Penehyclidine hydrochloride protects against oxygen and glucose deprivation injury by modulating amino acid neurotransmitters release

Abstract

Penehyclidine hydrochloride (PHC) is an anticholinergic agent, with only high degree of selectivity for M₁ and M₃ receptor subtypes. In this study, we investigated whether PHC could play a protective effect on hippocampal slice against oxygen and glucose deprivation (OGD), as well as related to the change of amino acid neurotransmitters release. Penehyclidine hydrochloride 2, 10, and 50 μM doses were adopted in the lactate dehydrogenase (LDH) leakage assay and triphenyl tetrazolium chloride (TTC) staining. The spontaneous miniature excitatory postsynaptic currents (mEPSCs) and amino acid neurotransmitters were detected by electrophysiology method and high-performance liquid chromatography (HPLC), respectively. Our study showed that PHC can lessen the LDH leakage ratio and tissue injury values according to TTC staining. Penehyclidine hydrochloride decreased the content of aspartate acid (Asp) and glutamate (Glu), and elevated the content of glycine (Gly) and gamma-aminobutyric acid (GABA). Ischemia increased the amplitude and frequency of the mEPSCs, but PHC obviously decreased the frequency and amplitude of mEPSCs. Thus, the study reveals the fact that PHC protects hippocampus slice against OGD injury by decreasing excitatory amino acids release and increasing inhibitory amino acids release.     

Calcium-dependent release of glutamate from human cerebral cortex.

The release of the excitatory amino acids glutamate and aspartate from human neocortex was investigated in vitro by utilizing brain tissue removed during anterior temporal lobectomies for tumor or epilepsy. Depolarization (50 mM K+) increased the glutamate release to 291% of control (809 pmol/mg/min) during blocked synaptic transmission and to 669% (1859 pmol/mg/min) when synaptic transmission was not blocked. Aspartate release increased to 141% (326 pmol/mg/min) and 178% (412 pmol/mg/min) respectively. The difference between release with and without blocked synaptic transmission was statistically significant only for glutamate (P less than 0.01). These data provides evidence for a Ca(2+)-dependent release of glutamate, supporting a possible role of this amino acid as a neurotransmitter in human neocortex.     

Protective effect of L-trans-pyrrolidine-2,4-dicarboxilic acid preload against cell death induced by oxygen/glucose deprivation in differentiated PC12 cells

It has been postulated that cellular glutamate is released into the extracellular fluid when the energy supply of the brain is compromised (i.e., anoxia or oxygen/glucose deprivation), and there the amino acid triggers the so-called excitotoxic cascade, causing neuronal death. Several mechanisms for this release have been postulated, and, by using glutamate transporter inhibitors, several authors have established that reversed uptake is the major mechanism through which glutamate is released in acute oxygen/glucose deprivation. We have studied the effect of the slowly transported glutamate analogue L-trans-pyrrolidine-2,4-dicarboxilic acid (PDC) preload on glutamate release and cell death in an in vitro model of oxygen plus glucose deprivation with differentiated PC12 cells. As expected, we found that PDC preload inhibits glutamate release induced by oxygen/glucose deprivation, supporting the conclusion that it occurs via reverse transport. In addition, we show that PDC preload but not the nontransportable glutamate uptake inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) protects cells against the death induced by oxygen/glucose deprivation, indicating that PDC entry into the cell is necessary for this protective effect. This protection does not correlate with the extracellular glutamate concentration or changes in proteins synthesis rate and eukaryotic initiation 2 phosphorylation. Oxygen/glucose deprivation induces a significant increase in glutathione levels in both unloaded and PDC-preloaded cells, but this increase is not due to up-regulation of glutamate cysteine ligase levels. Intracellular glutathione disulfide (GSSG) significantly increased after oxygen/glucose deprivation. It was also interesting that intracellular GSSG levels in PDC-preloaded cells under oxygen/glucose deprivation strongly correlate with the protection exerted by this compound against cell death.     

