Mitochondria are critical for cellular ATP production; however, recent studies suggest that these organelles fulfill a much broader range of tasks. For example, they are involved in the regulation of cytosolic Ca 2+ levels, intracellular pH and apoptosis, and are the major source of reactive oxygen species (ROS). Various reactive molecules that originate from mitochondria, such as ROS, are critical in pathological events, such as ischemia, as well as in physiological events such as long-term potentiation, neuronal-vascular coupling and neuronal-glial interactions. Due to their key roles in the regulation of several cellular functions, the dysfunction of mitochondria may be critical in various brain disorders. There has been increasing interest in the development of tools that modulate mitochondrial function, and the refinement of techniques that allow for real time monitoring of mitochondria, particularly within their intact cellular environment. Innovative imaging techniques are especially powerful since they allow for mitochondrial visualization at high resolution, tracking of mitochondrial structures and optical real time monitoring of parameters of mitochondrial function. Among the techniques discussed are the uses of classic imaging techniques such as rhodamine-123, the highly advanced semi-conductor nanoparticles (quantum dots), and wide field microscopy as well as high-resolution multi-photon imaging. We have highlighted the use of these techniques to study mitochondrial function in brain tissue and have included studies from our laboratories in which these techniques have been successfully applied.

Ischemic injury to the CNS results in loss of ionic homeostasis and the development of neuronal death. An increase in intracellular Ca

Nicotinamide adenine dinucleotide (NADH) imaging can be used to monitor neuronal activation and ascertain mitochondrial dysfunction, for example during hypoxia. During neuronal stimulation in vitro, NADH normally becomes more oxidized, indicating enhanced oxygen utilization. A subsequent NADH overshoot during activation or on recovery remains controversial and reflects either increased metabolic activity or limited oxygen availability. Tissue P(2) measurements, obtained simultaneously with NADH imaging in area CA1 in hippocampal slices, reveal that during prolonged train stimulation (ST) in 95% O(2), a persistent NADH oxidation is coupled with increased metabolic demand and oxygen utilization, for the duration of the stimulation. However, under conditions of either decreased oxygen supply (ST-50% O(2)) or enhanced metabolic demand (K(+)-induced spreading depression (K(+)-SD) 95% O(2)) the NADH oxidation is brief and the redox balance shifts early toward reduction, leading to a prolonged NADH overshoot. Yet, oxygen utilization remains elevated and is correlated with metabolic demand. Under these conditions, it appears that the rate of NAD(+) reduction may transiently exceed oxidation, to maintain an adequate oxygen flux and ATP production. In contrast, during SD in 50% O(2), the oxygen levels dropped to a point at which oxidative metabolism in the electron transport chain is limited and the rate of utilization declined.

The accumulation of reactive oxygen species during cellular injury leads to oxidative stress. This can have profound effects on ionic homeostasis and neuronal transmission. c-Aminobutyric acid (GABA) neurotransmission is sensitive to reactive oxygen species, but most studies have indicated that this is due to alterations in GABA release. Here, we determined whether reactive oxygen species can alter GABA A receptorgated Cl ± channels in the adult hippocampus. First, we measured the effects of hydrogen peroxide on intracellular Cl ± using UV laser scanning confocal microscopy and the Cl ± -sensitive probe, 6-methoxy-N-ethylquinolium iodide (MEQ). Superfusion of adult rat hippocampal slices with hydrogen peroxide for 10 min decreased MEQ fluorescence (elevation in [Cl ± ] i ) signi®cantly in area CA1 pyramidal cell soma. Alterations in [Cl ± ] i were prevented by the vitamin E analog Trolox, an antioxidant that scavenges free radicals. After exposure of slices to hydrogen peroxide, the ability of the GABA agonist muscimol to increase [Cl ± ] i was attenuated. To determine if GABA A receptors were sensitive to oxidative insults, the effect of hydrogen peroxide on the binding of [ 35 S]t-butylbicyclophosphorothionate (TBPS) to GABA-gated Cl ± channels was measured using receptor autoradiography and homogenate binding assays. Hydrogen peroxide inhibited [ 35 S]TBPS binding in a regionally selective manner, with the greatest inhibition in cerebral cortex, hippocampus and striatum, areas vulnerable to oxidative stress. Similarly, xanthine and xanthine oxidase, which generate superoxide radicals, reduced [ 35 S]TBPS binding in these regions. The effect of hydrogen peroxide on [ 35 S]TBPS binding was non-competitive and was prevented by Trolox and the iron chelator, deferoxamine. We conclude that reactive oxygen species may compromise GABA A -mediated neuronal inhibition via interaction with pre and postsynaptic sites. A reduction in GABA A -gated Cl ± channel function during periods of oxidative stress may contribute to the development of neuronal damage.

Benzodiazepines protect hippocampal neurons when administered within the first few hours after transient cerebral ischemia. Here, we examined the ability of diazepam to prevent early signals of cell injury (before cell death) after in vitro ischemia. Ischemia in vitro or in vivo causes a rapid depletion of ATP and the generation of cell death signals, such as the release of cytochrome c from mitochondria. Hippocampal slices from adult rats were subjected to 7 min of oxygen-glucose deprivation (OGD) and assessed histologically 3 h after reoxygenation. At this time, area CA1 neurons appeared viable, although slight abnormalities in structure were evident. Immediately following OGD, ATP levels in hippocampus were decreased by 70%, and they recovered partially over the next 3 h of reoxygenation. When diazepam was included in the reoxygenation buffer, ATP levels recovered completely by 3 h after OGD. The effects of diazepam were blocked by picrotoxin, indicating that the protection was mediated by an influx of Cl Ϫ through the GABA A receptor. It is interesting that the benzodiazepine antagonist flumazenil did not prevent the action of diazepam, as has been shown in other studies using the hippocampus. Two hours after OGD, the partial recovery of ATP levels occurred simultaneously with an increase of cytochrome c (ϳ400%) in the cytosol. When diazepam was included in the reoxygenation buffer, it completely prevented the increase in cytosolic cytochrome c. Thus, complete recovery of ATP and prevention of cytochrome c release from mitochondria can be achieved when diazepam is given after the loss of ATP induced by OGD.

