Chemogenetic Activation of PV+ Neurons in mPFC

Adeno-associated virus (AAV) designer receptors exclusively activated by designer drug (DREADD) vectors (Addgene) were stereotaxically infused in the ventromedial PFC (including the central part of the PrL and IL cortices) of male PV:Cre mice. Briefly, AAV2/hSyn-DIO-hm3D(Gq)-mCherry (“hM3DGq”) was injected bilaterally (0.5 μl/side, ∼10¹² viral genomes/ml) into mPFC using the following coordinates: anteroposterior, +1.7 mm; mediolateral, ±0.2 mm; dorsoventral, −2.6 mm. AAV2/hSyn-DIO-mCherry was used as the control virus. Previous work demonstrated that chemogenetic activation of PV⁺ neurons from both PrL and IL regions increases anxiety-like behaviors in female mice (Page et al., 2019 (link)). To be able to compare our findings with this study, we opted to use a similar strategy. Mice remained undisturbed for at least 21 d after surgery to allow full expression of the DREADD virus in PV⁺ cells before the commencement of behavioral testing. All mice received a daily intraperitoneal injection of clozapine-N-oxide (CNO; 0.5 mg/kg) 30 min before daily handling or stressor throughout the UCMS period. After the completion of behavioral assays, viral injection sites were verified using a rabbit anti-DsRed antibody (1:1000; TaKaRa Bio) followed by an Alexa Fluor anti-rabbit 555 secondary antibody (1:500; Thermo Fisher Scientific) to target mCherry in prefrontal brain sections.