Table 4. Metabolic responses of synaptosomes to activating conditions.
| Substratea and treatmentb | Response magnitudec | Tissue source and Reference |
|---|---|---|
| Electrical stimulation | 20–65% ↑respiration rate, 10–70% ↑ glycolysis | Seven brain regions from adult rat, sheep, or rabbit |
| Glucose + 50 mmol/L K+ | 15% ↑ respiration rate, 30% ↑ glycolysis | Bradford, 1975 |
| 10 mmol/L glucose + 24 mmol/L KCl | 137% ↑ respiration rate | Cerebral cortex, adult rat |
| 85% ↑ [1-14C]pyruvate decarboxylation | Schaffer and Olson, 1980 | |
| 10 mmol/L glucose + 40 mmol/L KCl | 69% ↑ respiration rate | Adult rat forebrain |
| Erecińska et al, 1991 | ||
| 5 mmol/L pyruvate + 40 mmol/L KCl | 63% ↑ respiration rate | |
| 9.5 mmol/L [U-14C]glucose + 72 mmol/L Na+ | 180% ↑ glucose oxidation to 14CO2 | Whole brain from adult or immature rats |
| + 100 μmol/L 2,4-dinitrophenol | 370% ↑ glucose oxidation to 14CO2 | Diamond and Fishman, 1973 |
| Developmental age from 1 to 90 days | 500%↑ Na+-stimulated glucose oxidation to 14CO2 | |
| Developmental age from 10 to 20 days | 200%↑ Na+-stimulated glucose oxidation to 14CO2 | |
| 1 mmol/L [U-14C]glucose + 100 μmol/L veratridine | 290% ↑ glucose oxidation to 14CO2 | Adult rat forebrain |
| Harvey et al, 1982 | ||
| 10 mmol/L glucose + 100 μmol/L veratridine | 300% ↑ respiration rate | Adult rat hippocampal mossy fiber synaptosomes |
| Terrian et al, 1988 | ||
| 10 mmol/L glucose + 0.5 μmol/L FCCP | Immediate 500% ↑ respiration rate | Cerebral cortex, 4–8-week-old guinea pig |
| 10 mmol/L glucose + 100 μmol/L veratridine, followed by 0.5 μmol/L FCCP | Immediate veratridine-induced 175% ↑ respiration, followed by an immediate additional 65% FCCP-evoked ↑ respiration | Scott and Nicholls, 1980 |
| 1.5 mmol/L glucose + [3,4-14C]glucose + 100 μmol/L 2,4-dinitrophenol | 120% ↑ glucose oxidation to 14CO2 | Adult rat forebrain Ksiezak and Gibson, 1981a,1981b |
| 1.5 mmol/L glucose, severe hypoxia (O2 tension 19 torr → <1 torr) | 250% ↑ lactate production rate | |
| 5 mmol/L glucose + 1 mmol/L cyanide or 6 μmol/L rotenone | Block respiration, 9-fold increase glycolysis | Cerebral cortex, adult guinea pig Kauppinen and Nicholls, 1986a |
| 5 mmol/L glucose + Nitrogen atmosphere (anoxia ) | Ten-fold increase in glycolysis | |
| 5 mmol/L glucose + A23187 (divalent cation ionophore) | Three-fold stimulation respiration, 5-fold stimulation glycolysis | |
| 10 mmol/L glucose + anoxia | 335% ↑ lactate amount produced | Adult rat forebrain |
| White et al, 1989 | ||
| 10 mmol/L glucose + 1 μmol/L FCCP | 900% ↑ glycolysis maintained for at least 30 minutes 500% ↑ respiration | Cerebral cortex, adult guinea pig Kauppinen and Nicholls, 1986b |
| 10 mmol/L glucose + 1 mmol/L arsenite (inhibit pyruvate oxidation) | 35% inhibition respiration, 3-fold increase in glycolysis | |
| 5 mmol/L glucose + 45 mmol/L KCl | 55% ↑ glycolysis, 47% ↑pyruvate decarboxylation | Cerebral cortex, 6–10-week-old guinea pig Kauppinen et al, 1989 |
| 5 mmol/L glucose + 75 μmol/L veratridine | 250% ↑ glycolysis, 290% ↑pyruvate decarboxylation | |
| 5 mmol/L glucose + 1 μmol/L Cl-CCP | 650% ↑pyruvate decarboxylation | |
| 10 mmol/L glucose + anoxia | 2,100% ↑ lactate synthesis rate | Adult rat cerebral cortex |
| 10 mmol/L glucose + 10 μmol/L veratridine | 160% ↑ respiration rate, 75% ↓ lactate synthesis rate | Gleitz et al, 1993 |
| 10 mmol/L glucose + 1 mmol/L nitroprusside or 100 μmol/L S-nitrocysteine | Aerobic conditions: 20–30% ↑ lactate synthesis rate; 70% ↑ lactate amount at 15 minutes | Adult rat forebrain Erecińska et al, 1995 |
