A Combinatorial Viral Vector Strategy for Targeting Direct Striatal Medium Spiny Neurons in Wildtype Rats
Bruno, Michael J.
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Medium spiny neurons account for over 90% of all neurons in the striatum and are classified as either direct (dMSNs) or indirect (iMSN) projecting to the substantia nigra. Direct MSNs can be identified based on the expression of dynorphin or Substance P, while iMSNs are enkephalin-positive. Recent evidence suggests that dMSNs and iMSNs may mediate different aspects of drug associated behaviors. However, it has been difficult to study their respective roles because these two populations of neurons are tightly intermingled. Recently our laboratory has created a combinatorial AAV approach to target either dMSN or iMSNs in rats. The first viral construct delivers Cre-recombinase under the control of either the dynorphin (dyn) or enkephalin (enk) promoter, which will drive expression only in dMSN or iMSNs, respectively. The second virus delivers a designer receptor exclusively activated by a designer drug (DREADD) fused with a fluorescent tag. While the DREADD transgene is driven by a strong, generalized promoter (e.g. EF1α), it is in an inverse orientation (double inverse open reading frame, DIO), and will not express unless CRE is present to flip the DREADD gene to the proper orientation. This dual technique compensates for the relatively small size of the AAV and the weak activity of the dynorphin and enkephalin promoters. When used together the DREADDs should be expressed exclusively either in the dMSN or iMSN populations. The DREADDs can then be used to activate or silence these neurons to study their function behavioral assays. The goal of this master’s thesis project is to determine whether the Dyn-Cre-AAV + EF1a-DIO-DREADD-mCherry-AAV combination will specifically target DREADDs to dMSNs. We injected rats with the virus combination and performed immunohistochemistry (IHC) for Substance P (a dMSN marker), enkephalin (an iMSN marker), and mCherry (the fluorescent tag fused to the DREADD) on free floating sections and imaged these using confocal microscopy. We found that ~ 80% of DREADD positive neurons were also dynorphin (Substance P) positive, while ~ 17% did not have discernable dynorphin expression. In addition, the DREADD-mCherry positive cells did not co-localize with the enkephalin IHC signal in preliminary experiments. Using this combinatorial AAV system we have successfully targeted DREADDs to dMSNs in the striatum. In addition, we have identified specific issues associated with performing co-localization studies for enkephalin containing neurons and have devised strategies to overcome these.