Measurement of the oxygen consumption rate (OCR)

Oxygen consumption is an excellent read-out for mitochondrial respiratory activity. Measurement of the oxygen consumption rate (OCR) is the current experiment of choice to determine underlying mitochondrial dysfunction [36, 37]. Although Complex IV is the only oxygen consumer within the proton circuit, appropriate pharmacological manipulation can isolate different respiratory states and facilitate modularised assessment of the complete circuit, including basal respiration, maximal respiration, proton (H⁺) leak, and ATP turnover (Fig. 2a).

a Mitochondrial function can be thoroughly investigated in intact cells by measuring the OCR during sequential addition of mitochondrial respiratory inhibitors (marked with grey triangles). The different stages of the experiment (i)–(iv) and the measured parameters (a)–(f) are described in Protocol 1. The addition of pharmacological compounds or fuel substrates prior to oligomycin (not shown here) can capture further detail regarding the OCR. b Illustration of the effects of relevant pharmacological compounds on the mitochondrial respiratory chain, proton (H⁺) leak across the mitochondrial inner membrane, and the F₁F_o ATP synthase, during the experimental stages marked as (i)–(iv) in A. O₂ in the mitochondria is consumed by the respiratory chain through the activity of Complex IV. (i) In the basal state, mitochondrial O₂ consumption is predominantly driven by H⁺ flux through the F₁F_o ATP synthase. (ii) Inhibition of the F₁F_o ATP synthase with oligomycin reduces mitochondrial O₂ consumption, with the OCR in this phase predominantly driven by the H⁺ leak (but also by substrate oxidation). (iii) Addition of an uncoupler such as FCCP or CCCP increases the H⁺ leak across the inner membrane, creating a H⁺ short circuit and facilitating the measurement of maximal OCR. The optimal FCCP/CCCP concentration to induce maximal respiration should be determined for each experimental setting (details in Protocol 1), and it is advisable to also assess maximal respiratory capacity in the absence of oligomycin. (iv) Inhibition of respiratory chain activity with Rotenone and/or Antimycin A ablates mitochondrial O₂ consumption. Any O₂ consumption measured in this phase is due to non-mitochondrial O₂ consumption. Rot rotenone, AA antimycin A, Oligo oligomycin, H⁺ proton, IMS intermembrane space. *Respiration in stage (b) is predominantly driven by H⁺ leak, but also by substrate oxidation

The OCR has been extensively studied in various cellular models of NDs [7, 38, 39]. It can be measured in isolated mitochondria or permeabilised cells following a slightly altered protocol to the one described below (initial addition of ADP, phosphate, and substrate to initiate pure ‘State 3’ respiration [36]), or in intact cells or brain slices. Mitochondria isolation is a delicate procedure that provides a precise and controllable model at the expense of physiological relevance, while brain slices, which maintain intact neuronal networks, constitute a more complete biological system. In whole cells, optimised permeabilisation of the plasma membrane allows controlled supply of substrate to mitochondria, providing a more controllable model without complete loss of the cytosolic milieu [40–42]. The measurement of OCR in permeabilised cells or isolated mitochondria provided with different substrates (e.g. the provision of glutamate/malate to drive flux through Complex I, or succinate to drive flux through Complex II) can isolate specific complex activity and help to identify the molecular origin of a mitochondrial defect [41].