Potassium-stimulated release of taurine from cultured cerebellar granule neurons is associated with cell swelling

Effects of increased concentrations of potassium and of hyposmolar conditions on release of taurine were investigated in cerebellar granule neurons cultured from mice. It was found that increases in the external potassium concentration as well as decreases in osmolarity dose-dependently increased release of exogenously supplied [3H]-taurine and endogenous taurine from the neurons. The release of endogenous taurine elicited by a reduction of the osmolarity of the incubation media to 70% or 50% was much more pronounced than that of other amino acids, particularly glutamine, the release of which was not affected at all. The potassium-stimulated release of [3H]-taurine was strictly chloride dependent and it was inhibited by an increase of the osmolarity of the media as well as by 4,4'-diisothyocyanostilbene-2,2'-disulfonate (DIDS) (100 microM). Moreover, a similar increase in the potassium concentration led to an increase in intracellular volume (swelling), a process which was also chloride dependent. It is concluded that potassium-stimulated taurine release from cerebellar granule neurons is associated with cell volume changes and that taurine is likely to play a role as an osmotically active substance in these neurons.     

Exocytotic release of alanine from cultured cerebellar neurons

Many neurons release a variety of amino acids in response to depolarizing stimuli. Although some of these amino acids, namely, glutamate, aspartate, and gamma-aminobutyric acid (GABA), have been qualified as neurotransmitters, functional roles of the other amino acids including alanine remain obscure. We investigated the mechanism and the origin of alanine release from cultured rat cerebellar cells. High-K(+)-induced depolarization produced a considerable amount (139+/-8 pmol/2 min/dish) of alanine release, comparable to that of glutamate (103+/-7 pmol/2 min/dish). Other depolarizing agents including veratridine or 4-aminopyridine also induced alanine release, suggesting that the major source is excitable neurons, rather than non-excitable glial cells. Depolarization-evoked alanine release was suppressed in the absence of extracellular Ca(2+), and was almost abolished by treating the cells with botulinum type B neurotoxin (BoNT/B), indicating that alanine is released by Ca(2+)-dependent exocytosis of vesicle-associated membrane protein-2 (VAMP-2)-containing vesicles. The properties of alanine release were different from those of glutamate and GABA in several aspects: (a) Depolarization-dependent alanine release appeared as early as 7 days in vitro, much earlier than that of GABA. (b) Fifty microM kainate, which causes selective cell death of GABAergic neurons in the culture, only partially reduced alanine release, whereas it had no effect on glutamate release. (c) Alanine release was not affected by phorbol ester, which enhanced glutamate and GABA release in a kinase-dependent manner. We therefore conclude that alanine release occurs via exocytosis of a pool of synaptic vesicles distinct from those containing glutamate or GABA.     

Differential effects of hypertonic mannitol and glycerol on rat brain metabolism and amino acids

Intraperitoneal injections in rats of two different dosages of hypertonic solutions containing mannitol or glycerol caused complex and differential changes in brain amino acids. When plasma osmolalities were elevated to toxic levels of 397–432 mOsm/kg H₂O, brain sodium was increased, whereas plasma sodium was decreased. Brain potassium was not affected. Brain water decreased significantly, concomitant with elevation of plasma osmolality. Both brain lactic acid and [¹²⁵I]albumin space rose significantly. Brain amino acids (mostly aliphatic and basic amino acids) as well as GABA and glycine (putative inhibitory neurotransmitters) increased after both mannitol and glycerol. Ammonia was stimulated by mannitol but was unaffected by glycerol. Plasma amino acids, which generally increased after mannitol, were decreased by glycerol. When the plasma osmolalities were elevated only to moderate levels (about 350 mOsm/kg H₂O, only glycerol induced a significant increase in brain taurine, aspartic acid, alanine, leucine and lysine. Thus, with moderate hyperosmolality, glycerol has striking effects on brain amino acid metabolism that are not observed with mannitol.     

