Terry C. Pellmar

Electrophysiological correlates of peroxide damage in guinea pig hippocampus in vitro

To study the effects of active oxygen on neuronal electrophysiology, hippocampal brain slices were exposed to hydrogen peroxide plus ferrous sulfate which react to produce hydroxyl free radicals. Analysis of extracellularly recorded somatic and dendritic responses to orthodromic stimulation indicated a decrease in both synaptic efficacy and impairment of action potential generation.

Use of brain slices in the study of free-radical actions

To understand the neuropathological roles of free radicals we investigate their actions in a model neuronal system, the hippocampal brain slice. Free radicals can be generated through a number of methods: hydrogen peroxide to produce hydroxyl radicals, dihydroxyfumarate to generate superoxide and ionizing radiation producing a variety of radical species. We find that free radicals have a number of profound effects in this system, which can be prevented by free-radical scavengers and antioxidants. With exposure to free radicals, the ability to generate spikes and synaptic efficacy are impaired. Decreased spike generating ability is correlated with lipid peroxidation. No change in membrane potential, membrane resistance, or many of the potassium currents can account for the effect on spike generation. Protein oxidation is likely to underlie synaptic damage. Both inhibitory and excitatory synaptic potentials are reduced by free-radical exposure. Presynaptic mechanisms are implicated. Lower concentrations of radicals prevent the maintenance of long-term potentiation, perhaps through oxidation of the NMDA receptor. The actions of the free radicals are often reversible because of the presence of repair mechanisms, such as glutathione, in hippocampal slices. The brain slice preparation has allowed us to begin to understand the electrophysiological and biochemical consequences of free-radical exposure.

Free Radicals Enhance Basal Release of D‐[3H]Aspartate from Cerebral Cortical Synaptosomes

Abstract: Excessive generation of free radicals has been implicated in several pathological conditions. We demonstrated previously that peroxide‐generated free radicals decrease calcium‐dependent high K+‐evoked l‐[3H]‐glutamate release from synaptosomes while increasing calcium‐independent basal release. The present study evaluates the nonyesicular release of excitatory amino acid neurotransmitters, using d‐[3H]aspartate as an exogenous label of the cytoplasmic pool of l‐glutamate and l‐aspartate. Isolated presynaptic nerve terminals from the guinea pig cerebral cortex were used to examine the actions and interactions of peroxide, iron, and desferrioxamine. Pretreatment with peroxide, iron alone, or peroxide with iron significantly increased the calcium‐independent basal release of d‐[3H]aspartate. Pretreatment with desferrioxamine had little effect on its own but significantly limited the enhancement by peroxide. High K+‐evoked release in the presence of Ca2+ was enhanced by peroxide but not by iron. These data suggest that peroxide increases nonvesicular basal release of excitatory amino acids through Fenton‐generated hydroxyl radicals. This release could cause accumulation of extracellular excitatory amino acids and contribute to the excitotoxicity associated with some pathologies.

Role of glutathione in repair of free radical damage in hippocampus in vitro

Depletion of glutathione (GSH), an intrinsic antioxidant, increases vulnerability to free radical damage in a number of cell systems. This study investigates the role of GSH in limiting electrophysiological damage and/or recovery from free radical exposure in slices of guinea pig hippocampus. Synaptic potentials (PSPs) and population spikes (PSs) were recorded from field CA1. Free radicals were generated from 0.006% peroxide through the Fenton reaction. Analysis of the input-output curves showed that peroxide treatment decreased PSPs and impaired ability of the PSPs to generate PSs as previously reported. Recovery was nearly total within a half hour. Treatment with 5 mM buthionine sulfoximine (BSO) for 2 h depleted hippocampal GSH to 79.2% of control values. The extent of free radical damage was not increased. Recovery, however, was only partial. GSH was further depleted by oxidation with diamide or covalent bonding with dimethyl fumarate (DMF) immediately before and during the peroxide treatment. Neither diamide nor DMF treatment in BSO-incubated tissue enhanced peroxide-induced electrophysiological deficits. Following these treatments, however, tissue showed little recovery from free radical damage. We conclude that glutathione is essential for repair processes in hippocampal neurons exposed to oxidative damage.

Peroxide effects on [3H]l-glutamate release by synaptosomes isolated from the cerebral cortex

Basal (non-depolarized) and high K(+)-stimulated [3H]L-glutamate release in the presence and absence of Ca2+ were assessed using presynaptic nerve terminals (synaptosomes) isolated from the cerebral cortex of the guinea pig. Basal glutamate release was found to be Ca(2+)-independent and was significantly increased following treatment with hydrogen peroxide (H2O2). On the other hand, depolarization-induced release had both a Ca(2+)-dependent and Ca(2+)-independent component. Both components of stimulated release were suppressed by H2O2. In fact, Ca(2+)-dependent evoked release was virtually eliminated by H2O2 pretreatment. The data suggest that H2O2 exerts a differential effect on the neurochemical mechanisms involved in basal and stimulated glutamate release at the presynaptic nerve terminal.