Conventional Clark-type oxygen electrode chambers (e.g. Hansatech Oxygraph), which can measure oxygen (O₂) in large numbers of cells, isolated mitochondria or tissue homogenates in suspension [43], have been replaced more recently by cell respirometers, which perfuse buffer over live, attached cells within a sealed chamber, a set-up more suitable for use with primary neurons [40, 44]. Chambers mounted on fluorescence microscopes allow simultaneous measurement of other fluorescent indicators [44]. The multi-well plate reader from Agilent Technologies (Seahorse XF Flux Analyser) can simultaneously measure both the OCR and the extracellular acidification rate (ECAR; a read-out that allows calculation of lactate release under certain conditions and therefore the rate of anaerobic glycolysis—more details are provided at the end of Section 5.2.2), although experiments are expensive and limited to non-perfused cell population measurements [45]. The Oroboros Oxygraph-2k system (O2k; Oroboros Instruments) can measure O₂ consumption in cell suspensions simultaneously to other parameters, such as the mitochondrial membrane potential or the ADP–ATP exchange rate mediated by the adenine nucleotide translocator (ANT) [46], but it is labour intensive and low-throughput. An overview of these two commercial systems is provided in [47]. Luxcel Bioscience’s MitoXpress^® Xtra plate-reader assay allows population-level O₂ measurements to be multiplexed with other reporters, such as indicators of cell viability [48].

Regardless of equipment, the OCR is generally inferred by measuring the levels of dissolved O₂ in the chamber/well over time, using polarographic O₂-sensing electrodes or fluorescent/phosphorescent reporters. The Seahorse system, for instance, utilises solid-state fluorescence-based sensors to measure extracellular O₂ levels within a sealed chamber for 2−6 min. The chamber is then unsealed (allowing the O₂ to re-equilibrate to atmospheric levels) and re-sealed to repeat the measurement. Intracellular O₂−sensing probes include nanoparticles based on the phosphorescent dye Pt(II)-tetrakis(pentafluorophenyl)porphine (PtTFPP; MitoXpress^®-Intra, Luxcel Biosciences), which require phosphorescence lifetime measurements and can be detected at single-cell level or on plate readers with time-resolved fluorescence/phosphorescence detection. This probe can provide quantitative intracellular O₂ measurements in neurons and brain slices [49, 50].

We here describe the most commonly deployed experimental protocol to thoroughly investigate mitochondrial bioenergetic function by measuring the OCR in intact primary neurons (Fig. 2 and Protocol 1). This protocol can be followed independently of the measurement technique.

Protocol 1: Investigating mitochondrial function in primary mouse cortical neurons by measuring the oxygen consumption rate in the presence of various inhibitors of the mitochondrial respiratory chain.

Primary cortical neurons preparation and culture

Prepare cortical neurons from post-natal (day 0–1) or embryonic (day 16–18) mice of either sex [21, 22].

Seed neurons at appropriate density on pre-washed, poly-D-lysine (and/or laminin)-coated dishes suitable for OCR measurements (e.g. 100,000–300,000 cortical neurons/well in 24-well Seahorse cell culture microplates if using Seahorse XF Flux Analyser).

Culture neurons in appropriate media. Neurobasal medium 21103-049 is commonly used, supplemented with 0.5 mM l-glutamine and 2% B27. It should be noted that this media contains supraphysiological glucose levels (25 mM).

Perform experiments after at least 8 days in vitro.

Performing the experiment

Exchange culture media for appropriate ‘experimental buffer’ (wash neurons once to ensure complete exchange). Example buffer composition (in mM): 120 NaCl, 3.5 KCl, 0.4 KH₂PO₄, 5 NaHCO₃, 20 HEPES, 1.2 Na₂SO₄, pH 7.4 (NaOH), supplemented with 1.2 CaCl₂, 1–2 MgCl₂ and desired substrate (e.g. 2.5–5 mM glucose). Equilibrate cells for 1 h at 37 °C with no CO₂.

Different components of the proton circuit exert varied control over mitochondrial O₂ consumption. Sequential addition of specific mitochondrial inhibitors isolates these components. Such an experiment involves several stages ((i)–(iv); refer to Fig. 2):

(i) The initial OCR is a measure of mitochondrial (a) and non-mitochondrial (f) O₂ consumption, and is predominantly driven by ATP turnover (H⁺ flow through the F₁F_o ATP synthase), and to a lesser extent by H⁺ leak and substrate oxidation (activity of the respiratory complexes). Differences in basal O₂ consumption (in the same cellular microenvironments) can suggest: (1) altered ATP consumption, (2) altered ATP synthesis (F₁F_o ATP synthase activity), (3) disrupted transport of adenine nucleotides (ANT) or phosphate between matrix and cytoplasm, (4) altered synthesis or consumption of reducing equivalents within the matrix by substrate oxidation or the respiratory chain, respectively, (5) disrupted substrate supply to the matrix, or (6) disrupted non-mitochondrial O₂ consumption. Measurement of the additional parameters below can further elucidate the contributing factors.