Amino acids acting as transmitters in amyotrophic lateral sclerosis (ALS)

Objectives - In amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of unknown origin, excitotoxic mechanisms are supposed to be involved. Divergent results are, however, presented either because of the heterogeneity of this disease, and/or different methodologies used to evaluate the excitotoxic amino acids content. The results of the most sensitive high performance liquid chromatography (HPLC) techniques with precolumn derivatization of fasting serum and CSF glutamate, aspartate, glycine and γ-aminobutyric acid (GABA) in mild and severely progressing ALS cases are presented here. Material and methods - We studied 25 ALS patients with different course of the disease and controls, which consisted of 10 cases with other motor neuron diseases and 20 healthy, age-matched subjects. Results - In the ALS patients with a mild course of the disease serum glutamate and aspartate content was either normal or slightly decreased, in all of these cases a rise in GABA and glycine was present. In the severely progressing ALS cases serum glutamate and aspartate was increased. The GABA content was either normal or increased, the glycine level appeared to be either normal or decreased. In CSF the amino acids changes in ALS were less pronounced as compared to serum. The most frequent finding was the increase in GABA concentration both in the mild and the severely progressing group. CSF glutamate in ALS patients with mild course of the disease was decreased, in the severely progressing cases the glutamate level appeared in a broad range from decreased to increased values. CSF aspartate was either normal or elevated, glycine values were present in a broad range from decreased to increased values. In the other tested motor neuron diseases no consistent changes in serum and CSF amino acids concentration was observed. Conclusions - The data from serum and CSF indicate that in ALS an imbalance between excitatory and inhibitory amino acids might be present in the brain, which may be induced in different ways in particular ALS patients. It may be an important factor for the mediation of neurons death.     

Neuroprotection Promoted by Guanosine Depends on Glutamine Synthetase and Glutamate Transporters Activity in Hippocampal Slices Subjected to Oxygen/Glucose Deprivation

Guanosine (GUO) has been shown to act as a neuroprotective agent against glutamatergic excitotoxicity by increasing glutamate uptake and decreasing its release. In this study, a putative effect of GUO action on glutamate transporters activity modulation was assessed in hippocampal slices subjected to oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia. Slices subjected to OGD showed increased excitatory amino acids release (measured by d-[³H]aspartate release) that was prevented in the presence of GUO (100 µM). The glutamate transporter blockers, DL-TBOA (10 µM), DHK (100 µM, selective inhibitor of GLT-1), and sulfasalazine (SAS, 250 µM, Xc^? system inhibitor) decreased OGD-induced d-aspartate release. Interestingly, DHK or DL-TBOA blocked the decrease in glutamate release induced by GUO, whereas SAS did not modify the GUO effect. GUO protected hippocampal slices from cellular damage by modulation of glutamate transporters, however selective blockade of GLT-1 or Xc- system only did not affect this protective action of GUO. OGD decreased hippocampal glutamine synthetase (GS) activity and GUO recovered GS activity to control levels without altering the kinetic parameters of GS activity, thus suggesting GUO does not directly interact with GS. Additionally, the pharmacological inhibition of GS activity with methionine sulfoximine abolished the effect of GUO in reducing d-aspartate release and cellular damage evoked by OGD. Altogether, results in hippocampal slices subjected to OGD show that GUO counteracts the release of excitatory amino acids, stimulates the activity of GS, and decreases the cellular damage by modulation of glutamate transporters activity.     

