Altered mitochondrial oxidative phosphorylation in hippocampal slices of kainate-treated rats

Mitochondria provide the main neuronal energy supply and are important organelles for the sequestration of intracellular Ca2+. This indicates a possible important role for mitochondria in modulating neuronal excitability in normal function as well as in disease. Therefore, we have investigated mitochondrial oxidative phosphorylation in the kainate model of epilepsy. We measured the oxygen consumption of single 400-micron rat hippocampal slices applying high resolution respirometry and determined mitochondrial NAD(P)H autofluorescence signal changes in single slices by laser-excited fluorescence spectroscopy. We observed an about 2-fold higher (p<0.001) basal glucose oxidation rate in slices from kainate-treated animals. This increased endogenous energy consumption was found to be unrelated to spontaneous activity since it was not sensitive to the inhibitors of the sodium-potassium ATPase ouabain and of the mitochondrial adenine nucleotide translocator atractyloside. This finding suggested an increased mitochondrial energy turnover in kainate-induced epilepsy. Furthermore, the uncoupler-stimulated oxygen consumption of the slices was approximately 1.3-fold higher (p<0.01) in the kainate model. In accordance with the respirometric data, fluorescence spectroscopy showed decreased reduction levels of the mitochondrial NAD-system in glucose oxidizing slices from kainate-treated rats. The preincubation of epileptic hippocampal slices with either BAPTA AM, ruthenium red or TPP+ increased the atractyloside sensitivity of glucose oxidation to about 1.4-fold (p<0.01). These observations indicate that the increased mitochondrial energy turnover in hippocampal slices from kainate-treated rats is most possibly caused by futile Ca2+-cycling.     

Glycolysis and oxidative phosphorylation in neurons and astrocytes during network activity in hippocampal slices

Network activation triggers a significant energy metabolism increase in both neurons and astrocytes. Questions of the primary neuronal energy substrate (e.g., glucose vs. lactate) as well as the relative contributions of glycolysis and oxidative phosphorylation and their cellular origin (neurons vs. astrocytes) are still a matter of debates. Using simultaneous measurements of electrophysiological and metabolic parameters during synaptic stimulation in hippocampal slices from mature mice, we show that neurons and astrocytes use both glycolysis and oxidative phosphorylation to meet their energy demands. Supplementation or replacement of glucose in artificial cerebrospinal fluid (ACSF) with pyruvate or lactate strongly modifies parameters related to network activity-triggered energy metabolism. These effects are not induced by changes in ATP content, pH_i, [Ca²⁺]_i or accumulation of reactive oxygen species. Our results suggest that during network activation, a significant fraction of NAD(P)H response (its overshoot phase) corresponds to glycolysis and the changes in cytosolic NAD(P)H and mitochondrial FAD are coupled. Our data do not support the hypothesis of a preferential utilization of astrocyte-released lactate by neurons during network activation in slices—instead, we show that during such activity glucose is an effective energy substrate for both neurons and astrocytes.     

Lithium-induced enhancement of mitochondrial oxidative phosphorylation in human brain tissue

Objectives:  Extensive preclinical and clinical evidence suggests mitochondrial dysfunction in bipolar disorder. Studies of brain energy metabolism in bipolar disorder suggest an impairment of energy generation by mitochondrial oxidative phosphorylation. Lithium is an effective drug widely used in treating bipolar disorder, but its mechanism of action has remained uncertain. The aim of this study was to clarify the effect of lithium on mitochondrial oxidative phosphorylation.
Methods:  We spectrophotometrically determined the activities of the respiratory chain complexes I + III [antimycin A-sensitive nicotinamide adenine dinucleotide (NADH) cytochrome c oxidorductase], complexes II + III (succinate cytochrome c oxidoreductase), succinate dehydrogenase, and complex IV [cytochrome c oxidase (COX)], and of the mitochondrial matrix enzyme citrate synthase in postmortem human brain cortex homogenates following exposure to lithium (up to 10 mM).
Results:  Activities of complexes I + III and of complexes II + III were dose-dependently increased by lithium with maximum values at 1 mM (165%, p = 0.03, and 146%, p = 0.00002, of controls). Activity of succinate dehydrogenase remained unchanged up to 2 mM, but was raised at higher drug concentrations (maximum 220%, p = 0.01, of controls). In contrast, activity of COX was not significantly affected by the drug (decrease of 12% at 1 mM, p = 0.4).
Conclusions:  Our study suggests that lithium stimulates mitochondrial respiratory chain enzyme activities at clinically relevant concentrations. Lithium's effect on the mitochondrial respiratory chain presents further evidence of the pathophysiological significance of mitochondrial dysfunction in bipolar disorder. The effect may be relevant to the therapeutic efficacy of the drug by potentially reversing a disease-related alteration.     

