Distinct differences in rates of oxygen consumption and ATP synthesis of regionally isolated non‐synaptic mouse brain mitochondria

Brain mitochondrial dysfunction has been implicated in several neurodegenerative diseases. The distribution and efficiency of mitochondria display large heterogeneity throughout the regions of the brain. This may imply that the selective regional susceptibility of neurodegenerative diseases could be mediated through inherent differences in regional mitochondrial function. To investigate regional cerebral mitochondrial energetics, the rates of oxygen consumption and adenosine‐5′‐triphosphate (ATP) synthesis were assessed in isolated non‐synaptic mitochondria of the cerebral cortex, hippocampus, and striatum of the male mouse brain. Oxygen consumption rates were assessed using a Seahorse XFe96 analyzer and ATP synthesis rates were determined by an online luciferin‐luciferase coupled luminescence assay. Complex I‐ and complex II‐driven respiration and ATP synthesis, were investigated by applying pyruvate in combination with malate, or succinate, as respiratory substrates, respectively. Hippocampal mitochondria exhibited the lowest basal and adenosine‐5′‐diphosphate (ADP)‐stimulated rate of oxygen consumption when provided pyruvate and malate. However, hippocampal mitochondria also exhibited an increased proton leak and an elevated relative rate of oxygen consumption in response to the uncoupler carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP), showing a large capacity for uncoupled respiration in the presence of pyruvate. When the complex II‐linked substrate succinate was provided, striatal mitochondria exhibited the highest respiration and ATP synthesis rate, whereas hippocampal mitochondria had the lowest. However, the mitochondrial efficiency, determined as ATP produced/O2 consumed, was similar between the three regions. This study reveals inherent differences in regional mitochondrial energetics and may serve as a tool for further investigations of regional mitochondrial function in relation to neurodegenerative diseases.


| INTRODUC TI ON
The mammalian brain is a complex and energy-demanding organ. It is estimated that 70%-80% of the total energy expenditure of the brain is accounted for by processes related to neuronal signaling (Attwell & Laughlin, 2001;Harris, Jolivet, & Attwell, 2012). This means that a constant production of energy is crucial for sustaining cerebral function. Mitochondria are cellular structures responsible for the majority of energy production through oxidative phosphorylation, which is the oxygen-dependent production of adenosine-5′-triphosphate (ATP) (Figure 1a) (Nunnari & Suomalainen, 2012). Inside the mitochondrial matrix, metabolic substrates are oxidized in the tricarboxylic acid (TCA) cycle, generating energy rich electrons, transported through the reduced carriers NADH and FADH 2 . These electrons are subsequently transferred through the electron transport chain (ETC), consisting of distinct protein complexes (I-IV), to generate a proton motive force by pumping protons out of the mitochondrial matrix, which drives the ATP synthase (also known as complex V) to synthesize ATP (Papa, Petruzzella, & Scacco, 2007).
A growing body of evidence suggests that brain mitochondrial dysfunction can mediate or accelerate the development of several neurodegenerative diseases (Correia et al., 2012;Johri & Beal, 2012;Nunnari & Suomalainen, 2012;Pathak, Berthet, & Nakamura, 2013). The brain consists of several well-defined areas, each handling different cerebral functions and with distinct susceptibility to neurodegenerative diseases (Camandola & Mattson, 2017;Masters et al., 2015;Poewe et al., 2017;Ross & Tabrizi, 2011). For example, Alzheimer's disease primarily affects the cerebral cortex and hippocampus, whereas Huntington's disease manifests predominantly in the striatum (Masters et al., 2015;Ross & Tabrizi, 2011). Furthermore, mitochondria found throughout the different brain regions and cell types display a large degree of heterogeneity in terms of distribution and efficiency (Dubinsky, 2009;Sonnewald, Hertz, & Schousboe, 1998). Collectively, these observations may imply that inherent regional differences in mitochondrial function could be the basis of regional susceptibility to brain diseases. An in-depth understanding of regional differences in mitochondrial bioenergetics may elucidate possible mechanisms for the selective neurodegeneration.
Cerebral mitochondrial function can be investigated using several experimental approaches and a common preparation is isolated mitochondria (Clark & Nicklas, 1970;Lai, Walsh, Dennis, & Clark, 1977). The use of isolated mitochondria allows a high degree of control and manipulation by circumventing cellular pathways.
Previous studies have primarily been performed on isolated mitochondria from the rat brain, due to the larger amount of starting material when compared to mice (Clark & Nicklas, 1970;Dagani, Marzatico, & Curti, 1988;Lai et al., 1977;Sims, 1990). However, recent technological advances have allowed high-throughput microplate-based assessment of small quantities of mitochondria, making it possible to run several conditions in parallel (Barros et al., 2018;Connolly et al., 2018;Rogers et al., 2011;Sauerbeck et al., 2011). Here, we apply two such assays to investigate the rate of oxygen consumption and importantly link this to the rate of ATP production, in isolated non-synaptic mitochondria of the mouse brain from three areas often affected by neurodegeneration: the cerebral cortex, hippocampus, and striatum. In addition, using two different mitochondrial fuels, pyruvate and succinate, we dissect regional mitochondrial function mediated through complex I and complex II of the ETC.
All other reagents were of the purest grade available from regular commercial sources.

