Gene expression profiling of inputs to the mesolimbic dopamine circuit

PRV-Circuit-TRAP of DAT-cre mice injected with PRV-Introvert-GFP in the nucleus accumbens These studies identify important inputs to the mesolimbic dopamine pathway and further show that PRV circuit-directed translating ribosome affinity purification (PRV-Circuit-TRAP) can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits. Overall design: The current study combined neuroanatomical tracing and molecular profiling of inputs to the mesolimbic dopamine pathway using pseudorabies virus whose replication and exression of GFP are dependent on Cre recombinase (PRV-Introvert-GFP). To molecularly profile this circuit, we mated DAT-Cre to SYN-NBL10 mice and injected PRV-Introvert-GFP into the NAc. GFP expressed from PRV-Introvert-GFP binds to the NBL10-tagged polysomes and is then precipitated using anti-GFP monoclonal antibodies. Immunoprecipitated RNA was purified (IP RNA) along with total RNA collected before immunoprecipitation (Input RNA) was purified and sequenced with an Illumina HiSeq 2000 system. The differential expression of transcripts in our IP fraction was calculated. Final values for fold enrichment were weighted for the effects of PRV infection on transcript abundance.