| 10 mmol/L glucose + 10 μmol/L rotenone | 900% ↑ lactate synthesis rate | |
| 10 mmol/L glucose + 40 μmol/L veratridine, 5 minutes | 210% ↑ respiration, 515% ↑ lactate production | Adult rat forebrain Erecińska et al, 1996 |
| 15 minutes | 135% ↑ respiration, 620% ↑ lactate production | |
| 10 mmol/L glucose + 10 μmol/L monensin, 5 minutes | 73% ↑ respiration, 1,100% ↑ lactate production | |
| 15 minutes | 70% ↑ respiration, 580% ↑ lactate production | |
| 10 mmol/L glucose + 5 μmol/L nigericin, 5 minutes | 58% ↑ respiration, 465% ↑ lactate production | |
| 15 minutes | 8% ↑ respiration, 200% ↑ lactate production | |
| 10 mmol/L glucose + 100 (young rats) or 50 (old rats) μmol/L veratridine | 120 or 110% ↑ respiration in synaptosomes from young or old rats, respectively | Whole brain from 3-month (young) or 24 (old)-month rats Joyce et al, 2003 |
| 10 mmol/L glucose + 240 nmol/L FCCP | 150 or 88% ↑ respiration in synaptosomes from young or old rats, respectively | |
| 15 mmol/L glucose + 4 μmol/L FCCP | 300% ↑ respiration rate | Cerebral cortex, 17–20 day-old mouse |
| 10 mmol/L pyruvate + 4 μmol/L FCCP | 230% ↑ respiration | Choi et al, 2009 |
| 15 mmol/L glucose + 10 mmol/L pyruvate + 4 μmol/L FCCP | 225% ↑ respiration rate | |
| 15 mmol/L glucose + 2–5 μmol/L veratridine | 100–135% ↑respiration, 200–300%, ↑extracellular acidification (i.e., ↑glycolysis with lactate production and release) | |
| 15 mmol/L glucose + 10 mmol/L pyruvate + 2–5 μmol/L veratridine | 210–165% ↑respiration, 180–250% ↑extracellular acidification | |
| 15 mmol/L glucose + 10–100 μmol/L AOAA | No effect on basal respiration rate | |
| 15 mmol/L glucose + 4 μmol/L FCCP + 10 μmol/L AOAA | 15% inhibition of maximal FCCP-evoked rate | |
| + 30 μmol/L AOAA | 30% inhibition of maximal rate | |
| + 100 μmol/L AOAA | 45% inhibition of maximal rate | |
| 15 mmol/L glucose + 10 mmol/L pyruvate + 0.05–1 mmol/L 4-aminopyridine | 35% ↑respiration rate compared with without 4-aminopyridine | |
| 15 mmol/L glucose + 10 mmol/L pyruvate + 4 μmol/L FCCP | Values normalized by number of bioenergetically competent synaptosomes: | Cerebral cortex or striatum from 3 to 4 month-old mice |
| 447 or 452 ↑ respiration rate in dopamine transporter-enriched synaptosomes from the striatum or cortex, respectively | Choi et al, 2011 | |
| 538 or 542 ↑ respiration rate in residual nondopaminergic synaptosomes from the striatum or cortex, respectively |
Synaptosomes are heterogeneous populations of presynaptic nerve endings that contain mitochondria and are capable of glycolytic and oxidative metabolism of glucose and other substrates. Glycolysis generates ATP plus pyruvate when reducing equivalents are transferred from cytoplasmic NADH to the mitochondria by the malate–aspartate shuttle (MAS). NADH can also be oxidized to regenerate NAD+ by production of lactate, which is sometimes used as a surrogate marker for glycolytic pathway flux. Oxidation of pyruvate through the tricarboxylic acid cycle generates NADH and FADH2. The electron transport chain transfers electrons from NADH and FADH2 to oxygen, along with extrusion of protons from the mitochondrial matrix. Proton reentry into the matrix through ATP synthetase drives ATP synthesis. In well-coupled mitochondria, respiration rate (oxygen consumption or uptake) is coupled to ATP synthesis, and ‘respiratory control' is exerted by energy demand, i.e., ADP availability. Respiration in the absence of ATP synthesis is low and arises from proton leakage into the matrix.