Profiles of extracellular amino acid changes in focal cerebral ischaemia: Effects of mild hypothermia

Abstract

Mild hypothermia has been recently proposed as a therapeutic approach for ameliorating ischaemic cerebral damage. The protective potential of mild hypothermia, however, may be dependent on its ability to reduce the efflux of potentially excitotoxic amino acids and the severity of ischaemia. In this study, we examined the effects of mild brain hypothermia (33 °C) in a rabbit model of permanent focal ischaemia. In vivo microdialysis was used to measure extracellular amino acids in central and peripheral regions of the ischaemic cortex. In normothermic ischaemia (n = 7), glutamate, alanine, taurine, and phosphoethanolamine increased above baseline levels by about 2 h post-ischaemia. Mild hypothermia (n = 7) reduced glutamate efflux only in the central regions and increased alanine efflux in the peripheral regions of ischaemia. There were no significant differences in other amino acid levels between the two temperature groups. Haematoxylin-eosin histology did not demonstrate hypothermic protection in the ischaemic hemisphere. The lack of neuroprotection in this study may correspond with the sustained release of glutamate in the peripheral regions of ischaemia even with lowered brain temperature. These results suggest that hypothermic reduction of excitotoxic perturbations may be more important in the ischaemic periphery than the core. [Neurol Res 1993; 15: 281-287]     

Patterns of endogenous amino acid release from slices of rat and guinea-pig olfactory cortex

A study has been made of the effects of depolarizing stimuli on the release of endogenous amino acid neurotransmitter candidates (aspartate, glutamate, GABA and taurine) from in vitro preparations of rat and guinea pig olfactory cortex. Exposure of small cubes of olfactory cortex tissue from either species to potassium chloride (50 mM) was accompanied by a calcium-dependent release of aspartate, glutamate and GABA. A similar release pattern was evoked by protoveratrine A (100 μM) although the release was largely calcium-independent. Neither agent led to increased release of taurine. Electrical stimulation of the excitatory input (lateral olfactory tract) of freshly prepared, synaptically intact olfactory cortex slices of both species induced significant release of aspartate and GABA from the uncut pial surface and of aspartate, GABA and glutamate from the cut surface. Evoked taurine release occurred from both surfaces of rat olfactory cortex slices but no release was detected from guinea pig olfactory cortex slices. These patterns of release were unaffected by changes in stimulus frequency and were mimicked by protoveratrine A (100 μM) applied to one or other surface. Preincubation of slices from rats for 2 h led to loss of tissue amino acids and to changes in their release patterns; the presence of glutamine (5 mM) during preincubation prevented the loss of amino acids but did not alter their pattern of release. Because of the close similarities between both the electrophysiological properties and the patterns of amino acid release it is concluded that there is probably an identity of amino acid neurotransmitters (aspartate, glutamate and GABA) in rat and guinea pig olfactory cortex. The role of taurine in the rat olfactory cortex is unknown but would seem unlikely to be that of a neurotransmitter. The results are discussed: (i) in terms of the cellular origins of the released amino acids; and (ii) with respect to apparent experimental discrepancies which have appeared in the literature^3,10.     

Glutamate antagonists in therapy of stroke

It is well established that excitotoxicity is a key mechanism of tissue destruction in focal cerebral ischemia (stroke). Very soon after onset of a critical perfusion deficit energy failure leads to neuronal depolarization and release of excitatory amino acids, most notably glutamate. At the same time, energy dependent reuptake of excitatory amino acids is impede. Overstimulation of glutamate receptors (NMDA, AMPA/kainate, metabotropic) induces dramatically increased intracellular Ca2+ concentrations, release of K+ into the extracellular space, and cell swelling due to the passive movement of water with Na+ influx. The massively increased intracellular second messenger Ca2+ triggers numerous deleterious processes, including free radial formation and membrane degradation, mitochondrial dysfunction, inflammation, DNA-damage and apoptosis. A plethora of experimental studies have convincingly demonstrated the relevance of excitotoxicity in focal cerebral ischemia, and pointed to very effective experimental treatment strategies, many of which involve the blockade of glutamate receptors. Unfortunately, large clinical studies were so far unable to replicate the animal data in human stroke patients. This article, by reviewing excitotoxic damage of focal cerebral ischemia in the context of a complex pathophysiological cascade, aims at explaining this failure and stimulating further efforts in drug design and clinical evaluation to establish the first neuroprotective therapy of human stroke.     

