AAV FLEX-switch delivery system promotes selective Cre and hM3D(Gq) expression in primary cortical neurons.

The current experiment utilized 2 viral vectors shown at the top of panel B. At the C-terminal of the AAV-CMV-GFP/Cre (serotype 9) virus, Cre was fused with green fluorescent protein (GFP) that can be observed in infected cells using a confocal microscope. The AAV-hSyn-DIO-hM3D(Gq)-mCherry virus contains a double inverted coding sequence promoted by the neuron-specific promoter, hSyn, and the mCherry red fluorescent marker that can similarly be observed in infected cells using a confocal microscope. A) A representative confocal image of GFP/Cre and hM3D(Gq)-mCherry expression in a primary cortical neuron is shown. Here, the GFP green fluorescence indicates the expression of Cre and is localized in the nuclei of cells. mCherry red fluorescence indicates the expression of hM3D(Gq) receptors after the vector was reoriented by GFP/Cre recombinase in vitro. 3-D reconstruction based on Z-stack scanning verified that mCherry fluorescence exhibited a membrane-specific distribution. B) When injected into the AcbSh of Fisher 344 rats, GFP/Cre is retrogradely transported along the axons of VTA neurons to the soma where it reorients hM3D(Gq)-mCherry as represented in the drawing.