Previous studies established that treatment with 25–50 µg/ml saponin permeabilizes neuronal plasma membrane without compromising mitochondrial structure 
. We measured O2
consumption by intact neurons in either a typical, nominally calcium-free KCl-based intracellular assay medium or a standard artificial cerebrospinal fluid (aCSF) solution and evaluated the response to the co-injection of saponin (25 µg/ml), mitochondrial complex I-linked substrates, ADP, and excess phosphate. EGTA (5 mM) was additionally co-injected with saponin for aCSF-incubated neurons to yield ~107 nM free calcium, approximating free calcium in the cytoplasm (calculated using Theo Schoenmakers' Chelator software, http://www.stanford.edu/~cpatton/CaMgATPEGTA-TS-Plot.htm
. Basal or ADP-stimulated state 3 (phosphorylating) respiration did not differ between aCSF and KCl medium (). Addition of saponin and substrate increased OCRs by >300% compared to non-permeabilized neurons injected with control solution (). As expected, injection of saponin to aCSF-incubated neurons without EGTA caused a rapid and complete loss of respiration (data not shown), consistent with calcium-mediated mitochondrial damage.
The ATP synthase inhibitor oligomycin was added after ADP to evaluate mitochondrial coupling. When H+ flux through the ATP synthase is inhibited, phosphorylating respiration stops and residual O2 consumption is primarily due to proton leak across the mitochondrial inner membrane. Oligomycin decreased OCR to the same level in intact and permeabilized neurons assayed side-by-side on the same microplate (). This finding indicates that mitochondria within permeabilized neurons remain coupled in both sodium and potassium-based assay medium. Next, we injected the uncoupler FCCP to examine maximal respiratory capacity. FCCP dissipates the H+ gradient across the mitochondrial inner membrane, uncoupling electron transport from oxidative phosphorylation. In the presence of FCCP, OCR increases to the maximum extent supported by the electron transport chain and substrate supply. FCCP stimulated respiration to a greater degree in intact neurons than in neurons permeabilized in either KCl or aCSF (). However, the ability of FCCP to stimulate respiration in aCSF medium was similar to that observed in KCl medium. Because bioenergetic function was relatively well-preserved in neurons permeabilized in aCSF, subsequent optimization was performed using this assay medium to allow for sequential measurements from intact and permeabilized cells.
Excessive concentrations of saponin can permeabilize the mitochondrial outer membrane as well as the plasma membrane. Mitochondrial outer membrane permeabilization leads to loss of cytochrome c
, the electron carrier between complex III and complex IV, which causes a reduction in respiratory capacity 
. To confirm that mitochondrial outer membrane integrity was not compromised in permeabilized neurons, leading to a limitation in ADP or FCCP-stimulated OCR, exogenous purified cytochrome c
was co-injected with saponin. Cytochrome c
had no effect on either state 3 or uncoupled respiration in neurons permeabilized by 25 µg/ml saponin (). However, injection of a ten-fold higher saponin concentration reduced both ADP-stimulated and uncoupled respiration and these reductions were completely prevented by exogenous cytochrome c
(). These results indicate that 250 µg/ml saponin, but not 25 µg/ml saponin, results in partial mitochondrial outer membrane permeabilization that limits OCR due to cytochrome c
release. In addition, they demonstrate that cytochrome c
loss was not responsible for the reduced FCCP response in neurons permeabilized by 25 µg/ml saponin compared to intact cells.
Optimized plasma membrane permeabilization by saponin does not compromise mitochondrial outer membrane integrity.
Some studies used digitonin in place of saponin to achieve selective plasma membrane permeabilization 
. We tested a range of saponin and digitonin concentrations and found that although digitonin effectively permeabilized neurons at ≥50 µg/ml, we were unable to increase ADP or FCCP-stimulated OCRs beyond those achieved by 25 µg/ml saponin ( and data not shown). In the experiments described in and , ADP was co-injected with saponin and substrate but FCCP was injected after nearly 20 minutes. To test the hypothesis that the impaired FCCP response in permeabilized cells relative to intact cells was time-dependent and/or due to the prior injection of oligomycin, we either injected FCCP simultaneous to saponin permeabilization and measured respiration continuously, or injected FCCP after oligomycin addition nearly 30 minutes later. FCCP stimulated respiration to the same level as ADP, and nearly to the level of FCCP-treated intact cells, when injected simultaneous to the saponin addition (, injection a
, open triangles). However, the FCCP response was greatly reduced when FCCP was added >20 minutes after the saponin injection (injection c
, filled triangles). ADP-stimulated respiration was stable over a 20 minute period (injection a
, filled triangles). However, FCCP-stimulated respiration declined progressively in permeabilized (open triangles) but not in intact cells (open squares). Injection of additional FCCP () or additional mitochondrial substrate (data not shown) failed to improve uncoupled respiration.
