Ctb 488

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.

Three female ferrets were used for tracing studies. To determine the projections from V1 to LPl, anterograde tracer was injected in V1. One animal was microinjected with 0.6 μl of AAV5-CaMKII-ArchT-GFP (7.5 × 10¹² vg/ml) in two sites of V1 (8.5 mm and 9 mm lateral from the midline and 3 mm anterior from lambda). To explore the layer-specific connections between V1 and LPl, two different strategies were used. In one animal, AAV5-CaMKII-ArchT-mCherry (1 × 10¹² vg/ml) was microinjected (0.3 μl) into LPl (13 mm anterior from lambda and 3.8 mm lateral from the midline) for anterograde labeling of projections in V1. Another animal was injected with 0.4 μl of Alexa 488–conjugated cholera toxin subunit B (2.5μg/μl CTB-488, Invitrogen) into LPl for retrograde labeling of projecting cells in V1. All the injections were conducted using a 1 μL Hamilton syringe with an infusion rate of 0.1μl/min. All viral constructs were packaged and titered by the UNC Vector Core Facility. Adequate pain relief and post-surgery monitoring was provided as described previously (Sellers et al., 2013 (link)). Animals were euthanized for histology 12 days after injection for CTB tracing and nine (ArchT-GFP) and eleven (ArchT-mCherry) weeks after the procedure for viral injections. All procedures were approved by the UNC – Chapel Hill IACUC and exceed the guidelines set forth by the NIH and USDA.