Quantifying Neuronal Gene Expression

Animals were killed 2 h post a single Flx (15 mg/kg) or saline injection by decapitation; the PFC was dissected out and snap-frozen in liquid nitrogen. RNA was extracted using Tri reagent (Sigma) and reverse transcribed using a cDNA synthesis kit (PrimeScript First Strand cDNA Synthesis Kit, Takara Bio). qPCR was performed with primers for the genes of interest (Supplementary Table S1) using a Bio-Rad CFX96 real-time PCR machine. Data were analyzed using the ΔΔC_t method, as described previously [17 (link)]. Normalization was done using hypoxanthine guanine phosphoribosyl transferase (Hprt), whose level was unaltered across experimental groups. Gene expression of IEGs analyzed included activity regulated cytoskeleton associated protein (Arc), brain-derived neurotrophic factor (Bdnf), early growth response gene 1-4 (Egr 1-4), FBJ osteosarcoma gene (Fos), FBJ murine osteosarcoma viral oncogene homolog B (Fosb), Fos-like antigen 2 (Fosl2), Homer scaffolding protein 1-3 (Homer 1-3), Jun proto-oncogene (Jun), Jun B proto-oncogene (Junb), Jun D proto-oncogene (Jund). qPCR analysis was also performed for the 5-HT_2A gene (Htr2a) to further confirm our genotyping results.