A novel approach to using combinatorial AAV to knockdown the D2 autoreceptor from specific pathways
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Mesolimbic dopamine neurotransmission is largely implicated in the motivation to abuse drugs. A number of studies have indicated that dopamine D2 receptor expression and function are altered in drug addicts. However, understanding the role of the D2 receptor in addiction is complicated by their presence in many brain regions and location on both pre and post-synaptic sites. For example, dopamine neurons that project from the ventral tegmental area (VTA) to other brain regions, including the prefrontal cortex, hippocampus, amygdala and the nucleus accumbens, contain D2 receptors in the terminals, but D2R is also found on post-synaptic sites in these regions. Although structurally very similar, the pre and post-synaptic receptors have entirely different functions. Activation of the presynaptic D2R decreases both the synthesis and release of dopamine, and thus acts as an autoreceptor. To better understand dopamine's role in drug abuse, we have been examining how depletion of the D2 autoreceptor from VTA terminals affects various behaviors related to addiction. Specifically we deliver a virus expressing a short hairpin RNA (shRNA) targeting the D2R to the VTA, which knocks down expression in the terminals. However, since the VTA has diffuse projections, it has not been possible to specifically target the presynaptic D2R knockdown to one specific brain region or projection, which would greatly aid our understanding of how this system impacts drug seeking behaviors. In order to tackle this problem we have developed a combinatorial approach to target shRNA to specific projections. This dual system employs a conditional expression vector (SICO-AAV) that generates shRNA only after a recombination event occurs. The enzyme Cre-Recombinase is capable of recognizing loxP sites that flank a target sequence and excises the nucleotide sequence that is located between them. The SICO system is designed such that Cre recombinase will remove a stuffer sequence (CMV-GFP) and brings the shRNA cassette in proximity to the U6 promoter. Thus, expression of the shRNA is dependent on Cre recombinase. We will then combine the SICO-AAV system with a virus that expresses a Wheat Germ Agglutinin (WGA)-Cre-Recombinase fusion protein. The WGA acts as a retrograde carrier for the Cre recombinase. By injecting the WGA-Cre-AAV in the projection site, such as the NAc, and the shD2R-SICO-AAV into the VTA, we expect retrograde transport of Cre to the soma of the subset of VTA neurons that project primarily to the NAc. The Cre will then act on the shD2R-SICO cassette, bringing the shRNA portion in line with the promoter, and drive D2R shRNA expression only in those neurons that project from the VTA to the NAc. This conditional knockdown system will allow us to study the D2 presynaptic receptor in individual projections, while avoiding disruption of the D2R in other neural circuits involving the VTA.