Stereotaxic surgery procedures

Adult mice (∼8 weeks of age) were anesthetized with isoflurane (initial dose = 3%, maintenance dose = 1.5%), and surgery was performed using an Angle Two stereotaxic frame (Leica Microsystems) to intracranially inject adeno-associated virus (AAV) into the dlBNST based on the Paxinos and Franklin (2004) mouse brain atlas (from bregma: AP 0.14, ML ±0.88, DV −4.24, 15.03° tilt) at a rate of 100 nl/min. For behavior studies using the GluN2D^flx/flx line, male mice were bilaterally injected with ∼300 nl of recombinant AAV5-CMV-eGFP or AAV5-CMV-Cre-eGFP (UNC Vector Core), and given a 4 week recovery period. GluN2D^flx/flx/Crf-Flp mice of either sex used for physiology studies were bilaterally injected with ∼300 nl of a 1:1 mixture of an AAV9-Ef1a-fDIO-tdTomato virus (Stanford Vector Core) and the AAV5-CMV-Cre-eGFP virus, and similarly given 4 weeks recovery before producing slices for ex vivo recordings.

For photometry studies, GluN2D^−/−/Crf-Cre mice of either sex were injected with ∼300 nl of AAV9-Syn-FLEX-jGCaMP7f-WPRE (Addgene) into the right dlBNST, and then given a 3 week recovery period to allow for full expression of the virus. Following this period, mice underwent a second round of surgery to place a fiberoptic cable implant (4.1 mm fiber, Doric Lenses) just above the injection site of the virus within the dlBNST (∼0.1 mm above DV coordinate listed above). After placing the fiberoptic into position, a system consisting of a light-curable Optibond primer and adhesive agent (Henry Schein Medical) and Herculite XRV dental composite enamel (Kerr) was applied to the mouse's skull to firmly affix and hold the fiberoptic in position. Briefly, after exposure of the skull, gel etchant was used to clean the skull, a screw was placed rostral to the craniotomy hole, and the implant was slowly lowered through the previously made craniotomy hole. Optibond FL Primer was applied around the implant, followed by Optibond FL light-curable adhesive, and last the light-curable Herculite enamel. Mice were given a minimum 1 week recovery period following implant surgery to allow for the surgery sites to fully heal before the start of in vivo calcium signal recording.