| Animals
Six male NMRI mice (Envigo, Cambridgeshire, United Kingdom) of 12 weeks of age (weight: 42.2 g ± 3.4 g) were housed in a pathogen-free, temperature and humidity-controlled environment at the Department of Drug Design and Pharmacology, University of Copenhagen. The mice were acclimatized for 2 weeks before experiments and were single-housed in individually ventilated cages with free access to chow and water. The experiments were approved by the Danish National Ethics Committee and performed in accordance with the European Convention (ETS 123 of 1986). Mitochondrial function is affected by sex steroids (Gaignard et al., 2017). To avoid introducing this potential confounding variation, all experiments were performed on male mice and hence sex specific aspects of the work could not be evaluated.

Significance
Brain mitochondrial dysfunction is implicated in several neurodegenerative diseases and might be linked to inherent regional differences in mitochondrial function. This study reports several differences in the rates of oxygen consumption and ATP synthesis of mitochondria isolated from the mouse cerebral cortex, hippocampus, and striatum. These findings may serve as a foundation for further investigations of the link between regional mitochondrial function and neurodegeneration.

| Cerebral mitochondrial isolation
Isolation of non-synaptic mitochondria from the cerebral cortex, hippocampus, and striatum was performed as described in Chinopoulos, Zhang, Thomas, Ten, and Starkov (2011) with slight modifications. All procedures were performed on ice or at 4°C. Mice were euthanized by cervical dislocation, decapitated and the brain quickly excised from the cranial vault onto a cooled surface covered by a damp filter paper. The brain was dissected into the three distinct regions: cerebral cortex, hippocampus, and striatum as described by Spijker (2011). Tissue was dissected from both hemispheres with a total dissection time of approximately 3 min. After dissection, the tissue was submerged in isolation buffer (MSHE), containing in mM: mannitol 210, sucrose 70, HEPES 5, EGTA 1 and 0.5% BSA (fatty acid free), pH 7.2, and gently homogenized using a Teflon on glass douncer at 500 revolutions/min for 7-8 strokes. The homogenate was centrifuged (500 g × 5 min) to pellet cell remnants. The supernatant was centrifuged (14.000 g × 10 min) and the pellet re-suspended in 12% Percoll MSHE solution. This suspension was gently layered on a 21% Percoll MSHE solution and centrifuged (18.000 g × 15 min).
The supernatant was discarded and the pellet was washed with two subsequent centrifugations (18.000 g × 5 min and 14.000 g × 5 min) by suspending the pellet in MSHE buffer and discarding the supernatant after each centrifugation. The final pellet, containing the isolated mitochondria, was re-suspended and diluted in MSHE and protein amounts were determined using the Bradford protein assay with BSA as standard protein.
The applied experimental template is known as the mitochondrial coupling assay, investigating the coupling between the ETC and oxidative phosphorylation (Rogers et al., 2011). Each experiment consisted of nine measurements cycles, with intermediate injections of different mitochondrial modulators as depicted in Figure 1b. Each measurement cycle lasted 3 min and was followed by 2 min of mixing and 1 min of rest. Firstly, three baselines measurements were performed before addition of pharmacological compounds. After injection of a compound, 1 min of mixing was performed before the measurement cycle was initiated. The compounds added were (all in final concentrations): ADP (4 mM), oligomycin A (2.5 µg/ml), FCCP (4 µM) and antimycin A (4 µM). Firstly, ADP was injected, which stimulates ATP synthesis and thus complex V-driven respiration. Subsequently, oligomycin A, an inhibitor of complex V, was added to terminate the ADPmediated respiration. The uncoupler FCCP allows protons from the intermembrane space to flow into the matrix (Figure 1a), stimulating uncoupled respiration. Finally, antimycin A was injected to inhibit the flow of electrons from complex III to complex IV, thus leaving only residual non-mitochondrial oxygen consumption, which was measured for three measurement cycles. The Seahorse assay works by forming a transient micro-chamber by bringing oxygen-sensitive probes close to the mitochondria in the wells. Due to this, concentrations of respiratory substrates and mitochondrial modulators may seem high when compared to other mitochondrial assays. The applied amount of mitochondria, concentrations of respiratory substrates, and injected compounds (saturating concentrations) were all verified as optimal in our setup. It was assured that O 2 was not depleted during the measurement period and that the O 2 levels reached baseline values before the next measurement cycle. Data were collected and extracted using the Wave software (Agilent Technologies, CA, USA).

