Faculty Bibliography

In many cells, ATP-sensitive K+ channels (KATP channels) couple metabolic state to excitability. In pancreatic beta cells, for example, this coupling regulates insulin release. Although KATP channels are abundantly expressed in the brain, their physiological role and the factors that regulate them are poorly understood. One potential regulator is H2O2. We reported previously that dopamine (DA) release in the striatum is modulated by endogenous H2O2, generated downstream from glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor activation. Here we investigated whether H2O2-sensitive KATP channels contribute to DA-release modulation by glutamate and gamma-aminobutyric acid (GABA). This question is important because DA-glutamate interactions underlie brain functions, including motor control and cognition. Synaptic DA release was evoked by using local electrical stimulation in slices of guinea pig striatum and monitored in real time with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. The KATP-channel antagonist glibenclamide abolished the H2O2-dependent increase in DA release usually seen with AMPA-receptor blockade by GYKI-52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride] and the decrease in DA release seen with GABA-type-A-receptor blockade by picrotoxin. In contrast, 5-hydroxydecanoate, a mitochondrial KATP-channel blocker, was ineffective, as were sulpiride, a D2-receptor antagonist, and tertiapin, a G protein-coupled K+-channel inhibitor. Diazoxide, a sulfonylurea receptor 1 (SUR1)selective KATP-channel opener, prevented DA modulation by H2O2, glutamate, and GABA, whereas cromakalim, a SUR2-selective opener, did not. Thus, endogenous H2O2 activates SUR1-containing KATP channels in the plasma membrane to inhibit DA release. These data not only demonstrate that KATP channels can modulate CNS transmitter release in response to fast-synaptic transmission but also introduce H2O2 as a KATP-channel regulator

Decreased cerebral blood flow, hence decreased oxygen and glucose, leads to ischemic brain injury via complex pathophysiological events, including excitotoxicity, mitochondrial dysfunction, increased intracellular Ca2+, and reactive oxygen species (ROS) generation. Each of these could also contribute to cerebral edema, which is the primary cause of patient mortality after stroke. In vitro brain slices are widely used to study ischemia. Here we introduce a slice model to investigate ischemia-induced edema. Significant water gain was induced in coronal slices of rat brain by 5 min of oxygen and glucose deprivation (OGD) at 35 degrees C, with progressive edema formation after return to normoxic, normoglycemic medium. Edema increased with increasing injury severity, determined by OGD duration (5-30 min). Underlying factors were assessed using glutamate-receptor antagonists (AP5/CNQX), blockade of mitochondrial permeability transition [cyclosporin A (CsA) versus FK506], inhibition of Na+/Ca2+ exchange (KB-R7943), and ROS scavengers (ascorbate, Trolox, dimethylthiourea, Tempol). All agents except KB-R7943 and FK506 significantly attenuated edema when applied after OGD; KB-R7943 was effective when applied before OGD. Significantly, complete prevention of ischemia-induced edema was achieved with a cocktail of AP5/CNQX, CsA and Tempo applied after OGD, which demonstrates the involvement of multiple, additive mechanisms. The efficacy of this cocktail further shows the potential value of combination therapies for the treatment of cerebral ischemia

How glutamate regulates dopamine (DA) release in striatum has been a controversial issue. Here, we resolve this by showing that glutamate, acting at AMPA receptors, inhibits DA release by a nonclassic mechanism mediated by hydrogen peroxide (H(2)O(2)). Moreover, we show that GABA(A)-receptor activation opposes this process, thereby enhancing DA release. The influence of glutamate and GABA on DA release was assessed in striatal slices using carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Modulation by both transmitters was prevented by H(2)O(2)-metabolizing enzymes. In addition, the influence of GABA(A)-receptor activation was lost when AMPA receptors were blocked with GYKI-52466. Together, these data show that modulation of DA release by glutamate and GABA depends on H(2)O(2) generated downstream from AMPA receptors. This is the first evidence that endogenous glutamate can lead to the generation of reactive oxygen species under physiological conditions. We also show that inhibition of DA release by H(2)O(2) is mediated by sulfonylurea-sensitive K(+) channels: tolbutamide blocked DA modulation by glutamate and by GABA. The absence of ionotropic glutamate or GABA receptors on DA terminals indicates that modulatory H(2)O(2) is generated in non-DA cells. Thus, in addition to its known excitatory actions in striatum, glutamate mediates inhibition by generating H(2)O(2) that must diffuse from postsynaptic sites to inhibit presynaptic DA release via K(+)-channel opening. These findings have significant implications not only for normal striatal function but also for understanding disease states that involve DA and oxidative stress, including disorders as diverse as Parkinson's disease and schizophrenia

