Cunningham adaptor

An isoflurane precision vaporizer (Smiths Medical PM) was used to anesthetize mice. Mice were then placed on a stereotaxic frame (David Kopf Instruments), with a Cunningham adaptor (Harvard Apparatus) to maintain anesthesia delivery during surgery. The skull was exposed, and a small hole was drilled at the desired injection site. The following stereotaxic coordinates were used: SNc, 3.1 and 1.3 mm posterior and lateral to bregma, at a depth of 4.5 mm from dura; dorsolateral striatum, 0.34 and 2.5 mm anterior and lateral to bregma, at a depth of 3 mm from dura. The Allen Mouse Brain Atlas, online version 1, 2008 (https://atlas.brain-map.org/) was used as a reference for the coordinates and generating diagrams.
For each mouse, the distance between bregma and lambda was calculated and used to adjust the coordinates. For AAV injections, ~350 nl of viral vector was delivered using a glass micropipette (Drummond Scientific) pulled with a P-97 glass puller (Sutter Instruments). Surgery for imaging experiments was performed unilaterally, and surgeries for RiboTag tissue collection were executed bilaterally. Experiments were performed after at least 10 postoperative days; tissue collection for RiboTag was performed 4 weeks after injection.

To examine mitochondrial oxidant stress, adeno-associated virus expressing the redox biosensor roGFP targeting the mitochondrial matrix (Sabharwal et al., 2013 (link)) under double-floxed inverse orientation (AAV9-DIO-mito-roGFP) was generated by the University of Minnesota Viral Vector and Cloning Core. Mice between 6 and 8 weeks of age were anesthetized using isoflurane and placed in a stereotaxic frame (Kopf Instruments) with a Cunningham adaptor (Harvard Apparatus). Anesthesia was maintained at a flow rate of 1.5–2.5% and anesthetic depth was monitored throughout the surgery. Meloxicam (2 mg/kg) was administered subcutaneously after which an incision was made to expose the skull. A small hole was drilled with a micro drill and 400 nL AAV9-DIO-mito-roGFP was injected into the locus coeruleus (LC, coordinates: AP: −5.45 mm, ML: 1.25 mm, and DV: 3.65 mm from bregma) in TH-Cre mice and into the dorsal raphe (DR, coordinates: AP: −4.5 mm, ML: 0 mm, and DV: 3 mm from bregma) in SERT-Cre mice using a thin glass pipette pulled on a P-1000 micropipette puller (Sutter Instruments). Mice received 2 mg/kg meloxicam subcutaneously for 3 days post-op and were allowed at least 10 days of recovery before being euthanized and ex vivo brain slices generated to examine mitochondrial oxidant stress.