Seizure-dependent modulation of mitochondrial oxidative phosphorylation in rat hippocampus

Mitochondrial function is a key determinant of both excitability and viability of neurons. Here, we demonstrate seizure-dependent changes in mitochondrial oxidative phosphorylation in the epileptic rat hippocampus. The intense pathological neuronal activity in pilocarpine-treated rats exhibiting spontaneous seizures resulted in a selective decline of the activities of NADH-CoQ oxidoreductase (complex I of the respiratory chain) and cytochrome c oxidase (complex IV of respiratory chain) in the CA3 and CA1 hippocampal pyramidal subfields. In line with these findings, high-resolution respirometry revealed an increased flux control of complex I on respiration in the CA1 and CA3 subfields and decreased maximal respiration rates in the more severely affected CA3 subfield. Imaging of mitochondrial membrane potential using rhodamine 123 showed a lowered mitochondrial membrane potential in both pyramidal subfields. In contrast to the CA1 and CA3 subfields, mitochondrial oxidative phosphorylation was unaltered in the dentate gyrus and the parahippocampal gyrus. The changes of oxidative phosphorylation in the epileptic rat hippocampus cannot be attributed to oxidative enzyme modifications but are very likely related to a decrease in mitochondrial DNA copy number as shown in the more severely affected CA3 subfield and in cultured PC12 cells partially depleted of mitochondrial DNA. Thus, our results demonstrate that seizure activity downregulates the expression of mitochondrial-encoded enzymes of oxidative phosphorylation. This mechanism could be invoked during diverse forms of pathological neuronal activity and could severely affect both excitability and viability of hippocampal pyramidal neurons.     

Control mechanisms in mitochondrial oxidative phosphorylation

Distribution and activity of mitochondria are key factors in neuronal development,synaptic plasticity and axogenesis.The majority of energy sources,necessary for cellular functions,originate from oxidative phosphorylation located in the inner mitochondrial membrane.The adenosine-5’triphosphate production is regulated by many control mechanism-firstly by oxygen,substrate level,adenosine-5’-diphosphate level,mitochondrial membrane potential,and rate of coupling and proton leak.Recently,these mechanisms have been implemented by second control mechanisms, such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes,allosteric inhibition of cytochrome c oxidase,thyroid hormones,effects of fatty acids and uncoupling proteins.Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia.Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5’-triphosphate in response to energy demands.Large amounts of reactive oxygen species are released by defective mitochondria,similarly,decline of antioxidative enzyme activities(e.g.in the elderly) enhances reactive oxygen species production.We reviewed data concerning neuroplasticity,physiology,and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.     

Biochemical and genetic studies in a family with mitochondrial myopathy

We present a family with severe exercise intolerance, progressive proximal weakness, and lactic acidemia. Fifteen of 24 family members in five generations were affected. Since the affected males do not have offspring at this time, the family pedigree is consistent with either maternal or autosomal dominant inheritance. Muscle histochemistry showed ragged-red fibers and electron microscopy showed globular mitochondrial inclusions. Biochemical analysis showed reduced muscle activities of mitochondrial NADH-cytochrome c reductase (1 of 2 patients), succinate-cytochrome c reductase (2 patients), and cytochrome c oxidase (2 patients). For 1 patient, sequence analysis of 44% of the muscle mitochondrial DNA including all 22 transfer RNA regions showed no point mutation with pathogenic significance. Southern blot analysis showed no deletion. Six affected members of the family were treated with methylprednisolone (0.25 mg/kg) for 3 months. Muscle strength, serum lactate, and energy metabolism at rest (measured by ³¹P magnetic resonance spectroscopy) significantly improved with treatment. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1219–1224, 1997     