Ground squirrels tolerate up to 90% reductions in cerebral blood flow during hibernation as well as rapid reperfusion upon periodic arousal from torpor without apparent neurological damage. Thus, hibernation is studied as a model of tolerance to cerebral ischemia and other types of brain injury. Metabolic suppression likely plays a primary adaptive role that allows hibernating species to tolerate dramatic fluctuations in blood flow. Several other aspects of hibernation physiology are also consistent with tolerance to ischemia and reperfusion suggesting that multiple neuroprotective adaptations may work in concert during hibernation. The purpose of the present work is to review evidence for enhanced antioxidant defense systems during hibernation, with a focus on ascorbate, and discuss potential roles of these antioxidants during hibernation. In concert with dramatic decreases in blood flow, nutrient and oxygen delivery, plasma concentrations of the antioxidant ascorbate [(Asc)p] increase 3-5-fold during hibernation. In contrast, during re-warming, [Asc]p declines at a relatively rapid rate that peaks at the time of maximal O(2) consumption. This peak in O(2) consumption also coincides with a brief rise in plasma urate concentration consistent with a surge in reactive oxygen species production. Overall, data suggest that elevated concentration of plasma ascorbate is poised for distribution to metabolically active tissues during the surge in oxidative metabolism that accompanies re-warming during hibernation. This pool of ascorbate, as well as increased expression of other antioxidant defense systems, may protect vulnerable tissues from oxidative stress during hibernation and re-warming from hibernation. Better understanding of the role of ascorbate in hibernation may guide use of ascorbate and other antioxidants in treatment of stroke, head trauma and neurodegenerative disease

Reactive oxygen species (ROS) generated by mitochondrial respiration and other processes are often viewed as hazardous substances. Indeed, oxidative stress, defined as an imbalance between oxidant production and antioxidant protection, has been linked to several neurological disorders, including cerebral ischemia-reperfusion and Parkinson's disease. Consequently, cells and organisms have evolved specialized antioxidant defenses to balance ROS production and prevent oxidative damage. Research in our laboratory has shown that neuronal levels of ascorbate, a low molecular weight antioxidant, are ten-fold higher than those in much less metabolically active glial cells. Ascorbate levels are also selectively elevated in the CNS of anoxia-tolerant reptiles compared to mammals; moreover, plasma and CSF ascorbate concentrations increase markedly in cold-adapted turtles and in hibernating squirrels. Levels of the related antioxidant, glutathione, vary much less between neurons and glia or among species. An added dimension to the role of the antioxidant network comes from recent evidence that ROS can act as neuromodulators. One example is modulation of dopamine release by endogenous hydrogen peroxide, which we describe here for several mammalian species. Together, these data indicate adaptations that prevent oxidative stress and suggest a particularly important role for ascorbate. Moreover, they show that the antioxidant network must be balanced precisely to provide functional levels of ROS, as well as neuroprotection

Endogenous reactive oxygen species (ROS) can act as modulators of neuronal activity, including synaptic transmission. Inherent in this process, however, is the potential for oxidative damage if the balance between ROS production and regulation becomes disrupted. Here we report that inhibition of synaptic transmission in rat hippocampal slices by H2O2 can be followed by electrical hyperexcitability when transmission returns during H2O2 washout. As in previous studies, H2O2 exposure (15 min) reversibly depressed the extracellular population spike (PS) evoked by Schaffer collateral stimulation. Recovery of PS amplitude, however, was typically accompanied by mild epileptiform activity. Inclusion of ascorbate (400 microM) during H2O2 washout prevented this pathophysiology. No protection was seen with isoascorbate, which is a poor substrate for the stereoselective ascorbate transporter and thus remains primarily extracellular. Epileptiform activity was also prevented by the N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonopentanoic acid (AP5) during H2O2 washout. Once hyperexcitability was induced, however, AP5 did not reverse it. When present during H2O2 exposure, AP5 did not alter PS depression by H2O2 but did inhibit the recovery of PS amplitude seen during pulse-train stimulation (10 Hz, 5 s) in H2O2. Inhibition of glutamate uptake by l-trans-2,4-pyrrolidine dicarboxylate (PDC; 50 microM) during H2O2 washout markedly enhanced epileptiform activity; coapplication of ascorbate with PDC prevented this. These data indicate that H2O2 exposure can cause activation of normally silent NMDA receptors, possibly via inhibition of redox-sensitive glutamate uptake. When synaptic transmission returns during H2O2 washout, enhanced NMDA receptor activity leads to ROS generation and consequent oxidative damage. These data reveal a pathological cycle that could contribute to progressive degeneration in neurological disorders that involve oxidative stress, including cerebral ischemia

Midbrain dopamine (DA) cells of the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) exhibit somatodendritic release of DA. To address how somatodendritic release is regulated by synaptic glutamatergic and GABAergic input, we examined the effect of ionotropic-receptor antagonists on locally evoked extracellular DA concentration ([DA]o) in guinea pig midbrain slices. Evoked [DA]o was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. In SNc, evoked [DA]o was 160% of control in the presence of the AMPA-receptor antagonist, GYKI-52466, or the NMDA-receptor antagonist, AP5. Similar increases were seen with the GABAA-receptor antagonist, picrotoxin, or the GABA(B)-receptor antagonist, saclofen. The increase seen with GYKI-52466 was prevented when both picrotoxin and saclofen were present, consistent with normal, AMPA-receptor mediated activation of GABAergic inhibition. The increase with AP5 persisted, however, implicating NMDA-receptor mediated activation of another inhibitory circuit in SNc. In the VTA, by contrast, evoked [DA]o was unaffected by GYKI-52466 and fell slightly with AP5. Neither picrotoxin nor saclofen alone or in combination had a significant effect on evoked [DA]o. When GABA receptors were blocked in the VTA, evoked [DA]o was decreased by 20% with either GYKI-52466 or AP5. These data suggest that in SNc, glutamatergic input acts predominantly on GABAergic or other inhibitory circuits to inhibit somatodendritic DA release, whereas in VTA, the timing or strength of synaptic input will govern whether the net effect on DA release is excitatory or inhibitory