| Mitochondrial ATP synthesis-Luciferinluciferase assay
The rate of ATP synthesis was measured in the isolated mitochondria with an online luciferin-luciferase coupled luminescence assay using a NOVOstar microplate reader at 28°C for optimal luciferinluciferase response (BMG Labtech GmbH, Ortenberg, Germany) (Jakobsen et al., 2018). Mitochondria were diluted in respiration buffer, containing in mM: sucrose 250, K 2 PO 4 15, MgSO 4 2, EDTA 0.5 and 0.5% BSA (fatty acid free), pH 7.2. 10 µl of mitochondrial suspension containing 2.5 µg of protein was added to a clear bottom 96-well plate, containing the respiratory substrates: 10 mM pyruvate in combination with 2 mM malate or 10 mM succinate and 2 µM rotenone (all final concentrations and pH 7.2). To inhibit adenylate cyclase activity, all wells also contained 0.2 µM P 1 ,P 5 -Di(adenosine-5′)pentaphosphate pentasodium. A luciferin-luciferase cocktail and hexokinase-purified ADP (final concentration 0.6 mM) was added to each well and luminescence was assayed for 36 s followed by injection of 9.62 µM ATP (final concentration) as internal standard. Each condition was performed in four technical replicates. As a negative control, some wells were added 2.5 µg/ml oligomycin A, to inhibit ATP synthesis (data not shown).
Mitochondrial ATP synthesis is presented as pmol ATP produced/ min/µg protein. As a measure of mitochondrial efficacy, that is, ATP production in relation to O 2 consumption, an ATP/O 2 ratio was calculated as the rate of ATP production divided by the ADP-stimulated OCR determined by Seahorse analyses.

| Mitochondrial membrane potential-TMRM assay
To monitor the mitochondrial membrane potential (ΔΨ m ), tetramethylrhodamine methyl ester (TMRM) was added (1 µM, final concentration) to 1 µg of regionally isolated mitochondria and measured in quench mode (530 nm excitation/590 nm emission) in a NOVOstar microplate reader at 28°C (BMG Labtech GmbH, Ortenberg, Germany) (Jakobsen et al., 2018;Nicholls & Ward, 2000). After a stable baseline was reached (30 min), oligomycin A (5 µg/ml µM, final concentration) was added to assess the mitochondrial proton leak ( Figure 1b). Measurements were performed in a clear bottom 96well plate, in the same respiration buffer used for the ATP synthesis assay, containing 10 mM pyruvate in combination with 2 mM malate or 10 mM succinate and 2 µM rotenone, both conditions with 0.2 µM P 1 ,P 5 -Di(adenosine-5′)pentaphosphate pentasodium (all final concentrations and pH 7.2). Traces of mitochondrial membrane potential are presented as ΔΨ m (F 0 /F), in which data points were normalized to the baseline levels obtained within the first 5 min of measurements.