Activity-dependent plasticity of inhibitory and excitatory amino acid transmitter systems in cultured rat cerebral cortex

Chronic suppression of spontaneous bioelectric activity in cultures of dissociated fetal rat cerebral cortex increases neuronal cell death and results in electrophysiological changes which indicate an altered balance between excitatory and inhibitory neurotransmission in culture. To delineate whether alterations in neurotransmitter release could underlie this imbalance, we investigated the effects of chronic tetrodotoxin (TTX) treatment on the content and release of glutamate, aspartate and γ-aminobutyric acid (GABA) in culture. Chronic TTX treatment decreased the content of all amino acids investigated. However, only GABA was decreased relative to the neuronal marker NSE (neuron-specific enolase), indicating a disproportionate loss of GABA production following chronic silencing. Depolarization-induced release of GABA, glutamate and aspartate increased about 10-fold between 7 and 21 days in control cultures. Chronic TTX treatment significantly increased the depolarization-induced release of glutamate and aspartate at 7 days in vitro relative to control levels. At all ages it caused a two-fold increase in the ratio of evoked excitatory amino acid release to that of GABA. These observations suggest that chronic silencing of developing neocortex cell cultures increases the ratio of excitatory to inhibitory synaptic activity either by differential cell death or by reduced synaptic efficiency, on which a decrease in GABA neurotransmission appears to play a major role. Since similar mechanisms may be involved in activity-dependent plasticity in vivo, these cultures provide a useful model to analyse this phenomenon at the cell biological and molecular level.     

Peripheral sensory stimulation and the release of transmitter amino acids in vivo from specific regions of cerebral cortex

The effects of sensory stimulation on the release of amino acids from sensorimotor and visual cortex have been studied using a superfusion technique. Electrical stimulation of the brachial plexus contralateral to the superfusion cannula increased significantly the release of glutamate and glutamine from the sensorimotor cortex of anaesthetized rats. No clear effect was observed with the other amino acids. Stimulation of the ipsilateral plexus had no effect on glutamate and glutamine release. In unanaesthetized animals stimulation of the contralateral brachial plexus raised the levels of all the amino acids in sensorimotor cortex superfusate. Weak photic stimulation of the eyes of dark-adapted rats increased glutamate release from the visual cortex but caused no significant change in the release of other amino acids. All evoked increases in amino acids release were reversible at the cessation of the stimuli.     

Cytotoxic effect of Ca++ released from intracellular stores during cerebral energy deprivation

Abstract

The increase in cytoplasmatic calcium concentration during cerebral ischemia has been proposed as a key event leading to neuronal death. In order to investigate a possible role of calcium-release from intracellular stores in ischemic neuronal injury; intracellular calcium pools were depleted prior to ischemia by the use of thapsigargin. Evoked activity (population spike) in rat hippocampal slices was monitored during a 30 min control period[ 9 min of energy deprivation and 60 min of recovery. The population spike recovered to 27% (17-33) (median and 95% confidence interval) following energy deprivation in normal calcium, to 56% (50-58) in calcium-free incubation fluid and to 83% (75-88) in slices pretreated with 1 fiM thapsigargin. Combining calcium removal and thapsigargin pretreatment did not improve recovery further. Both removal of extracellular calcium and emptying intracellular calcium stores prior to energy deprivation thus improved functional recovery following energy deprivation, however the latter was more effective. These results suggest that calcium release from intracellular stores maybe of major importance in calcium-related neuronal injury during cerebral ischemia. [Neurol Res 1996; 18: 499-504]     

Electrophysiological and neurochemical study of the rat geniculo‐cortical pathway. Evidence for glutamatergic neurotransmission

