Figure 5.

PRV-Circuit-TRAP of hypothalamic and midbrain neurons projecting to the mesolimbic dopamine circuit. A, Strategy for PRV-Circuit-TRAP. PRV-Introvert-GFP was injected bilaterally into the NAc of DAT-Cre × SYN-NBL10 mice. Translating ribosomes tagged with GFP binding nanobody (NBL10) were immunoprecipitated from a section of midbrain 3 days after injection of PRV-Introvert-GFP. B, Results of qPCR of input and IP samples from PRV-Circuit-TRAP for Gfp (p < 0.05), Dat/Slc6a3 (p < 0.01), Anxa1 (p < 0.01), and Gfap (p < 0.05). Fold enrichment for each gene from the IP or input sample is normalized to the housekeeping gene Rpl23. C, Electropherogram of representative control, input, and IP RNA samples measured on an Agilent 2100 Bioanalyzer before sequencing on an Illumina HiSeq 2000. D, Differential enrichment of immunoprecipitated transcripts. Dotted lines indicate twofold enrichment or depletion in IP RNA compared with input RNA. Colors of dots indicate the significance of enrichment: red, q < 0.005; orange, q < 0.05; blue, q ≥ 0.05. *p < 0.05; **p < 0.01.