Ionic Mechanism of a Voltage-Dependent Current Elicited by Cyclic AMP

Intracellular pressure injection of cyclic AMP induces a slow voltage-dependent inward current in some neurons of Aplysia californica.The time course, voltage dependence, and ionic sensitivities of this response are nearly identical to those of the voltage-dependent calcium current induced by serotonin in the same preparation. The response to cyclic AMP is unaffected by changes in the extracellular concentration of chloride or potassium. The current is slowly but minimally reduced by a sodium-free solution. The calcium channel blocker, cadmium, blocks the current elicited by injection of cyclic AMP. The data presented here suggest that cyclic AMP can induce a voltage-dependent calcium current.

Effect of oxidative stress on excitatory amino acid release by cerebral cortical synaptosomes

Previous studies in our laboratory have suggested that an oxidation reaction is responsible for the actions of free radicals to decrease synaptic potentials. Recently we observed that free radicals both decreased depolarization-induced vesicular release and enhanced basal, nonvesicular release of the excitatory amino acid, [3H]L-glutamate. In order to evaluate the contribution of oxidative reactions to this latter effect, we evaluated the actions of the oxidizing agent chloramine-T on synaptosomal release of excitatory amino acids, using [3H]D-aspartate as the exogenous label. Basal and depolarization evoked [3H]D-aspartate release were calcium-independent and nonvesicular. Chloramine-T pretreatment significantly increased basal release, while having no effect on high K(+)-evoked release. These data suggest that an oxidative process can mimic the free radical increase of basal release, as well as the decrease in synaptic potentials. On the other hand, the calcium-independent-evoked release may involve a different mechanism. Our results demonstrate that under basal, nondepolarizing conditions, oxidative stress exerts an adverse effect on the presynaptic nerve terminal, resulting in an increased release of potentially damaging excitatory amino acid neurotransmitters.

Oxidative damage in the guinea pig hippocampal slice.

Free radicals and active oxygen compounds are implicated in brain ischemia and head trauma. Previous studies have shown that free radicals, generated by radiation and through the Fenton reaction, produce both synaptic and postsynaptic damage in the hippocampal brain slice. To evaluate the contribution of oxidation to the observed damage, the actions of the oxidants, chloramine-T and N-chlorosuccinimide (NCS), were studied on electrophysiological responses in the hippocampal slice isolated from the brains of guinea pigs. Electrical stimulation of afferents to neurons of the CA1 region of hippocampus evoked a population postsynaptic potential (population PSP) in the dendritic layer and a population spike in the cell body layer. Chloramine-T (25-500 microM) and NCS (750-4000 microM) decreased the population spike in a dose-dependent manner (ED50 congruent to 125 microM and 1100 microM, respectively). Input/output curves revealed that both the population PSP were significantly reduced with both oxidants; but, the ability of the population PSP to produce a population spike was not impaired. These studies suggest that oxidation reactions can account for the synaptic component of the damage produced by free radicals but can not account for the postsynaptic effects.

Reactive Oxygen Species on Neural Transmissiona

Normal function of the central nervous system requires an intricate balance of numerous electrical and chemical processes. Neurons process both excitatory and inhibitory inputs that govern their level of excitability. Synthesis, breakdown, uptake, and release mechanisms control the availability of neurotransmitters. Neuronal excitability is modulated through membrane currents, second messengers, and the extracellular microenvironment. Glial cells play an important role in maintaining the equilibrium of the nervous system. We are now finding that many of these processes are vulnerable to reactive oxygen species (ROS). ROS are generated in excess in many nervous system pathologies, such as Alzheimers disease, Parkinsons disease, Downs syndrome, ischemia/reperfusion injury, and amyotrophic lateral sclerosis, which have very different etiologies and symptoms. ROS could be a primary factor, targeting different processes and causing slightly different perturbations of the nervous systems balance. Disruptions of the homeostasis of the nervous system by free radicals can manifest itself in any number of ways, including hyperexcitability, impaired neuronal plasticity, and even cell death. Our laboratory has been characterizing the effects of ROS on neural transmission in an effort to elucidate the consequences of freeradical exposure and to understand the functional interactions that underlie the diversity of observed symptoms.

Dithiothreitol elicits epileptiform activity in CA1 of the guinea pig hippocampal slice

Dithiothreitol (DTT) is a sulfhydryl reducing agent used as a radioprotectant. Exposure of hippocampal slices for 30 min to 0.5 mM DTT irreversibly increased the orthodromic population spike amplitude, promoted repetitive firing and induced spontaneous epileptiform activity in the CA1 subfield. The same concentration of the oxidized form of DTT did not increase hippocampal excitability. Although the slope of the population synaptic response to afferent stimulation (popPSP) was unchanged by DTT, the duration of the popPSP was prolonged. Recurrent inhibition was unaffected. DTT probably exerts its effects through an irreversible chemical reaction with cellular components. Possible mechanisms of DTT-induced epileptiform activity are discussed.