Stereotaxic viral injections in rodent brains

Viral injections were performed using previously described procedures^{6 (link)} at the following stereotaxic coordinates: pPVT, –1.34 mm from Bregma, 0.05 mm lateral from midline, and 3.03 mm vertical from cortical surface; CeL, −1.22 mm from Bregma, 2.9 mm lateral from midline, and 4.6 mm vertical from cortical surface; BLA, –1.80 mm from Bregma, 3.4 mm lateral from midline, and 5.4 mm vertical from cortical surface. For pPVT injections we used a 6.5° angle to avoid damage of the superior sagittal sinus. Animals were kept on a heating pad throughout the entire surgical procedures and were brought back to their home cages after 24 h post-surgery recovery and monitoring. Postoperative care included intraperitoneal injection with 0.3–0.5 ml of lactated Ringers solution and metacam (meloxicam, 1–2 mg/kg) for analgesia and anti-inflammatory purposes. All AAVs and the CAV2-Cre were injected at a total volume of approximately 1 μl (except for the monosynaptic rabies viral tracing, see below), and were allowed at least two weeks for maximal expression. For retrograde tracing of amygdala-projecting pPVT cells, CTB-555 or CTB-488 (0.1-0.3 μl, 0.5% in PBS) (Invitrogen) was injected into CeL and BLA and allowed 3-5 days for sufficient retrograde transport.