| Study design and handling of data
This study was designed as an exploratory study with no allocation of subjects into experimental groups, hence no blinding or randomization were applied. Data shown represent means ± standard deviations (unless other is noted), with individual data points plotted as well. The data are based on six individual experiments (biological replicates, i.e., from individual animals), except for the ATP measurements with pyruvate/malate where only four data points were obtained due to an experimental defect in the enzymatic assay. In some instances, negative values were obtained due to methodological errors. All negative values were removed prior to analysis. If the amount of negative values exceeded more than half of the technical replicates the data point was omitted (one incidence: RCR and proton leak with pyruvate/malate). Sample sizes were estimated based on a pilot study and previous experiments from which it has been observed that the variability and validity of data is optimal as indicated by standard errors generally below 10% of the mean values.

| Pyruvate/malate-Complex I-driven respiration and ATP synthesis
Pyruvate is the end-product of glycolysis and a major mitochondrial fuel (Sonnewald et al., 1998). To investigate NADH-linked, that is, complex I-mediated, respiration and ATP synthesis, isolated mitochondria were provided pyruvate and malate as respiratory substrates. The provided malate enables formation of oxaloacetate needed for oxidation of acetylCoA, generated through activity of pyruvate dehydrogenase (PDH), in the TCA cycle ( Figure 1a).
Furthermore, the OCR of cerebral cortical mitochondria was lower than striatal mitochondria (F 2,10 = 88.17, p = 0.0015), which had the highest basal OCR. When ADP was added, it was again observed that hippocampal mitochondria had a lower OCR when compared to cerebral cortical (F 2,10 = 70.03, p < 0.0001) and striatal (F 2,10 = 70.03, F I G U R E 1 Mitochondrial bioenergetics. (a) Mitochondrial overview. Mitochondria are double membrane enclosed structures, consisting of an inner and an outer membrane, separating the cytosol, the intermembrane space and the mitochondrial matrix. In the tricarboxylic acid (TCA) cycle, located inside the mitochondrial matrix, substrates can be oxidized to produce the reduced electron carriers NADH and FADH 2 . In the inner mitochondrial membrane, protein complexes (designated I-IV) are embedded, collectively known as the electron transport chain (ETC). Electrons from NADH are transferred to complex I, whereas electrons from FADH 2 are transferred to complex II. The enzyme converting succinate to fumarate, succinate dehydrogenase (SDH), is an integral part of complex II. The flow of electrons through the ETC, drives the expulsion of protons (H + ), through complex I, III, and IV, out of the mitochondrial matrix into the intermembrane space, creating a proton motive force. In complex IV, electrons are neutralized by O 2 . The proton gradient, generated by the ETC, can be utilized by the ATP synthase (complex V) to convert ADP into ATP. The entire process with oxidative metabolism of substrates, ETC activity, oxygen consumption, and ATP synthesis is known as oxidative phosphorylation. To investigate mitochondrial function in isolated mitochondria, pyruvate in combination with malate or succinate can be provided as substrates. Pyruvate is converted into acetylCoA by activity of pyruvate dehydrogenase (PDH). The provided malate will form oxaloacetate needed to oxidize acetylCoA units in the TCA cycle. Since most of the reactions in the TCA cycle form NADH, oxidation of pyruvate in combination with malate, is mediated primarily via complex I. Succinate can also be provided as a metabolic substrate and will generate FADH 2 directly for the ETC via SDH activity. Since oxaloacetate is a powerful inhibitor of SDH, it is customary to add rotenone, a complex I inhibitor, to the media, to ensure that succinate respiration is not inhibited and only reflects complex II activity. In coupled mitochondria it is the level of ATP that decides the activity of the ETC, that is, there is a coupling between ATP synthesis and ETC activity. However, protons can also cross the inner mitochondrial matrix independently of complex V, which is known as the proton leak. Certain compounds known as mitochondrial uncouplers, can greatly increase this flow of protons into the mitochondrial matrix bypassing complex V, which will induce uncoupled respiration. (b) Seahorse coupling assay. The Seahorse coupling assay can be used to assess critical mitochondrial parameters including coupled and uncoupled oxygen consumption rates (OCR) and proton leak. Firstly, three basal measurements are performed (1-3) in the presence of the respiratory substrates. ADP is then added to the mitochondria which will induce coupled respiration by stimulating ATP synthesis (4). To inhibit coupled respiration, oligomycin A, a complex V inhibitor is added to the mitochondria (5). FCCP is a mitochondrial uncoupler, allowing protons to cross the inner mitochondrial membrane and induce uncoupled respiration, circumventing complex V (6). Finally, antimycin A, an inhibitor of complex III, is added to assess non-mitochondrial respiration (7-9). Arrows indicate addition of the respective compounds. The data shown are from cerebral cortical mitochondria provided succinate (in the presence of rotenone) as respiratory substrate, mean ± standard deviation, n = 6 TA B L E 1 Oxygen consumption rates (OCRs), ATP synthesis rates, and respiratory ratios of regionally isolated mitochondria with pyruvate and malate as substrates p < 0.0001) mitochondria (Figure 2b). Due to the larger basal respiration of the striatal mitochondria, a significant decrease in relative OCR, in response to ADP, was observed when compared to cerebral cortical mitochondria (F 2,10 = 6.729, p = 0.0118).