(ii) Inhibiting the F₁F_o ATP synthase with oligomycin allows the measurement of oligomycin-sensitive respiration driven by ATP turnover. Following oligomycin addition, the remaining mitochondrial O₂ consumption is predominantly controlled by the H⁺ leak across the inner membrane, and to a lesser extent by substrate oxidation. As H⁺ leak itself is voltage-dependent and oligomycin generally hyperpolarises mitochondria, these measurements will tend to over/underestimate the contribution of the H⁺ leak/ATP turnover, respectively, to O₂ consumption. Such errors may significantly impact findings if comparing systems with only subtle differences between them [36, 37]. Differences in the oligomycin-resistant respiration rate can indicate: (1) disrupted H⁺ leak (accompanied by mitochondrial membrane depolarisation), (2) altered substrate oxidation (accompanied by mitochondrial membrane hyperpolarisation), or (3) disrupted non-mitochondrial O₂ consumption.

(iii) Addition of an uncoupler (such as FCCP, CCCP or DNP) creates a H⁺ short-circuit across the mitochondrial inner membrane, decreasing the proton-motive force and allowing respiration to increase. In this state, substrate oxidation is the dominant controller of O₂ consumption. This is considered to be maximal respiratory capacity, although this measurement critically depends on the concentration of the uncoupler. Excess uncoupler can inhibit respiration and collapse the proton-motive force, disrupting transport processes facilitated by Δψ_m (e.g. the malate/aspartate shuttle, Ca²⁺ transport) or ΔpH (uptake of several metabolites). Cell density and buffer composition can also affect the concentration required to induce maximal respiration (albumin, for instance, can sequester FCCP). Vitally, therefore, the uncoupler concentration should be optimised for each experimental set-up – adding just enough to stimulate uncontrolled respiration while limiting the decrease in Δψ_m (Table 2; [36, 51]). Discrepancies in maximal respiratory capacity can indicate dysfunction in the respiratory complexes, or in cellular or mitochondrial substrate uptake/supply (processes upstream of the respiratory chain) if measured in intact cells. Be aware that cellular function relies heavily on adequate mitochondrial ATP production, and that the switch to glycolysis on addition of oligomycin can induce energy failure to such an extent that, regardless of uncoupler concentration, subsequent respiration levels are not an accurate measure of maximum respiratory capacity [52]. It is therefore advisable to also assess maximal respiratory capacity in the absence of oligomycin.

Drugs targeting the mitochondrial bioenergetic machinery

^aOligomycin concentrations are often listed as μg/ml, as commercial preparations are a mixture of compounds with different individual molecular weights

Concentrations are guidelines only for primary neurons, and should be optimised for each cell type or experimental setting. Changes to the experimental buffer, such as the inclusion of bovine serum albumin, can alter some of the effective drug concentrations by more than four-fold [40, 44]. High protonophore concentrations collapse the mitochondrial membrane potential (and may also depolarise the plasma membrane potential [62]), while low concentrations induce maximal respiration (this requires titration to determine the optimal concentration for each experimental set-up [36, 37]). References for concentrations were obtained from experiments in primary neurons: [53, 64, 102, 153, 154, 192–194]

3-NP 3-nitropropionic acid, FCCP carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, CCCP carbonyl cyanide m-chlorophenylhydrazone, DNP 2,4-dinitrophenol

(iv) Finally, inhibition of the respiratory complexes (commonly rotenone+antimycin A to inhibit complexes I and III, respectively, although antimycin A is likely sufficient [37]) measures O₂ consumption driven by non-mitochondrial processes, such as cytoplasmic NAD(P)H oxidases.

