Female CD-1 mice (29-32 g, Charles River) were used for the experiments. They were kept in standard cages, under 60% humidity, 22° C temperature, and a 12-h light/dark cycle. Food and water were available ad lib. In accordance with GV-Solas guidelines, all procedures were designed to minimize the suffering of the experimental animals. In total, 90 mice were used for this study.

After decapitation, the brain was immediately dissected from the skull, the cerebellum was removed, and the remaining brain tissue (ca. 300 mg) was homogenized in 2-mL MiR05 buffer (Schwarzkopf et al. 2015; Gnaiger 2020). In addition, a protease inhibitor cocktail was added to the medium (cOmplete Tablets EASY pack, Roche, Mannheim, Germany). The homogenate was centrifuged twice to remove all cell debris (1.400 g, 7 min, 4 °C). The purified supernatant was then centrifuged again (10.000 g, 5 min, 4 °C), and the resulting pellet containing the mitochondria was resuspended in 1-mL MiR05 + PI and centrifuged once again (1.400 × g, 3 min, 4 °C). Finally, the supernatant was centrifuged one more time (10.000 g, 5 min, 4 °C) and the pellet resuspended in 250-µL MiR05 + PI.

Mitochondria from two hemispheres of the same mouse brain were put into parallel chambers of the respirometer (Oroboros® Instruments, Innsbruck, Austria). Each chamber was filled with 2.4-mL MiR05 medium according to manufacturer’s instructions and kept at 37 °C with constant stirring (750 rpm). After 30 min equilibration and subsequent air calibration, 80 µL of the mitochondrial suspensions was injected into the closed chamber. The remaining mitochondria were frozen in liquid nitrogen for protein determination with the Bradford assay. After equilibration, a solution containing pyruvate (5 mM) and malate (1 mM), two substrates linked to complex I (CI), was injected into the chamber (LEAK-state, non-phosphorylating resting state). Then, ADP (2 mM) was added to stimulate oxidative phosphorylation (OXPHOS-CI; ADP-stimulated and CI-linked respiration). To induce the full ADP-stimulated respiration, succinate (10 mM), a CII-linked substrate, was injected (total OXPHOS capacity). To verify the integrity of the outer mitochondrial membrane, cytochrome c (10 µM) was added; mitochondria whose respiration increased by more than 15% upon cytochrome c addition were discarded. The maximum capacity of the electron transfer system (ETS) was determined by the stepwise titration of the uncoupler FCCP (state E). To measure CII respiration, the complex I inhibitor rotenone (2.5 µM) was added (CII-linked substrate state, uncoupled). After inhibition of complex III by antimycin A (2.5 µM), the residual oxygen consumption (ROX; ROX-state) remains, which is used to correct the mitochondrial respiration states. Ascorbate (2 mM) and tetramethyl-phenylendiamine (TMPD, 0.5 mM) are artificial electron donors that induce maximum cytochrome c- oxidase (complex IV, CIV) respiration by reducing cytochrome c. Ascorbate regenerates TMPD and is injected first. At the end of the experimental run, CIV is inhibited by a high concentration of sodium azide (120 mM). The chemical background as well as ROX remains. To obtain the CIV activity, this value has to be subtracted from the total measured oxygen flux (for further details, see Schwarzkopf et al. 2015). A shortened version of the titration protocol is shown in Fig. 1.

Exemplary readout of the respirometer: oxygen consumption in isolated brain mitochondria, here induced by addition of pyruvate and malate (P + M), ADP, addition of pentobarbital (PB), succinate (Succ), and addition of cytochrome c (CyC) to check for CyC loss induced by pentobarbital. Mauve line, control; green line, pentobarbital

Isoflurane, sevoflurane, and halothane are volatile. Therefore, the incubation of the sample suspension was carried out in closed glass vials to prevent evaporation of the drugs during incubation. The concentrations of the volatile anesthetic drugs can be calculated from the minimal alveolar concentration and the blood/gas coefficient and was taken from the literature (see legend of Table 1).