Ontogenetic differences in energy metabolism and inhibition of protein synthesis in hippocampal slices during in vitro ischemia and 24 h of recovery

The present study was designed to clarify whether ontogenetic differences in the vulnerability of the brain towards hypoxic-ischemic insults are only caused by the low cerebral energy demand of immature animals or whether there are additional mechanisms, such as protein synthesis (PSR), that may be involved in this phenomenon. We therefore measured tissue levels of adenylates and PSR in hippocampal slices from immature (1340) and mature (1360) guinea pigs fetuses and from adult guinea pigs during in vitro ischemia and 24 h of recovery using a recently modified method. Hippocampal slices were incubated in a temperature controlled flow-through chamber, gassed with 95% 02/5% C02. In vitro ischemia was induced by transferring slices to a glucose-free artificial cerebrospinal fluid (aCSF) eqiilibrated with 95% N2/5% C02. The duration of ischemia ranged from 10 to 40 min. Adenylates were measured by HPLC after extraction with perchloric acid. PSR was evaluated as the incorporation rate of (t4C)leucine into proteins. Under control conditions, tissue levels in adenylates did not change, whereas PSR increased slightly in hippocampal slices from mature fetuses and adult animals during a 24-h control incubation period. In slices from immature fetuses ATP levels were only maintained for 2 h. During in vitro ischemia the decline in ATP, total adenylate pool, and adenylate energy charge was much slower in slices from immature fetuses than in slices from mature fetuses or adults. After in vitro ischemia, ATP and the total adenylate pool did not completely recover in mature fetuses and adults, whereas adenylate energy charge almost returned to control values independently of the developmental stage. Two hours after in vitro ischemia PSR was undisturbed in slices from immature fetuses, but severely inhibited in slices from mature fetuses and adults. With ongoing recovery, PSR in mature fetuses returned to control values, while in adults it was still inhibited even 24 h after in vitro ischemia. From these results we conclude that hippocampal slices prepared from mature guinea pig fetuses as well as from adult guinea pigs can be held metabolically stable during long-term incubation using a recently modified technique. However, in slices from immature fetuses a stable energy state could not be maintained for more than 2 h. We further conclude that postischemic disturbances in PSR closely reflect the ontogenetic changes in the vulnerability of the brain to ischemia and that low energy metabolism is certainly not the only cause of the increased vulnerability of the fetal brain to ischemia.     

Calcium-independent inhibition of GABA(A) current by caffeine in hippocampal slices

Although inhibitory postsynaptic currents (IPSCs) mediated by GABA(A) receptor is thought to be affected by intracellular calcium ion concentration ([Ca2+]i), origin or route of [Ca2+]i increment has not been well elucidated. Reports on the effect of [Ca2+]i elevation on GABA(A)ergic IPSCs per se are also controversial. In this study, effects of caffeine and several other [Ca2+]i-mobilizing drugs were examined on the IPSCs in acute slices of rat hippocampus. Using the patch clamp recording method, spontaneous and evoked currents were recorded from CA3 neurons. Caffeine strongly inhibited both extra-synaptic and synaptic GABAergic IPSCs, regardless of the presence or absence of extracellular Ca2+. This inhibition was not relieved by the intracellular application of EGTA or 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). This inhibition by caffeine was not prevented by preequilibration with caffeine. Ca2+ store depletion caused by thapsigargin or repetitive stimulation by caffeine could not prevent the inhibition. Moreover, ruthenium red and ryanodine could not overcome the inhibition. On the contrary, GABA(A)ergic currents were not inhibited by stimulation with several Ca2+-mobilizing agonists. Forskolin could not mimic the effect of caffeine on the IPSC, and caffeine inhibited the IPSC in the presence of adenosine. These results suggest that intracellular Ca2+ mobilization through ryanodine-sensitive store stimulation does not significantly affect GABAergic IPSCs, and most of the inhibitory effect of caffeine is independent of [Ca2+]i elevation under the present experimental conditions.     

