow some unforeseen alterations of the genetically modified cells. In addition, this virus-based approach opens up the possibility of modifying multiple cell types in distinct brain regions and performing alternative recombination-based intersectional genetic manipulations.