(A) Primary rat cortical neurons were control-treated in aCSF (con, filled squares) or permeabilized by saponin (sap, 25 µg/ml) in aCSF (open squares) or KCl-based assay medium (open triangles) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Respiration was stimulated by co-injection of ADP (1 mM) in the presence of the complex I substrates pyruvate and malate (P/M, 5 mM each). K₂HPO₄ was also co-injected with saponin to obtain a final concentration of 4 mM in each assay medium (see Methods) and EGTA (5 mM) was included for cells assayed in aCSF. Oligomycin (oligo, 0.3 µg/ml), FCCP, (F, 2 µM) and antimycin A (AA, 1 µM) were subsequently added as indicated (arrows). Pyruvate (10 mM) was included with FCCP for intact cells (filled squares) here and in subsequent experiments to insure that substrate supply was not rate-limiting for uncoupled respiration. OCRs were measured in triplicate (mean ± SD). (B) OCRs in (A) baseline-normalized to the point prior to sap or con addition.

(A) Primary rat cortical neurons were permeabilized by saponin (sap, 25 µg/ml, filled squares) or digitonin (25 µg/ml, open squares, 50 µg/ml, open triangles, 100 µg/ml, open circles) plus EGTA (5 mM) in aCSF medium after three baseline O₂ consumption rate (OCR) measurements (first arrow). Pyruvate and malate (P/M, 5 mM each), ADP (1 mM), and excess K₂PHO₄ (3.6 mM for 4 mM final) were co-injected with saponin to measure complex I-dependent ADP-stimulated respiration. Oligomycin (oligo, 0.3 µg/ml), FCCP, (F, 2 µM) and antimycin A (AA, 1 µM) were added as indicated (arrows). (B) OCRs were measured and at the injection marked a, neurons were control-treated in the absence (filled squares) or presence of FCCP plus pyruvate (2 µM and 10 mM, respectively, open squares) or permeabilized using saponin (triangles). Complex I-linked respiration (P/M) in permeabilized neurons was stimulated by ADP (1 mM, filled triangles) or FCCP (F, 2 µM, open triangles). Intact (open squares) and permeabilized (open triangles) cells treated with FCCP received a second FCCP injection (1 µM) at b to insure respiration was maximally uncoupled, followed by a control injection at c and finally antimycin A (AA, 1 µM) at d. Control-treated intact cells (filled squares) and ADP-treated permeabilized cells (filled triangles) received injections of oligo, FCCP, (F, 2 µM) and AA in ports b, c, and d, respectively, with pyruvate (10 mM) included with FCCP for intact cells. OCRs in (A) and (B) are mean ± SD in quadruplicate, normalized to the third measurement point and expressed as % baseline OCR.

(A) Primary rat cortical neurons were incubated in aCSF in the presence of glucose (15 mM, filled squares), no substrate (open circles), pyruvate (10 mM, filled triangles), or methyl succinate (10 mM, open squares). Cells were control-treated (con, filled squares) or treated with 2-deoxyglucose (2-DG, 2 mM, all other groups) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Rotenone (0.5 µM) was subsequently added (second arrow) to inhibit complex I. (B) Neurons were control-treated (con, filled squares) or permeabilized by saponin (sap, 25 µg/ml) plus EGTA (5 mM) in aCSF medium after three baseline O₂ consumption rate (OCR) measurements (first arrow). Succinate/rotenone (S/R, 5 mM and 0.5 mM respectively) or methyl succinate/rotenone (MeS/R, 5 mM and 0.5 mM respectively) as substrate (sub), ADP (1 mM), and excess K₂PHO₄ (3.6 mM for 4 mM final) were co-injected with saponin to measure complex II-dependent ADP-stimulated respiration. Subsequently, oligomycin (oligo, 0.3 µg/ml), FCCP, (F, 2 µM) and antimycin A (AA, 1 µM) were added as indicated (arrows). Pyruvate (10 mM) was included with FCCP for intact cells (filled squares) to insure that substrate supply was not rate-limiting for uncoupled respiration. OCRs in (A) and (B) are mean ± SD in triplicate, normalized to the third measurement point and expressed as % baseline OCR.

(A) Primary rat cortical neurons were control-treated (con, filled squares) or permeabilized by saponin (sap, 25 µg/ml) plus EGTA (5 mM) in aCSF medium in the absence (filled circles) or presence (open circles) of purified cytochrome c (100 µM) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Pyruvate and malate (P/M, 5 mM each), ADP (1 mM), and excess K₂PHO₄ (3.6 mM for 4 mM final) were co-injected with saponin to measure complex I-dependent ADP-stimulated respiration. Subsequently, oligomycin (oligo, 0.3 µg/ml), FCCP, (F, 2 µM) and antimycin A (AA, 1 µM) were added as indicated (arrows). Pyruvate (10 mM) was included with FCCP for intact cells (filled squares). (B) Neurons were permeabilized as described in (A) using 250 µg/ml saponin in the absence (filled triangles) or presence (open triangles) of purified cytochrome c (100 µM). Control-treated neurons (filled squares) and neurons permeabilized by 25 µg/ml saponin (filled circles) are duplicated from (A) for comparison. OCRs in (A) and (B) are mean ± SD in quadruplicate, normalized to the third measurement point and expressed as % baseline OCR. All treatments in (A) and (B) were assayed in parallel on an individual 24-well plate.

(A) Primary rat cortical neurons were control-treated (con, filled squares) or treated with KB-7943 (K-BR, 10 µM, open circles, 20 µM, filled triangles, or 30 µM, open triangles) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Subsequently, oligomycin (oligo, 0.3 µg/ml), FCCP plus pyruvate (F, 2 µM and 10 mM, respectively), and antimycin A (AA, 1 µM) were added as indicated (arrows). (B) Neurons were control-treated (con, filled squares) or treated with KB-7943 (30 µM, all other groups) as in (A). Other additions were as (A) except the FCCP+pyruvate injection after KB-R7943 addition also contained methyl succinate (MeS, 10 mM, open triangles), methyl succinate plus rotenone (10 mM/0.5 µM, MeS/R, open circles), or no additional substrate (open squares). OCRs in (A) and (B) are mean ± SD in triplicate, normalized to the third measurement point and expressed as % baseline OCR.

Primary rat cortical neurons were control-treated (filled symbols) or treated with KB-7943 (K-BR, 30 µM, open symbols) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Neurons were then permeabilized by saponin (sap, 25 µg/ml) plus EGTA (5 mM) in aCSF medium containing glutamate/malate (G/M, 3 mM and 1 mM, respectively, circles) or succinate/glutamate (S/G, 3 mM each, triangles) as substrates (sub), ADP (1 mM), and excess K₂PHO₄ (3.6 mM for 4 mM final) to measure ADP-stimulated respiration (second arrow). Neurons permeabilized in the presence of G/M received a subsequent succinate (5 mM) addition (third arrow, circles) while neurons already exposed to succinate received a control (con) injection (triangles). OCRs are mean ± SD in triplicate, normalized to the third measurement point and expressed as % baseline OCR.