Effects of acidosis on the post-hypoxic recovery of synaptic transmission in gerbil hippocampal slices

We investigated the effects of acidosis on the hypoxic neuronal damage using gerbil hippocampal slices. Acidosis has delayed the onset of harmful hypoxic depolarization, resulting in a decrease in the total hypoxic period and the hypoxic depolarization. This effect has been considered to be protective. However, the synaptic recovery after reoxygenation was attenuated when acidosis (pH: 6.2-6.9) was sustained. Conversely, the synaptic recovery was potentiated when the acidosis was restored to the physiological milieu during the reoxygenation period. These results suggest that acidosis plays a protective effect against the hypoxic neuronal damage only when rapid appreciable pH recovery is achieved during reoxygenation.     

Potassium-induced structural changes of the endoplasmic reticulum in pyramidal neurons in murine organotypic hippocampal slices

The endoplasmic reticulum (ER) structure is of central importance for the regulation of cellular anabolism, stress response, and signal transduction. Generally continuous, the ER can temporarily undergo dramatic structural rearrangements resulting in a fragmented appearance. In this study we assess the dynamic nature of ER fission in pyramidal neurons in organotypic hippocampal slice cultures stimulated by depolarizing concentration of potassium (50 mM). The slices were obtained from transgenic mice expressing fluorescent ER-targeted DsRed2 protein. We employed live tissue confocal microscopy imaging with fluorescence recovery after photobleaching (FRAP) to monitor the extent of structural rearrangements of the ER. In control slices, the ER structure was continuous. Potassium stimulation resulted in extensive fragmentation (fission), whereas return to basal potassium levels (2.5 mM) led to ER fusion and normalization of ER structure. This ER fission/fusion could be repeated several times in the same neuron, demonstrating the reversibility of the process. Blockade of the N-methyl-D-aspartate receptor (NMDAR) with the antagonist D-AP5 or removal of extracellular Ca(2+) prevented depolarization-induced ER fission. ER fission is sensitive to temperature, and decreasing temperature from 35°C to 30°C augments fission, implying that the altering of ER continuity may be a protective response against damage. We conclude that events that generate membrane depolarisation in brain tissue lead to the release of endogenous glutamate that may regulate neuronal ER continuity. The rapid and reversible NMDAR-mediated changes in ER structure reflect an adaptive, innate property of the ER for synaptic activation as well as response to tissue stress, injury, and disease.     

NADH in the pyramidal cell layer of hippocampal regions CA1 and CA3 upon selective inhibition and uncoupling of oxidative phosphorylation

N₂-laser-induced fluorescence in combination with the time and spectral resolution of fluorescent NADH molecules allows on-line measurement of relative NADH concentration with high spatial resolution (diameter of optical fibre 200 μm, λ_exc = 337 nm, λ_det = 460 nm). Energy metabolism was impaired in submerged rat hippocampal slices using the inhibitors amytal, 3-nitropropionate (3-np), sodium cyanide (1 mM each) and the uncoupling agent 2,4-DNP (200 μM). A microprocessor-controlled repeated positioning of the optical fibre in CA1 and CA3 pyramidal cell layers, and CA1 stratum radiatum (CA1_SR). Time-dependently, NADH fluorescence increased reversibly upon perfusion with amytal and cyanide. It was unchanged by perfusion with 3-np for 40 min and rapidly decreased upon perfusion with 2,4-DNP. The CAl/CA3 ratio of NADH fluorescence mildly decreased to 0.92 ± 0.04 (mean ± S.D.) at 10 min (P < 0.05) and 0.89 ± 0.05 at 211 min (P < 0.01) upon perfusion with amytal. The CA1/CA3 ratio increased to 1.56 ± 0.28 at 10 min (P < 0.01) and 1.29 ± 0.35 at 20 min (P < 0.05) upon application of 2,4-DNP. Fluorescence in CA1_SR, was similar to fluorescence in CA1 upon perfusion with 2,4-DNP and similar to CA3 upon perfusion with amytal. We conclude that NADH fluorescence can be measured with high regional selectivity and specificity in hippocampal slices. Selective inhibition of mitochondrial complex I and uncoupling of energy metabolism differentially impair NADH concentration in different hippocampal areas.     

