Figure 2.

DIO and DO rAAVs achieve Cre-On and Cre-Off expression but exhibit interfering interactions when co-infected. (A) Sagittal section through a D2-Cre mouse infected with DIO-GFP in the striatum. GFP⁺ axons emanating from the striatal infection innervate the globus pallidus (GP), the target of indirect pathway medium spiny neurons (iMSNs). No GFP⁺ expression is observed in the substantia nigra reticulata (SNr, inset). COR = cortex, STR = striatum. (B) Sagittal section through a D2-Cre mouse infected with DO-mCherry in the striatum such that mCherry expression is excluded from iMSNs. mCherry⁺ axons leaving the striatum innervate the SNr (inset), consistent with expression in direct pathway MSNs (dMSNs). (C) Antibody staining against Cre in D2-Cre striatum infected with DO-mCherry reveals that Cre efficiently turns off mCherry expression, indicative of Cre-Off behavior. Top, a single confocal plane showing mCherry expression excluded from cells with Cre⁺ nuclei. Bottom, quantification of co-expression of Cre and mCherry (N = 4 infections, N = 1187 cells) reveals that mCherry expression (N = 495 cells) was almost completely excluded from Cre⁺ cells (N = 1/691 cells double-positive for mCherry and Cre). (D) Co-infection of rAAVs encoding a Cre-mCherry fusion and DO-GFP into the striatum of a wild type (WT) mouse efficiently prevents GFP expression in Cre-mCherry expressing cells. Top, a single confocal plane containing interspersed mCherry⁺ and GFP⁺ cells. Note that the mCherry expression is nuclear due to nuclear targeting of Cre. The arrowhead indicates a cell co-expressing mCherry and GFP. Bottom, confocal imaging of infected tissue (N = 2 infections, N = 521 cells) reveals that cells expressing Cre-mCherry (N = 237) and cells expressing GFP (N = 284) are largely non-overlapping (N = 11/521 cells were double-positive for both fluorophores). (E) Sagittal section through a D2-Cre mouse infected with DIO-mCherry and DO-GFP in the striatum. Despite the physical intermixing of Cre positive and negative neurons in the striatum, the resulting GFP and mCherry expression is spatially segregated. Below, separate visualization of the green and red fluorescence channels demonstrates that mCherry is excluded from the striatal volume expressing GFP. (F) Normalized mean fluorescent values for GFP and mCherry quantified from boxed inset in E, bottom right. (G) Confocal imaging around the border regions (N = 2 infections, N = 865 cells) containing both GFP⁺ (N = 529) and mCherry⁺ (N = 336) cells revealed no overlap in expression.