2.5. High-Resolution Mitochondrial Respirometry

Hippocampal mitochondrial function was measured using an Oroboros O2k-Respirometer [31]. Briefly, oxygen polarography was performed at 37 °C, and oxygen flux per tissue mass (pmol O₂/second/mg) was recorded using Datlab software (OROBORO INSTRUMENTS, Innsbruck, Austria). A total of 5 mL oxygen was injected into each chamber for oxygenation at the beginning of each experiment until oxygen concentration reached 380 μM. Hippocampal tissue was homogenized in cold Miro5 media on ice containing: 0.5 mM EGTA, 3 mM MgCl₂, 60 mM K-lactobionate, 20 mM Taurine, 10 mM KH₂PO₄, 20 mM HEPES. 110 mM sucrose and 1.0 g/L fatty acid free BSA; pH 7.1. One milligram of lysate was loaded into each of the 2 mL respirometers chambers containing MiRO₅ and kept at 37 °C during the measurement. As shown in Figure 1, after 10 min of equilibration, OXPHOS—capacity of complex I (CI OX) linked activity was measured by adding CI-linked substrate pyruvate (5 mM), glutamate (10 mM), malate (0.5 mM), and ADP (2.5 mM); maximum oxidative phosphorylation capacity of CI and II (CI + II OX) linked activity was measured by addition of succinate (10 mM), maximum uncoupled capacity (CI + II mUC) were determined following stepwise titration of 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP). Additionally, CII coupled respiration capacity (CII) was determined in another O2k-Respirometer chamber by addition of CI inhibitor, rotenone (0.5 μM), CII substrate succinate (10 mM), and ADP (2.5 mM). Similarly, maximum uncoupled capacity was determined following stepwise titration of 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) (Figure 1B). Oxygen concentration in the chamber was kept at more than 150 μM till the end to avoid the limitation of respiration. Duplicate data of each sample were averaged to obtain the final readout. Oxygen flux was expressed as mass-specific respiration (per mg), or as Flux Control Respiration (FCR) by internal normalization for maximum ETS capacity (CI&II ETS, mUC value) [32]. Mass-specific oxygen consumption rate and FCR was obtained using DatLab 7 software.

Representative respirometry traces of hippocampal homogenates using the Oroboros O2k-Respirometer. (A) Complex I and II linked activity. (B) Complex II linked activity. Complexes substrates as described in the methods.