Monitoring Mitochondrial Oxidant Stress in Mice

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.