Neuronal activity, astrocytic responses to this activity, and energy homeostasis are linked together during baseline, conscious conditions, and short-term rapid activation (as occurs with sensory or motor function). Nervous system energy homeostasis also varies during long-term physiological conditions (i.e., development and aging) and with adaptation to pathological conditions, such as ischemia or low glucose. Neuronal activation requires increased metabolism (i.e., ATP generation) which leads initially to substrate depletion, induction of a variety of signals for enhanced astrocytic function, and increased local blood flow and substrate delivery. Energy generation (particularly in mitochondria) and use during ATP hydrolysis also lead to considerable heat generation. The local increases in blood flow noted following neuronal activation can both enhance local substrate delivery but also provides a heat sink to help cool the brain and removal of waste by-products. In this review we highlight the interactions between short-term neuronal activity and energy metabolism with an emphasis on signals and factors regulating astrocyte function and substrate supply.

Synaptic train stimulation (10 Hz · 25 s) in hippocampal slices results in a biphasic response of NAD(P)H fluorescence indicating a transient oxidation followed by a prolonged reduction. The response is accompanied by a transient tissue PO 2 decrease indicating enhanced oxygen utilization. The activation of mitochondrial metabolism and/or glycolysis may contribute to the secondary NAD(P)H peak. We investigated whether extracellular lactate uptake via monocarboxylate transporters (MCTs) contributes to the generation of the NAD(P)H response during neuronal activation. We measured the effect of lactate uptake inhibition [using the MCT inhibitor a-cyano-4-hydroxycinnamate (4-CIN)] on the NAD(P)H biphasic response, tissue PO 2 response, and field excitatory post-synaptic potential in hippocampal slices during synaptic stimulation in area CA1 (stratum radiatum). The application of 4-CIN (150-250 lmol/L) significantly decreased the reduction phase of the NAD(P)H response. When slices were supplemented with 20 mmol/L lactate in 150-250 lmol/L 4-CIN, the secondary NAD(P)H peak was restored; whereas 20 mmol/L pyruvate supplementation did not produce a recovery. Similarly, the tissue PO 2 response was decreased by MCT inhibition; 20 mmol/L lactate restored this response to control levels at all 4-CIN concentrations. These results indicate that lactate uptake via MCTs contributes significantly to energy metabolism in brain tissue and to the generation of the delayed NAD(P)H peak after synaptic stimulation.

Monitoring changes in the fluorescence of metabolic chromophores, reduced nicotinamide adenine dinucleotide and flavin adenine dinucleotide, and the absorption of cytochromes, is useful to study neuronal activation and mitochondrial metabolism in the brain. However, these optical signals evokedby stimulation, seizures and spreading depression in intact brain differ from those observed in vitro. The responses in vivo consist of a persistent oxidized state during neuronal activity followed by mild reduction during recovery. In vitro, however, brief oxidation is followed by prolonged and heightened reduction, even during persistent neuronal activation. In normally perfused, oxygenated and activated brain tissue in vivo, partial pressure of oxygen (P O2 ) levels often undergo a brief 'dip' that is always followed by an overshoot above baseline, due to increased blood flow (neuronal-vascular coupling). By contrast, in the absence of blood circulation, tissue P O2 in vitro decreases more markedly and recovers slowly to baseline without overshooting. Although oxygen is abundant in vivo, it is diffusion-limited in vitro. The disparities in mitochondrial and tissue oxygen availability account for the different redox responses. Changes in redox level of mitochondrial respiratory chain components can be monitored in live brain tissue by optical imagingChanges in fluorescence of metabolic cofactors [i.e. reduced nicotinamide adenine dinucleotide (NADH) ‡ and flavin adenine dinucleotide (FAD)] involved in energy processes, and in the absorption spectrum of cytochromes, provide a measure of their redox level. Mitochondrial energy metabolism is tightly coupled with neuronal activity, and changes in metabolic activity alter the redox level of these cofactors. Optical changes related to redox state have been used for many years to gauge the metabolic activity in brain and in other organs. Relating redox responses to electrical, mechanical or secretory processes has provided a rich source of data on the coupling of metabolic activity to their function. Such investigations in mammalian brain shed light on the mechanism of seizures, of spreading depression (SD) and of hypoxia and ischemia. Recently, however, a discrepancy has become apparent between the pattern of redox changes in intact, perfused brain 'in vivo' * , and isolated preparations of central neurons, tissue slices and slice cultures maintained 'in vitro' † in an organ bath. This essay aims at resolving the apparent discrepancy.Corresponding author: Turner, D.A. (dennis.turner@duke.edu). ‡ The term 'reduction' is used to denote shifts in redox state away from oxidation, not a decrease in a variable. * To avoid ambiguity, we use 'in vivo' to mean brain tissue in its anatomical location (in situ) and perfused by blood, usually studied in an anesthetized animal with functioning lungs, heart and autonomic reflexes. † 'In vitro' means isolated cells, tissue slices, cell cultures and organotypic slice cultures maintained in an oxygenated, saline-perfused organ bath. NI...