The addition of pharmacological compounds or fuel substrates prior to oligomycin can capture further detail [37]. Compounds to induce varying degrees of neuronal stimulation, such as gramicidin (permeabilises plasma membrane to monovalent cations), carbachol (acetylcholine receptor antagonist), or veratridine (inhibitor of Na⁺ channel inactivation) [53], increase ATP demand and introduce a ‘second hit’ that may be required to unveil underlying deficiencies not apparent in the basal, resting state. Addition of alternative fuel substrates such as ketone bodies or amino acids can help to investigate fuel dependence and metabolic flexibility [54].

Experiment analysis

Measurement of the OCR in this way allows calculation of several experimental parameters ((a)–(f) in Fig. 2a), detailed below.

The average OCR during stage (iv), non-mitochondrial O₂ consumption (f), is subtracted from the average OCR during all other stages to determine ‘basal respiration’ (a), ‘proton (H⁺) leak’ (b), and ‘maximal respiration’ (d). Subtraction of non-mitochondrial O₂ consumption also removes any background signal [40].

ATP turnover, assessed as oligomycin-sensitive respiration (c), is calculated as (a)–(b).

Spare respiratory capacity (e), also known as respiratory reserve, is calculated as (d)–(a), and is a measure of the cell’s ability to respond to an increase in energy demand.

The calculation of ratios from these parameters can be informative, and provides a form of internal normalisation. The coupling efficiency between ATP turnover and basal respiration is calculated as (c)/(a). The cell respiratory control ratio (RCR; similar but not identical to the RCR measured in isolated mitochondria) is calculated as (d)/(b) [36]. A higher RCR generally indicates more coupled mitochondria and more efficient ATP synthesis [40]. The bioenergetic health index is calculated as (c*e)/(b*f) [55].

Further guidelines for interpretation of OCR measurements can be found in [36, 37, 51, 56].

Data analysis

The measured OCR ([O₂]/time) can be normalised to cell number, total protein content or to levels of specific proteins of interest, giving final units of [O₂]/time/cell number, [O₂]/time/μg protein or [O₂]/time/band density [37, 41, 51]. The OCR can also be normalised to the activity of citrate synthase, a mitochondrial matrix TCA cycle enzyme commonly assumed to be a measure of mitochondrial abundance [38, 43, 57].

Changes in cell viability, mitochondrial density, or protein levels will impact the normalised OCR, and should be reported. In experiments where neurons are exposed to toxic manipulations (e.g. glutamate), it may be particularly important to normalise the OCR to cell viability throughout the experiment [44].

As a guideline for multi-well experiments (e.g. for Seahorse measurements), a minimum of three individual wells per condition should be included per plate, with experiments repeated in three independent cultures. Variability between wells and cultures may necessitate increased replicates to identify small effects.

One-way analysis of variance with repeated measures can be used to test for differences between OCR measurements at specific time-points.

For thorough investigation of the cellular metabolic state and bioenergetic capacity, OCR measurements can be coupled with those of the extracellular acidification rate (ECAR). While lactate release (specifically, protons co-transported with lactate) contributes to ECAR, CO₂ formation from mitochondrial oxidative decarboxylation and the oxidative pentose phosphate pathway also contributes. OCR and ECAR measurements can be combined to calculate lactate release under certain conditions (the buffering power of the media must be calculated) [56, 58, 59], but it should be noted that, even after correction, ECAR only determines anaerobic glycolysis (i.e. the portion of pyruvate metabolised to lactate), whereas the true rate of glycolysis (glucose metabolised to pyruvate) would also include pyruvate metabolised to acetyl CoA. Algorithms are also available that utilise OCR and ECAR measurements to accurately calculate mitochondrial and cytosolic ATP production and consumption, providing information on the cellular bioenergetic state, capacity and flexibility [56, 58, 59].