The mitochondrial CI activity was determined as reported elsewhere [52 (link)]. The tissue from an entire SN was homogenized in PBS pH 7.4 and subsequently centrifugated for 5 min at 2000 g. The supernatants were collected and used to perform the analyses. CI activity measurement is based on its ability to oxidize nicotinamide adenine dinucleotide + hydrogen (NADH) while reducing decylubiquinone (Dub) to dihydro-decylubiquinone (DUbH2), a molecule that is next oxidized by 2,6-Dichloroindophenol (DCPIP). This product can absorb light at 600 nm. In the meantime, 20 mM rotenone was included as a specific inhibitor of CI. All the measurements were completed employing a Synergy-Biotek (Biotek Instruments; Winooski, VT, USA) microplate reader at 37 °C. The CI activity was defined by subtracting the activity obtained with the inhibitor rotenone from the not inhibited activity and expressed as nmol/min/mg protein (data normalized by mean values of the not treated group).
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