Midbrain slices were obtained from mice between postnatal ages 21 and 30. Mice were handled according to guidelines established by the Northwestern University Animal Care and Use Committee, the National Institutes of Health and the Society for Neuroscience. Midbrain slices were visualized using infrared-differential interference contrast (IR-DIC) video microscopy system (for patch clamp recording) and imaged with 2PLSM to measure calcium transients, mitochondrial membrane potential using TMRM dye, or mito-roGFP signal. SNc or VTA neurons were filled with Alexa594 and Fluo-4 and calcium transients were imaged as described previously 6 (link). Mitochondrial membrane potential was calculated using a Nernst equation describing the distribution of TMRM 11 (link). Transgenic mice were generated by conventional approaches with a roGFP2 construct containing the TH promoter and a matrix mitochondria-targeting sequence. Relative oxidation of mito-roGFP was determined from fluorescence measurements after fully reducing mitochondria with dithiothreitol and then fully oxidizing with aldrithiol. Since the calibrated signal becomes independent of absolute expression level of mito-roGFP, this strategy allows cell-to-cell comparisons. Results in the main body of the paper were derived from a single line of mice showing strong mito-roGFP expression. In the presence of a strong reducing agent as an estimate of roGFP concentration, we inferred that there was not a significant difference in the expression level between SNc and VTA neurons (Supplementary Figure 7). The oxidation state of mitochondria was also verified in a second line of mice having lower mito-roGFP expression levels (Supplementary Figure 7) and in cultured cells expressing mito-roGFP (Supplementary Figure 6). Single-cell reverse transcription-polymerase chain reaction (scRT-PCR) has been described previously 4. Relative gene expression of UCPs was performed by reverse transcriptase reaction followed by quantitative PCR analysis. Mn-SOD immunostaining used standard approaches. Sample “n” represents number of mice. Statistical analysis was performed with non-parametric test with threshold for significance less than 0.05.