Hippocampus
Uncoupled respiration, that is, respiration circumventing complex V, induced by FCCP showed no significant differences between the three regions ( Figure 2c). However, the hippocampal mitochondria showed the greatest relative increase in OCR in response to FCCP when compared to mitochondria of the cerebral cortex (F 2,10 = 30.83, p = 0.0006) and striatum (F 2,10 = 30.83, p < 0.0001). To assess the overall mitochondrial function, the RCR was calculated. It was observed that hippocampal mitochondria had No changes were found in the ATP production rate of the three regions ( Figure 2g). To further evaluate the mitochondrial efficiency of substrate oxidation, that is, how much ATP is produced relative to oxygen consumption, a ratio between ATP production and oxygen consumption was calculated (Figure 2h). Here, it was observed that striatal mitochondria exhibited a slightly reduced ATP/O 2 ratio when compared to hippocampal mitochondria (F 2,6 = 5.749, p = 0.0342).

| Succinate-Complex II-driven respiration and ATP synthesis
Complex II-driven respiration and ATP synthesis can be assessed by providing the mitochondria with succinate as respiratory substrate.
Succinate is converted into fumarate by activity of succinate dehydrogenase (SDH), which is part of complex II and hereby directly provide FADH 2 to the ETC (Figure 1a). To ensure that succinate oxidation reflects complex II, that is, FADH 2 -mediated respiration, the complex I inhibitor rotenone was included in the respiration buffer. Rotenone inhibits the oxidation of NADH to NAD + , which will block the conversion of malate to oxaloacetate as this reaction reduces NAD + to NADH (Figure 1a). Since oxaloacetate is a powerful inhibitor of SDH, the addition of rotenone will increase the oxidation of succinate (Papa, Lofrumento, Paradies, & Quagliariello, 1969;Wojtczak, Wojtczak, & Ernster, 1969).
Furthermore, the hippocampal mitochondria exhibited a decreased OCR when compared to cerebral cortical mitochondria (F 2,10 = 30.63, p = 0.0204). The differences in relative OCR upon ADP stimulation were of a much smaller magnitude. However, it was observed that the relative OCR of the hippocampal mitochondria again was decreased when compared to mitochondria of the cerebral cortex (F 2,10 = 12.80, p = 0.0058) and striatum (F 2,10 = 12.80, p = 0.0024).
However, from measurements of the mitochondrial membrane potential, it was observed that hippocampal mitochondria exhibited a lower membrane potential in the presence of oligomycin A, indicative of an elevated proton leak, when compared to both mitochondria of the cerebral cortex and striatum (Figure 3f).
However, the ATP production was balanced by oxygen consumption, signified by equal ATP/O 2 ratios for all regions (Figure 3h).

| D ISCUSS I ON
Here, we investigated the rates of oxygen consumption and ATP synthesis, in isolated mitochondria of the mouse cerebral cortex, hippocampus, and striatum using different respiratory substrates. We provide functional evidence of heterogeneity in mitochondrial energetics between the three brain regions investigated. When pyruvate and malate were provided as substrates, hippocampal mitochondria exhibited the lowest basal and coupled OCR, however, with comparable efficiency of ATP synthesis to the other regions. Hippocampal mitochondria also exhibited a larger proton leak and elevated relative OCR in response to FCCP, showing a large capacity for uncoupled respiration. When the complex II-linked substrate succinate was provided, striatal mitochondria exhibited the highest respiration and ATP synthesis rate, whereas hippocampal mitochondria had the lowest. However, equal ATP/O 2 ratios between the regions suggests similar mitochondrial efficiency of succinate oxidation.