The projection from the dorsal lateral geniculate nucleus to the primary visual cortex of the rat was studied electrophysiologically. Electrical stimulation of the dorsal lateral geniculate nucleus and the optic tract produced three types of responses on neurons of area 17: excitation followed by inhibition, excitation and inhibition. These results extend and confirm, in adult rats, previous studies done in rat geniculate-visual cortex cocultures preparations in vitro. The role of glutamate in the neurotransmission of the rat geniculo-cortical pathway was also investigated. In a first set of experiments, the effects of kynurenate, an antagonist of glutamate receptors, on visual cortex neurons with a monosynaptic excitatory response to dorsal lateral geniculate nucleus stimulation were studied. Microiontophoresis of kynurenate in area 17 neurons selectively suppressed the excitatory response to dorsal lateral geniculate nucleus and optic tract stimulation. In a second set of experiments, the effects of electrical stimulation of the dorsal lateral geniculate nucleus and the optic tract on the release of amino acids in the rat visual cortex in vivo were studied. Using the push–pull method, we perfused a discrete region of the visual cortex with artificial cerebrospinal fluid (CSF), and the amino acid content of the perfusates was analysed by high performance liquid chromatography (HPLC). Stimulation of either the dorsal lateral geniculate nucleus or the optic tract significantly increased glutamate release in area 17. The rest of the amino acids studied did not show significant changes. The results provide evidence for the participation of glutamate in the neurotransmission of the geniculo-cortical pathway in the rat.     

Effects of d-amphetamine administration on the release of endogenous excitatory amino acids in the rat nucleus accumbens

• 1.1. The effects of acute D-amphetamine administration to rats on the release of endogenous excitatory amino acids from nucleus accumbens slices were studied.
• 2.2. D-amphetamine (5 mg/kg and 10 mg/kg; i.p.) significantly increased the spontaneous release of aspartate and glutamate from nucleus accumbens slices.
• 3.3. In contrast, D-amphetamine either produced no change or rather decreased K⁺ (40 mM)-evoked and N-methyl-D-aspartate (100 μM)-evoked release of aspartate and glutamate from the slices, respectively.
• 4.4. When D-amphetamine treated rats were pretreated with haloperidol, the effects of D-amphetamine on the spontaneous release of excitatory amino acids were not produced, whereas its effects on N-methyl-D-aspartate-evoked release remained unchanged.
• 5.5. These data suggest that amphetamine produces changes in excitatory amino acid-mediated transmission in the nucleus accumbens, that may play a role in amphetamine-induced behavioral or psychotomimetic effects.

     

Release of excitatory and inhibitory amino acids from the locus coeruleus of conscious rats by cardiovascular stimuli and various forms of acute stress

The release of amino acids in the locus coeruleus (LC) of conscious, freely moving rats was studied in time periods of 3 min by use of push-pull superfusion under basal conditions and during application of various experimental stimuli known to influence the activity of the LC-noradrenergic system. Tail pinch for 3 min led immediately to a pronounced tetrodotoxin-sensitive increase in the release rates of the excitatory amino acids (EAA) glutamate (Glu) and aspartate (Asp) and to moderate increases in GABA and taurine (Tau) outflow. Immobilization stress for 9 min elevated the release of the EAA Glu and Asp, as well as that of the inhibitory amino acid GABA to a similar extent. A fall of blood pressure (BP) by nitroprusside or haemorrhage slightly enhanced the release rates of Glu and Asp. Noradrenaline-induced rise in BP, as well as hypervolaemia increased the release rate of GABA, but did not influence the release rates of Glu, Asp, Tau and arginine (Arg). The results provide direct evidence that the amino acid release pattern in the LC of conscious rats differs in response to various stimuli, according to the modality of the stimulus. A functional significance of excitatory and inhibitory amino acids in the regulation of LC activity during stress and haemodynamic changes is suggested.     