We showed previously that dopamine (DA) release in dorsal striatum is inhibited by endogenously generated hydrogen peroxide (H(2)O(2)). Here, we examined whether endogenous H(2)O(2) can also modulate somatodendritic DA release in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), with companion measurements in DA terminal regions. Evoked DA release was monitored in brain slices using carbon-fiber microelectrodes with fast-scan cyclic voltammetry. Exogenous H(2)O(2) decreased DA release by 50-60% in SNc and VTA but only by 35% in nucleus accumbens. Whether endogenous H(2)O(2) also modulated somatodendritic release was examined using the glutathione peroxidase inhibitor, mercaptosuccinate (MCS), which should increase stimulation-evoked H(2)O(2) levels. In the presence of MCS, DA release was suppressed by 30-40% in SNc as well as in dorsal striatum and nucleus accumbens. In striking contrast, DA release in the VTA was unaffected by MCS. These data are consistent with stronger H(2)O(2) regulation or lower H(2)O(2) generation in VTA than in the other regions. Importantly, oxidative stress has been linked causally to Parkinson's disease, in which DA cells in SNc degenerate, but VTA cells are spared. The present data suggest that differences in oxidant regulation or generation between SNc and VTA could contribute to this

Huntington's disease is an autosomal dominant hereditary neurodegenerative disorder characterized by severe striatal cell loss. Dopamine (DA) has been suggested to play a role in the pathogenesis of the disease. We have previously reported that transgenic mice expressing exon 1 of the human Huntington gene (R6 lines) are resistant to quinolinic acid-induced striatal toxicity. In this study we show that with increasing age, R6/1 and R6/2 mice develop partial resistance to DA- and 6-hydroxydopamine-mediated toxicity in the striatum. Using electron microscopy, we found that the resistance is localized to the cell bodies and not to the neuropil. The reduction of dopamine and cAMP regulated phosphoprotein of a molecular weight of 32 kDa (DARPP-32) in R6/2 mice does not provide the resistance, as DA-induced striatal lesions are not reduced in size in DARPP-32 knockout mice. Neither DA receptor antagonists nor a N-methyl-d-aspartate (NMDA) receptor blocker reduce the size of DA-induced striatal lesions, suggesting that DA toxicity is not dependent upon DA- or NMDA receptor-mediated pathways. Moreover, superoxide dismutase-1 overexpression, monoamine oxidase inhibition and the treatment with the free radical scavenging spin-trap agent phenyl-butyl-tert-nitrone (PBN) also did not block DA toxicity. Levels of the antioxidant molecules, glutathione and ascorbate were not increased in R6/1 mice. Because damage to striatal neurons following intrastriatal injection of 6-hydroxydopamine was also reduced in R6 mice, a yet-to-be identified antioxidant mechanism may provide neuroprotection in these animals. We conclude that striatal neurons of R6 mice develop resistance to DA-induced toxicity with age

Somatodendritic release of dopamine (DA) in midbrain represents a novel form of intercellular signaling that inherently differs from classic axon-terminal release. Here we report marked differences in the Ca(2+) dependence and time course of stimulated increases in extracellular DA concentration ([DA](o)) between the substantia nigra pars compacta (SNc) and striatum. Evoked [DA](o) was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry in brain slices. In striatum, pulse-train stimulation (10 Hz, 30 pulses) failed to evoke detectable [DA](o) in 0 or 0.5 mm Ca(2+) but elicited robust release in 1.5 mm Ca(2+). Release increased progressively in 2.0 and 2.4 mm Ca(2+). In sharp contrast, evoked [DA](o) in SNc was nearly half-maximal in 0 mm Ca(2+) and increased significantly in 0.5 mm Ca(2+). Surprisingly, somatodendritic release was maximal in 1.5 mm Ca(2+), with no change in 2.0 or 2.4 mm Ca(2+). Additionally, after single-pulse stimulation, evoked [DA](o) in striatum reached a maximum (t(max)) in <200 msec, whereas in SNc, [DA](o) continued to rise for 2-3 sec. Similarly, the time for [DA](o) to decay to 50% of maximum (t(50)) was 12-fold longer in SNc than striatum. A delayed t(max) in SNc compared with striatum persisted when DA uptake was inhibited by GBR-12909 and D(2) autoreceptors were blocked by sulpiride, although these agents eliminated the difference in t(50). Together, these data implicate different release mechanisms in striatum and SNc, with minimal Ca(2+) required to trigger prolonged DA release in SNc. Coupled with limited uptake, prolonged somatodendritic release would facilitate DA-mediated volume transmission in midbrain