Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA A 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 ARγ2 77Ilox) in which the γ2 subunit of the GABA A receptor (GABA AR) was mutated to become zolpidem insensitive (γ2 77I) and used viral vectors to swap γ2 77I with wild-type, zolpidem-sensitive γ2 subunits (γ2 77F). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABA ARγ2 -/- 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 γ2 ( AU1γ2 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 AU1γ2 77F subunit (Cre-2A- AU1γ2 77F) into the neocortex of GABA ARγ2 77Ilox 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 AU1γ2 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 γ2 77I and AU1γ2 77F GABA ARs 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.