左旋多巴对帕金森病大鼠毒性作用的实验研究

目的　研究左旋多巴 (L dopa)对帕金森病 (PD)模型大鼠异常行为、黑质抗氧化系统、线粒体呼吸链功能和神经递质代谢的影响及其机制。方法　应用 6 羟基多巴胺 (6 OHDA)立体定向注射制作PD大鼠模型 ,给PD大鼠L dopa 2 5mg/ (kg·d)灌胃 ,共 4 5d。给药前后分别进行行为学测试 ,给药后测定黑质区谷胱甘肽过氧化物酶 (GSH Px)、丙二醛 (MDA)、活性氧 (ROS)及线粒体呼吸链酶复合体Ⅰ水平 ,测定尾状核头部多巴胺 (DA)、高香草酸 (HVA)、单胺氧化酶 B(MAO B)的水平。结果　 (1)L dopa组大鼠旋转速度给药前为(13.1± 1.5 )r/min ,给药后为 (7.2± 1.6 )r/min,给药前后比较差异有显著性 (P <0 .0 1) ;(2 )L dopa组GSH Px活性、呼吸链酶复合体Ⅰ水平降低 ,MDA含量、ROS活性升高 ,与对照组比较差异均有显著性 (均P <0 .0 1) ;(3)L dopa组MAO B活性、DA、HVA含量及DA/HVA比值与对照组比较均显著升高 (P <0 .0 5～ 0 .0 1)。结论L dopa能有效改善PD大鼠的旋转行为 ,但可加重黑质区氧化应激损伤 ,抑制线粒体呼吸链酶活性。     

Activation of metabotropic glutamate receptors increases endogenous protein kinase C substrate phosphorylation in adult hippocampal slices

We previously reported (Staak, S., Behnisch, T. and Angenstein, F., Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C (PKC) isoenzymes α and β, Brain Res., 682 (1995) 55–62) that Ca²⁺-dependent PKC isoenzymes α/β and γ are not translocated between subcellular compartments after stimulation of glutamate receptor subtypes in hippocampal slices. Extending our previous work in this study in situ phosphorylation of endogenous PKC substrates and the translocation of novel PKC isoenzymes δ and ε was analysed to detect PKC activation. Two proteins of approximately 94 kDa and 18 kDa were first characterised to be specific PKC substrates. As control of the technique carbachol was shown to increase in situ phosphorylation of the two substrates without any measurable translocation of PKC protein. Activation of metabotropic glutamate receptors by 50 μM DHPG also increased the in situ-phosphorylation by 43.9% (94 kDa) and 32.8% (18 kDa) compared to controls but did not induce a measurable subcellular redistribution of conventional and novel PKC isoenzymes. Stimulation by 50 μM trans-ACPD or 0.1 mM quisqualate enhanced the in situ phosphorylation in the same range, whereas 0.1 mM NMDA was ineffective. To our knowledge this is the first report showing a direct link between metabotropic glutamate receptor activation and increased endogenous PKC substrate phosphorylation in adult hippocampal slices. This PKC activation was not detectable by a redistribution of enzyme protein between subcellular compartments. We, therefore, conclude, that the failure to detect PKC translocation in physiological experiments is not an indicator for unchanged enzyme activity.     