Synaptosomal energetics can be modulated by treatment with compounds that inhibit specific metabolic or transport reactions, abolish respiratory control, or increase energy demand. The MAS can be blocked by aminooxyacetate (AOAA), which inhibits pyridoxal-dependent enzymes (e.g., aminotransferases), thereby reducing activity of the malate–aspartate shuttle, preventing reoxidation of cytosolic NADH, and blocking the ability of mitochondria to use glycolytic pyruvate. 4-CIN (α-cyanocinnamate) inhibits monocarboxylic acid transporters and pyruvate transport into the mitochondria. Electron transport can be inhibited at different sites, complex I (NADH dehydrogenase complex) by rotenone; complex II (succinate dehydrogenase flavoprotein complex) by 2-nitropropionate, malonate, or methylmalonate; complex III (cytochrome bc1 complex) by antimycin A; and complex IV (cytochrome a1a3 or cytochrome oxidase) by cyanide, azide, nitric oxide, carbon monoxide, or anoxia. Oligomycin inhibits proton reentry into the mitochondrial matrix through the ATP synthase and blocks ATP synthesis; the residual respiration reflects the proton leak into the matrix. FCCP (carbonylcyanide-p-trifluoromethoxy-phenylhydrazone), Cl-CCP (carbonylcyanide-m-chlorophenylhydrazone), and dinitrophenol are uncouplers or protonophores that allow proton reentry into the mitochondrial matrix and relieve respiratory control by diverting the proton current away from the ATP synthase and reducing the capacity to generate ATP. Uncouplers stimulate respiration, and FCCP treatment can reveal maximal respiratory capacity, i.e., the available capacity of nerve endings or cells for substrate delivery and electron transport to increase ATP synthesis in response to increased ATP demand. Ion movements can also be altered to increase ATP demand and stimulate metabolism. Increased extracellular K+ levels are a consequence of neuronal activity, and depolarization of cells or nerve endings with high concentrations of KCl stimulates energy production. Veratridine prevents inactivation of voltage-activated sodium channels and causes intracellular Na+ to increase, thereby increasing ATP use by Na+-K+-ATPase; veratridine also causes intracellular Ca2+ to increase, followed by glutamate release. 4-Aminopyridine (4-AP) is an inhibitor of A-type K+ channels that causes synaptosomes to fire repetitive action potentials. NMDA, N-methyl-D-aspartate, is an agonist for a class of ionotropic glutamate receptors. Nigericin and monensin are ionophores that exchange H+ for K+ or Na+, respectively, whereas A23187 exchanges a divalent cation (Ca2+ or Mg2+) for 2H+. For more details regarding synaptosomal bioenergetics and responses to various treatments, see Nicholls (2003, 2009, 2010) and Erecińska et al (1996).
Magnitude of response is expressed as approximate percentage change owing to treatment, 100[(treated−control)/control], or, when indicated (i.e., as ‘–fold' change), treated relative to control ratio.