Aav ef1a doubled floxed hchr2 h134r eyfp

Mice underwent stereotaxic surgery between P25 and P35. Following anaesthesia (1–2% isoflurane) animals were placed on a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). Injections were performed at the following coordinates measured from bregma, LH: AP −0.6 mm, ML ±1.2 mm, DV −5.3 mm; PAG: AP −3.15 mm, ML ±0.5 mm, DV −2.7 mm; PFC: AP +2.45 mm, ML ±0.5 mm, DV −1.8 mm. Using a Nanoject III (Drummond Scientific, Broomall, PA, USA), either AAV‐EF1a‐doubled floxed‐hChR2(H134R)‐mCherry (Addgene, Watertown, MA, USA, 20297‐AAV5) or AAV‐EF1a‐doubled floxed‐hChR2(H134R)‐EYFP (Addgene 20298‐AAV5) was injected at a volume of 100 nl for LH, 50 nl for PAG and 300 nl for PFC. As per nomenclature suggestions outlined in Laubach et al., 2018 , injections to the medial prefrontal cortex specifically targeted the prelimbic and infralimbic cortices, with the target plane corresponding to 1.78 mm anterior from bregma in the mouse brain atlas (Franklin & Paxinos, 2007 ). For simplicity, this afferent is referred to in the text as ‘PFC’. The volume of each injection site was optimized to the spread of the virus within that region, without spreading into neighbouring regions. Mice received carprofen (5 mg/kg, subcutaneous) at the beginning of surgery and 24 h post, after which they were monitored daily to ensure proper recovery.