Glass vials with MiRO5 medium were kept at 37 °C on an aluminum heating block. Malate (2 mM), pyruvate (5 mM), and ADP (2 mM) were added and vials were incubated for about 2 min. Afterward, isoflurane (1.20 mM), sevoflurane (3.42 mM), halothane (2.26 mM), ketamine (7.8 µM), pentobarbital (360.0 µM), propofol (252.0 µM), and sample suspension (60 µl) were added to the vials. Incubation was carried out for 15 min in a shaking water bath at 37 °C. Then, the reaction was stopped on ice, and ATP content was measured using an ATP Assay Kit (Lonza). For this purpose, ice-cold samples were diluted 1:1000 with MiRO5 buffer. 100 µl of diluted samples was shaken (Unimax 1010) with lysis buffer for 10 min at room temperature in black 96-well plates. Next, the ATP measurement reagent (100 µl) was added and the mixture was incubated for 15 min in a dark place at room temperature. Measurement of fluorescence was carried out with a Wallac Victor2 multi-label counter (PerkinElmer). Exposition time was 0.5 s per well. Each sample was measured in 3 different wells and each well plate was measured three times. Luminescence intensity of treated samples was compared with reference samples.

Data in Figs. 2, 3, 4, 5 and 6 were calculated as percentages of baseline levels (100%). All data are given as means ± S.E.M. of N experiments and were analyzed by one-way ANOVA (GraphPadR Prism 5.03) with Bonferroni’s post-test. One-way ANOVA followed by Dunnett’s test was used for the ATP data (Fig. 6). P values < 0.05 were considered to be statistically significant.

Oxygen consumption by complex I in isolated brain mitochondria in the presence of isoflurane, sevoflurane, and halothane. All data points are means ± S.E.M. of N = 4–5 experiments. Statistics: one-way ANOVA for each drug with Dunnett’s post-test vs. controls, all data points are significantly different from control incubations (100 ± 2.3%, N = 12)

Oxygen consumption by complex I in isolated brain mitochondria in the presence of ketamine, propofol, and pentobarbital. All data points are means ± S.E.M. of N = 4 experiments. Statistics: one-way ANOVA for each drug with Dunnett’s post-test vs. controls (100 ± 2.6%, N = 12). **, P < 0.01 vs. control incubations without drug

Oxygen consumption by complex II in isolated brain mitochondria in the presence of six anesthetic drugs. (A) Inhibition of respiration by propofol. (B) Non-significant effects of the remaining five anesthetic drugs. All data points are means ± S.E.M. of N = 4 experiments. Statistics: one-way ANOVA with Dunnett’s post-test vs. controls (100 ± 2.1%, N = 12). *, P < 0.05; **, P < 0.01 vs. control incubations without drug

Oxygen consumption in isolated brain mitochondria in the presence of six anesthetic drugs. (A) Increase of respiration after addition of cytochrome c. (B) Non-significant effects of the six anesthetic drugs on complex IV activity. In this graph, the following concentrations are shown: isoflurane 1.2 mM, sevoflurane 3.42 mM, halothane 4.26 mM, pentobarbital 360 µM, ketamine 7.8 µM, and propofol 254 µM. All data points are means ± S.E.M. of N = 4 experiments. Statistics: one-way ANOVA with Dunnett’s post-test vs. controls (100 ± 1.2%, N = 12). *, P < 0.05; **, P < 0.01 vs. control incubations without drug

Formation of ATP in isolated brain mitochondria in the presence of six anesthetic drugs. The ATP assay was performed as described in Methods. In this graph, the following concentrations are shown: isoflurane 1.2 mM, sevoflurane 3.42 mM, halothane 4.26 mM, pentobarbital 360 µM, ketamine 7.8 µM, and propofol 254 µM. All data points are means ± S.E.M. of N = 4 experiments. Statistics: one-way ANOVA with Dunnett’s post-test vs. controls (Ctr: 100 ± 1.2%, N = 12). *, p < 0.05; **, p < 0.01 vs. control incubations without drug