| Mitochondrial isolation and function
In this report, cerebral mitochondria were isolated using a Percoll density gradient, allowing separation of non-synaptic mitochondria from other cellular components, including myelin and cellular debris, but also unwanted metabolic active entities such as synaptosomes (Chinopoulos et al., 2011;Sims, 1990). Isolation of mitochondria allows tight experimental control and the possibility of direct manipulation, but may compromise other physiological factors, such as structure, intra-cellular connections, and signaling pathways (Brand & Nicholls, 2011;Picard, Taivassalo, Gouspillou, & Hepple, 2011). To assess the integrity of isolated mitochondria, the RCR is a commonly applied relative measure that describes overall mitochondrial function (Brand & Nicholls, 2011). We obtained RCRs values in the range of 3-7 with pyruvate/malate and 2-4 with succinate as substrate.
Compared to the literature, these values signify well-functioning mitochondria, validating the quality of our preparation (Burtscher, Zangrandi, Schwarzer, & Gnaiger, 2015;Cardoso, Santos, Seica, & Moreira, 2010;Czerniczyniec et al., 2015;Dagani et al., 1988;Lai et al., 1977;Sauerbeck et al., 2011;Sims, 1990;Varela, Schwartz, & Horvath, 2016). We observed that hippocampal mitochondria exhibited a larger proton leak, with both substrate conditions, resulting in reduced RCR values for this region. This is an important notion, as the choice of substrate should not alter the RCR (Brand & Nicholls, 2011). However, since succinate oxidation does not contribute to the generation of the proton motive force through complex I of the ETC, the RCR values often tends to be lower when compared to NADHlinked substrates, which was also the case in the present study (Brand & Nicholls, 2011). The isolation technique used for this study yields non-synaptic mitochondria originating from both neurons and glial cells. The Percoll isolation yields a mitochondrial fraction with a high purity (Chinopoulos et al., 2011;Sims, 1990), however, some contamination of membrane proteins and synaptosomes will still be present. It should therefore be noted that the isolated mitochondrial fractions are derived from heterogeneous cellular populations and might exhibit different degrees of contamination, which potentially could have influenced the results.

| Regional differences in mitochondrial OCR and ATP synthesis rate
Only few other studies, using Seahorse technology to assess mitochondrial function in regionally isolated brain mitochondria, have been performed. A study by Sauerbeck et al. showed that mitochondria isolated from the rat cerebral cortex, hippocampus, and striatum exhibited equal coupled OCRs with pyruvate/malate, and the same uncoupled OCRs with succinate in the presence of rotenone (Sauerbeck et al., 2011). Interestingly, the authors also reported a trend toward a larger residual respiration in the presence of oligomycin A, that is, proton leak, of striatal and hippocampal mitochondria (Sauerbeck et al., 2011). Here, we observed an increased proton leak in isolated hippocampal mitochondria of the mouse brain, however, this was not the case in the striatal mitochondria. Another recent study, likewise performed in rats, showed that regional mitochondrial function fluctuates with age (Pandya, Royland, MacPhail, Sullivan, & Kodavanti, 2016). The authors reported that cerebral cortical, striatal, and hippocampal mitochondria, isolated from young rats, exhibited comparable bioenergetics profiles, however, particularly hippocampal mitochondrial function declined with increasing age (Pandya et al., 2016). A relevant extension of this study could include assessment of how regional mitochondrial function in mice is related to age.
We found that mitochondria of the hippocampus, generally consumed oxygen and synthesized ATP at lower rates, when compared to mitochondria of the other two regions. These observations of low hippocampal oxygen consumption is in line with several other reports of isolated mitochondria (Burtscher et al., 2015;Cardoso et al., 2010;Czerniczyniec et al., 2015;Dagani et al., 1988;Sims, 1990;Varela et al., 2016) and brain homogenates (Sims, Finegan, & Blass, 1986;Sims & Pulsinelli, 1987). However, it was also observed that hippocampal mitochondria exhibited comparable ATP/O 2 ratios to the other regions, suggesting an overall general lower mitochondrial activity in this region (Dagani et al., 1988). A striking finding of this study is the hippocampal capacity of uncoupled respiration when provided pyruvate and malate. Even though both the basal and ADP-induced OCR of the hippocampal mitochondria were lower, the absolute uncoupled respiration was equal across the regions, leading to a much larger relative OCR increase. This elevated hippocampal uncoupling capacity has also been observed in hippocampal synaptosomes fueled by glucose (Lee et al., 2013), and shows that hippocampal mitochondria possess the same capacity for maximal uncoupled respiration as the other regions, which is not active during coupled respiration.
We also observed that the RCR of hippocampal mitochondria were significantly lower when compared to the other regions, due to an elevated proton leak across the inner mitochondrial membrane. The proton leak constitute a large fraction of the resting mitochondrial metabolic rate but the mechanisms and functions are still unclear (Jastroch, Divakaruni, Mookerjee, Treberg, & Brand, 2010). The proton leak is affected by the mitochondrial membrane composition, including fatty acid composition and expression of transporters and proteins (Brand, Chien, Ainscow, Rolfe, & Porter, 1994;Jastroch et al., 2010;Stuart, Cadenas, Jekabsons, Roussel, & Brand, 2001). One group of proteins, the uncoupling proteins (UCPs), allows protons to cross the inner mitochondrial membrane and are involved in the proton leak (Jastroch et al., 2010). The functions of the UCPs in the brain are still unclear, but they influence mitochondrial function, calcium handling, and production of reactive oxygen species (Andrews, Diano, & Horvath, 2005 (Beal et al., 1993;Brouillet et al., 1995).
Our results of elevated basal respiration and increased capacity of succinate oxidation of striatal mitochondria are in line with this and support that SDH impairments may be a sensitive target of neurodegeneration in the striatum.

