Under the same plane of anesthesia the Gq-DREADD (AAV2-hSyn-HA-hM3D(Gq)-IRES mCitrine, Bryan Roth, University of North Carolina, Chapel Hill, NC) was microinjected into the perirhinal cortex (coordinates measured from bregma at the skull surface, angled 10° laterally, as follows: anterior–posterior (AP) −4.8 mm, medial-lateral (ML) −5 mm, DV −7.5mm). Gq-DREADDs were allowed 3 wk to reach maximal expression levels before CNO testing began. AAV was microinjected in a volume of 0.75 µL/side (1012 IU/mL) at a rate of 0.1 µL/min, allowing 10 min for diffusion.
Following the last test session, rats were transcardially perfused with 10% buffered formalin, and brains were removed for immunohistochemistry on free-floating (40 µm) sections. Tissue was treated with peroxidase, then blocked with 2% normal donkey serum in PBS. Sections were incubated overnight at 4°C in primary antibody: mouse anti-hemagglutinin (HA) (1:1000; Covance #MMS101-P, RRID: AB_2314672). The secondary antibody was biotin-SP conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch Laboratories). The signal was amplified with an avidin-biotin complex (1:500), then reacted with diaminobenzidine (in 5% nickel). Tissue was mounted onto slides, dehydrated, cover-slipped, and examined under a microscope to visualize the Gq-DREADD.
Following the last test session, rats were transcardially perfused with 10% buffered formalin, and brains were removed for immunohistochemistry on free-floating (40 µm) sections. Tissue was treated with peroxidase, then blocked with 2% normal donkey serum in PBS. Sections were incubated overnight at 4°C in primary antibody: mouse anti-hemagglutinin (HA) (1:1000; Covance #MMS101-P, RRID: AB_2314672). The secondary antibody was biotin-SP conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch Laboratories). The signal was amplified with an avidin-biotin complex (1:500), then reacted with diaminobenzidine (in 5% nickel). Tissue was mounted onto slides, dehydrated, cover-slipped, and examined under a microscope to visualize the Gq-DREADD.