Effect of the permeabilizing agent and time on FCCP-stimulated respiration.
The ratio of ADP-stimulated, state 3 respiration to state 4, non-phosphorylating respiration is referred to as the respiratory control ratio (RCR). The greater the RCR, the better coupled the mitochondria, and the greater the efficiency of ATP synthesis. We estimated RCRs in permeabilized neurons using the substrates pyruvate/malate or glutamate/malate (complex I-linked respiration), or succinate in the presence of the complex I inhibitor rotenone (complex II-linked respiration). RCRs for permeabilized neurons oxidizing complex I or complex II substrates were comparable to or superior than previous estimates using isolated mitochondria from primary cortical neurons or rat brain () 
. State 4 respiration was significantly higher when the complex II substrate succinate was used compared to the utilization of complex I substrates, consistent with results obtained using mitochondria isolated from cortical neurons 
. FCCP-stimulated OCR (measured after oligomycin addition) was significantly lower with any of the substrate combinations compared to the FCCP rate in intact cells. FCCP-stimulated OCR was also significantly lower for the substrates glutamate/malate compared to pyruvate/malate.
Having demonstrated that coupled respiration can be measured using either complex I- or complex II-linked substrates in neurons permeabilized in aCSF, we used this technique to investigate the mechanism of neuronal respiratory inhibition by KB-R7943, a drug with multiple targets. First, we confirmed the ability of KB-R7943 to inhibit O2
consumption in intact neurons and investigated whether methyl succinate, a putative cell permeable complex II-linked substrate, could relieve respiratory inhibition. Treatment with KB-R7943 (10–30 µM) led to an immediate 25–40% inhibition of basal OCR and a dose-dependent attenuation of respiratory capacity in primary cortical neurons (), consistent with effects reported in hippocampal neurons 
. Although KB-R7943 was shown to inhibit complex I-linked but not complex II-linked respiration in isolated brain mitochondria 
, surprisingly KB-R7943-inhibited respiration in intact neurons was not rescued by methyl succinate (). To test whether methyl succinate could support respiration in neurons in the absence of other substrates, 2-deoxyglucose (2-DG), a competitive inhibitor of glycolysis, was added to glucose-deprived neurons. Neurons were able to support basal OCR for a short time in the absence of exogenous substrate (, open circles). However, 2-DG (2 mM) inhibited OCR by ~50% in the absence of glucose (open circles) and respiration was further reduced to ~10% of the basal rate by the addition of the complex I inhibitor rotenone (). Pyruvate, a cell permeable complex I substrate (filled triangles), but not methyl succinate (open squares) occluded the ability of 2-DG to inhibit respiration (). Residual respiration in the presence of methyl succinate and 2-DG was rotenone sensitive, indicating that methyl succinate was not able to support complex II-dependent respiration in intact neurons in the absence of glycolysis ().
Methyl succinate fails to rescue KB-R7943-inhibited respiration in intact neurons.
Methyl succinate is a poor substrate for complex II.
To test whether the inability of methyl succinate to support complex II-dependent respiration was due to its inability to penetrate cells, we compared state 3, state 4, and uncoupled respiration in neurons permeabilized by saponin in the presence of rotenone (0.5 mM) and an equimolar concentration of methyl succinate or succinate (5 mM). ADP and FCCP stimulated OCRs were greatly reduced when methyl succinate rather than succinate was supplied as the lone exogenous substrate (). These results indicate that methyl succinate is a poor complex II substrate, irrespective of its ability to permeate cells.
Brustovetsky et al. showed that KB-R7943 inhibited the respiration of isolated brain mitochondria oxidizing malate/glutamate but not of mitochondria oxidizing succinate/glutamate 
. We treated neurons with KB-R7943 (30 µM) to inhibit respiration and subsequently permeabilized cells in the presence of ADP and malate/glutamate or succinate/glutamate at the concentrations described 
. ADP-stimulated state 3 respiration was impaired in KB-R7943-treated permeabilized neurons in the presence of malate/glutamate but not in the presence of succinate/glutamate (). The subsequent addition of succinate to neurons oxidizing complex I substrates stimulated OCR to the level of the control, rescuing KB-R7943-mediated respiratory inhibition. These results suggest that the reduction of respiration by KB-R7943 in intact neurons is primarily due to the inhibition of complex I.
Inhibition of respiration by KB-R7943 is reversed by the complex II substrate succinate.