Glucose enhances recovery of potassium ion homeostasis and synaptic excitability after anoxia in hippocampal slices

Hippocampal slices exposed to brief anoxia combined with elevated glucose exhibit greater postanoxic recovery of synaptic transmission. Glucose may have improved recovery of synaptic transmission by enhancing the production of metabolic energy during and after anoxia. This enhancement should provide more ATP for energy-requiring ion transport processes, and lead (1) to a delayed onset of complete depolarization of CA1 pyramidal cells during anoxia (anoxic depolarization) and (2) to greater ion transport activity following anoxia. A delay in anoxic depolarization would protect neurons from damage if the duration of anoxic depolarization was shortened. Greater postanoxic ion transport would allow the re-establishment of ion gradients supportive of neuronal and synaptic excitability. The effects of glucose and anoxia on ion homeostasis and synaptic transmission were examined in rat hippocampal slices exposed to different glucose concentrations (5–20 mM). The duration of anoxic depolarization was held constant so that postanoxic damage related to this duration was controlled. We found that K⁺ transport and recovery of synaptic transmission after anoxia in hippocampal slices improved as glucose concentration increased. Also, anoxic depolarization was delayed as glucose concentration increased. Thus, added glucose may improve postanoxic recovery of synaptic transmission by better supporting ion transport.     

Transient depression of excitatory synaptic transmission induced by adenosine uptake inhibition in rat hippocampal slices

The transient property of the dipyridamole-induced depression of excitatory synaptic transmission was analyzed using field EPSPs (fEPSPs) recorded from the CA1 region in rat hippocampal slices. The fEPSPs were depressed by 1 microM dipyridamole and then gradually recovered to the control level. The depression was antagonized by aminophylline or DPCPX, although it was not significantly affected by DMPX. The results suggest that the fEPSP depression is induced by a mechanism through the A(1) receptor.     

Mitochondrial calcium content and oxidative phosphorylation in heart and skeletal muscle of dystrophic mice

Mitochondrial calcium overloading was investigated in the genetically dystrophic mouse (strains $\text{129}\text{ReJ}\text{dy}\text{dy}$) as a possible contributing factor to the development of muscle fiber necrosis. Mitochondrial calcium concentrations were significantly elevated in both skeletal muscle and heart organelles. Because mitochondria were isolated in the presence of ruthenium red this finding was not the result of an artefact of isolation. State 3 respiration rates and concomitantly the respiratory control ratios were slightly decreased in skeletal muscle, but not in heart mitochondria. This abnormality could result from calcium overloading in a small fraction of the mitochondria. Fractionation of skeletal muscle mitochondria on sucrose gradients gave two distinct populations of dystrophic organelles, one with high calcium, whereas normal skeletal muscle mitochondria and heart organelles showed only one broad band on the gradient. The results support the idea that both skeletal muscle and heart are affected in dystrophic mice, strain $\text{129}\text{ReJ}\text{dy}\text{dy}$ and also that in the dystrophic mouse the process of cell necrosis is associated with cellular calcium overloading.     

Calcium-sensitive recovery of extracellular potassium and synaptic transmission in rat hippocampal slices exposed to brief anoxia

We examined the possibility that Ca2+-sensitive inhibition of synaptic transmission following anoxia involves compromise of ion transport activity. Rat hippocampal slices were superfused with artificial cerebrospinal fluids containing different concentrations of CaCl2, and subjected to short anoxia. Durations of anoxia were sufficient to provoke anoxic depolarization, indicated by a sudden rise in extracellular K+ (K+o). Following anoxia, apparent K+ transport was assessed by measuring the magnitude of subnormal K+o (the K+o undershoot) in hippocampal region CA1. Recovery of synaptic transmission 1 h after anoxia was determined by evaluation of the magnitudes of the orthodromically stimulated population spike recorded from CA1 pyramidal cells. K+o undershoots and recovery of synaptic transmission decreased as CaCl2 or the duration of anoxic depolarization increased. These data suggest: (1) that increased artificial cerebrospinal fluid CaCl2 compromised K+ reaccumulation after anoxia; and (2) that ion transport dysfunction may inhibit recovery of synaptic transmission.