| Complex I-and II-mediated OCR and ATP synthesis rate
When comparing the applied respiratory substrate conditions, pyruvate/malate versus succinate, reflecting complex I and complex II-linked respiration, respectively, the absolute differences in OCRs are striking. The OCRs were found to be approximately fourfold higher for succinate when compared to pyruvate/malate for all regions. This observation is in accordance with previous reports (Clark & Nicklas, 1970;Iuso et al., 2017;Rogers et al., 2011), and is explained by succinate providing electrons for the ETC with only one enzymatic reaction through SDH with subsequent FADH 2 generation. Pyruvate, on the other hand, must undergo several consecutive enzymatic steps in order to provide NADH for the ETC.
However, the ATP synthesis rate was not increased to the same extent as the OCR, which became apparent by the ATP/O 2 ratios, which were threefold reduced for succinate when compared to pyruvate/malate. This notion is in line with the classic phosphate/ oxygen (P/O or ADP/O) ratios, describing how many moles ATP are synthesized/0.5 moles O 2 consumed (Hinkle & Yu, 1979). The standard P/O ratios are estimated to be 2.5 for NADH-linked substrates (such as pyruvate in combination with malate) and 1.5 for succinate (Hinkle, 2005;Hinkle & Yu, 1979

| Conclusions
This study reveals clear differences in OCR and ATP synthesis rates, mediated through complex I and complex II of the ETC, in isolated mitochondria from cerebral cortex, hippocampus, and striatum of the mouse brain. We found that mitochondrial efficiency, that is, ATP synthesis/O 2 consumed, was comparable between the three regions. Hippocampal mitochondria exhibited a larger proton leak, whereas striatal mitochondria had an elevated basal respiration and increased capacity for succinate oxidation. This study underlines the importance of investigating cerebral mitochondrial function regionally, as regional differences may by masked when multiple regions are pooled. Furthermore, we advocate to asses OCR in combination with ATP synthesis rate, as either one alone can be misleading.

D ECL A R ATI O N O F TR A N S PA R EN C Y
The authors, reviewers, and editors affirm that in accordance to the policies set by the Journal of Neuroscience Research, this manuscript presents an accurate and transparent account of the study being reported and that all critical details describing the methods and results are present.

ACK N OWLED G M ENTS
JVA gratefully acknowledge the financial support of the Scholarship of Peter & Emma Thomsen.

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflict of interests.

DATA ACCE SS I B I LIT Y
Data can be provided via e-mail upon request.