Evidence for a direct action of N-methylaspartate on non-neuronal cells

The effects of N-methylaspartate (NMA) on extracellular amino acids and purine catabolites in the hippocampus were studied with brain dialysis in rats with unilateral hippocampal NMA lesions. In the lesioned side, an increased basal output of glutamine was observed while glutamate was significantly decreased. NMA evoked a drop in extracellular glutamine. The effect was not observed in the lesioned hippocampus. NMA markedly enhanced the release of taurine and phosphoethanolamine (PEA). This response was unchanged in NMA-lesioned hippocampus. Analysis of the tissue content of endogenous amino acids revealed decrements in glutamate and GABA whereas other amino acids were not significantly altered. The resting and NMA-stimulated efflux of inosine was higher in the intact hippocampus. However, the extracellular concentrations of the inosine break-down products hypoxanthine and xanthine were not influenced by a prior NMA lesion, neither before nor after NMA administration. The present findings indicate that NMA releases amino acids (mainly taurine and PEA) from non-neuronal cells. The depression of extracellular glutamine elicited by NMA is probably a neuronal event. A direct stimulation of the energy metabolism of non-neuronal cells by NMA appears to exist as measured by the efflux of purine catabolites. I propose that non-neuronal cells, possibly glia, possess NMA receptors which, upon stimulation, initiate biochemical changes. The physiological significance of these responses remains to be elucidated.     

Stress-induced increase in extracellular dopamine in striatum: role of glutamatergic action via N-methyl- -aspartate receptors in substantia nigra

There is considerable support for an influence of excitatory amino acids released from corticofugal neurons on dopaminergic activity in the basal ganglia. However, the relative importance of cortico-striatal and cortico-mesencephalic projections remains unclear, particularly with respect to the nigro-neostriatal pathway. We have therefore examined the influence of endogenous excitatory amino acids in substantia nigra on stress-induced dopaminergic activity in neostriatum. Microdialysis probes were implanted unilaterally into substantia nigra and ipsilateral neostriatum, and dopamine release in neostriatum was monitored by measuring changes in extracellular dopamine. In separate animals, neostriatal dopamine synthesis was assessed by measuring extracellular DOPA in the presence of 3-hydroxylbenzylhydrazine (NSD-1015; 100 μM), an inhibitor of aromatic amino acid decarboxylase. Thirty minutes of intermittent foot shock increased both dopamine release (+41%) and synthesis (+37%) in neostriatum. Infusion of 2-amino-5-phosphonovalerate (APV; 100 μM), an inhibitor of N-methyl- -aspartate (NMDA) receptors, into substantia nigra greatly attenuated the stress-induced increase in neostriatal dopamine release, while having no effect on the apparent increase in stress-induced dopamine synthesis. These data suggest that excitatory amino acids such as glutamate act on NMDA receptors in substantia nigra to increase striatal dopamine release produced by exposure to stress, but that the increase in dopamine synthesis is mediated through a separate mechanism.     

Aging,energy, and oxidative stress in neurodegenerative diseases

The etiology of neurodegenerative diseases remains enigmatic; however, evidence for defects in energy metabolism, excitotoxicity, and for oxidative damage is increasingly compelling. It is likely that there is a complex interplay between these mechanisms. A defect in energy metabolism may lead to neuronal depolarization, activation of N-methyl-D-aspartate excitatory amino acid-receptors, and increases in intracellular calcium, which are buffered by mitochondria. Mitochondria are the major intracellular source of free radicals, and increased mitochondrial calcium concentrations enhance free radical generation. Mitochondrial DNA is particularly susceptible to oxidative stress, and there is evidence of age-dependent damage and deterioration of respiratory enzyme activities with normal aging. This may contribute to the delayed onset and age dependence of neurodegenerative diseases. There is evidence for increased oxidative damage to macromolecules in amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease. Potential therapeutic approaches include glutamate release inhibitors, excitatory amino acid antagonists, strategies to improve mitochondrial function, free radical scavengers, and trophic factors. All of these approaches appear promising in experimental studies and are now being applied to human studies.