CaMKIIα promoter-controlled circuit manipulations target both pyramidal cells and inhibitory interneurons in cortical networks.

A key assumption in studies of cortical functions is that excitatory principal neurons, but not inhibitory cells express calcium/calmodulin-dependent protein kinase II subunit α (CaMKIIα) resulting in a widespread use of CaMKIIα promoter-driven protein expression for principal cell manipulation and monitoring their activities. Using neuroanatomical and electrophysiological methods we demonstrate that in addition to pyramidal neurons, multiple types of cortical GABAegic cells are targeted by adeno-associated viral vectors (AAV) driven by the CaMKIIα promoter in both male and female mice. We tested the AAV5 and AAV9 serotype of viruses with either Channelrhodopsin 2-mCherry or Archaerhodopsin-T-GFP constructs, with different dilutions. We show that in all cases, the reporter proteins can visualize a large fraction of different interneuron types, including parvalbumin (PV), somatostatin (SST), neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY) and cholecystokinin (CCK)-containing GABAergic cells, which altogether cover around 60% of the whole inhibitory cell population in cortical structures. Importantly, the expression of the excitatory opsin Channelrhodopsin 2 in the interneurons effectively drive spiking of infected GABAergic cells even if the immunodetectability of reporter proteins is ambiguous. Thus, our results challenge the use of CaMKIIα promoter-driven protein expression as a selective tool in targeting cortical glutamatergic neurons using viral vectors. Significance Statement Studies exploring the mechanisms underlying cortical functions apply refined tools to selectively manipulate individual network elements to reveal their role in circuit operations. A widely used approach to specifically target cortical principal cells is to utilize the calcium/calmodulin-dependent protein kinase II subunit α (CaMKIIα) promoter. In this study we show that the CaMKIIα promoter drives expression in at least five types of inhibitory cells in various cortical regions, which altogether cover around 60% of the whole inhibitory cell population in cortical structures. The infected interneurons can be functionally activated using optogenetic approaches, a finding that challenges the interpretation of earlier results and calls for the use of other principal cell-targeting methods, e.g., exploiting Vglut1-Cre mice.