Changes In Extracellular Glutamate Research Articles

Corticostriatal network dysfunction in Huntington's disease: Deficits in neural processing, glutamate transport, and ascorbate release.

This review summarizes evidence for dysfunctional connectivity between cortical and striatal neurons in Huntington's disease (HD), a fatal neurodegenerative condition caused by a single gene mutation. The focus is on data derived from recording of electrophysiological signals in behaving transgenic mouse models. Firing patterns of individual neurons and the frequency oscillations of local field potentials indicate a disruption in corticostriatal processing driven, in large part, by interactions between cells that contain the mutant gene rather than the mutant gene alone. Dysregulation of glutamate, an excitatory amino acid released by cortical afferents, plays a key role in the breakdown of corticostriatal communication, a process modulated by ascorbate, an antioxidant vitamin found in high concentration in striatum. Up-regulation of glutamate transport by drug administration or viral-vector delivery improves ascorbate homeostasis and neurobehavioral processing in HD mice. Further analysis of electrophysiological data, including the use of sophisticated computational strategies, is required to discern how behavioral demands modulate the flow of corticostriatal information and its disruption by HD. Long before massive cell loss occurs, HD impairs the mechanisms by which cortical and striatal neurons communicate. A key problem identified in transgenic animal models is dysregulation of the dynamic changes in extracellular glutamate and ascorbate. Improved understanding of how these neurochemical systems impact corticostriatal communication is necessary before an effective therapeutic strategy can emerge.

Personalized approach in brain protection by hypothermia: individual changes in non-pathological and ischemia-related glutamate transport in brain nerve terminals.

BackgroundBoth deep and profound hypothermia are effectively applied in cardiac surgery of the aortic arch, when the reduction of cerebral circulation facilitates operations, and for the prevention of ischemic stroke consequences. Neurochemical discrimination of the effects of deep and profound hypothermia (27 and 17 °C, respectively) on non-pathological and pathological ischemia-related mechanisms of presynaptic glutamate transport with its potential contribution to predictive, preventive and personalized medicine (PPPM) was performed.MethodsExperiments were conducted using nerve terminals isolated from rat cortex (synaptosomes). Glutamate transport in synaptosomes was analyzed using radiolabel l-[14C]glutamate. Diameter of synaptosomes was assessed by dynamic light scattering.ResultsSynaptosomal transporter-mediated uptake and tonic release of l-[14C]glutamate (oppositely directed processes, dynamic balance of which determines the physiological extracellular level of the neurotransmitter) decreased in a different range in deep/profound hypothermia. As a result, hypothermia-induced changes in extracellular l-[14C]glutamate are not evident (in one half of animals it increased, and in other it decreased). A progressive decrease from deep to profound hypothermia was shown for pathological mechanisms of presynaptic glutamate transport, that is, transporter-mediated l-[14C]glutamate release (*) stimulated by depolarization of the plasma membrane and (**) during dissipation of the proton gradient of synaptic vesicles by the protonophore FCCP.ConclusionsTherefore, the direction of hypothermia-induced changes in extracellular glutamate is unpredictable in “healthy” nerve terminals and depends on hypothermia sensitivity of uptake vs. tonic release. In affected nerve terminals (e.g., in brain regions suffering from a reduction of blood circulation during cardiac surgery, and core and penumbra zones of the insult), pathological transporter-mediated glutamate release from nerve terminals decreases with progressive significance from deep to profound hypothermia, thereby underlying its potent neuroprotective action. So, alterations in extracellular glutamate during hypothermia can be unique for each patient. An extent of a decrease in pathological glutamate transporter reversal depends on the size of damaged brain zone in each incident. Therefore, test parameters and clinical criteria of neuromonitoring for the evaluation of individual hypothermia-induced effects should be developed and delivered in practice in PPPM.

Roles of the NMDA Receptor and EAAC1 Transporter in the Modulation of Extracellular Glutamate by Low and High Affinity AMPA Receptors in the Cerebellum in Vivo: Differential Alteration in Chronic Hyperammonemia.

The roles of high- and low-affinity AMPA receptors in modulating extracellular glutamate in the cerebellum remain unclear. Altered glutamatergic neurotransmission is involved in neurological alterations in hyperammonemia, which differently affects high- and low-affinity AMPA receptors. The aims were to assess by in vivo microdialysis (a) the effects of high- and low-affinity AMPA receptor activation on extracellular glutamate in the cerebellum; (b) whether chronic hyperammonemia alters extracellular glutamate modulation by high- and/or low-affinity AMPA receptors; and (c) the contribution of NMDA receptors and EAAC1 transporter to AMPA-induced changes in extracellular glutamate. In control rats, high affinity receptor activation does not affect extracellular glutamate but increases glutamate if NMDA receptors are blocked. Low affinity AMPA receptor activation increases transiently extracellular glutamate followed by reduction below basal levels and return to basal values. The reduction is associated with transient increased membrane expression of EAAC1 and is prevented by blocking NMDA receptors. Blocking NMDA receptors with MK-801 induces a transient increase in extracellular glutamate which is associated with reduced membrane expression of EAAC1 followed by increased membrane expression of the glutamate transporter GLT-1. Chronic hyperammonemia does not affect responses to activation of low affinity AMPA receptors. Activation of high affinity AMPA receptors increases extracellular glutamate in hyperammonemic rats by an NMDA receptor-dependent mechanism. In conclusion, these results show that there is a tightly controlled interplay between AMPA and NMDA receptors and an EAAC1 transporter in controlling extracellular glutamate. Hyperammonemia alters high- but not low-affinity AMPA receptors.

Endothelial Nitric Oxide Synthase (eNOS) Blockade Within the Ventrolateral Medulla Differentially Modulates the Exercise Pressor Reflex in Stroked‐Rats

We have previously shown that blockade of endothelial nitric oxide synthase (eNOS) within the rostral (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulates cardiovascular responses which was associated with changes in extracellular glutamate and GABA concentrations during static skeletal muscle contraction, the Exercise Pressor Reflex. In this study using left‐sided stroked‐rats we determined if microdialyzing a specific eNOS antagonist into the left RVLM and/or the left CVLM would alter cardiovascular responses during the Exercise Pressor Reflex. Rats were stroked by employing a transient (90‐minute) left‐sided middle cerebral artery occlusion (MCAO) followed by 24 hr reperfusion. In Protocol 1, microdialysis of a selective eNOS antagonist, L‐N(5)‐(1‐iminoethyl)ornithine (L‐NIO; 10 µM) for 120 minutes into the left RVLM significantly potentiated cardiovascular responses during a static muscle contraction compared to those observed in intact rats. In Protocol 2, application of L‐NIO into the left CVLM significantly attenuated the cardiovascular responses during muscle contraction. Finally, in Protocol 3, administration of L‐NIO simultaneously into both the left RVLM and left CVLM produced results similar to those observed with Protocol 1. These results demonstrate that blockade of eNOS within the two regions of the ventrolateral medulla differentially modulates cardiovascular responses during static exercise in stroked‐rats.

Ammonia Mediates Methamphetamine-Induced Increases in Glutamate and Excitotoxicity

Ammonia has been identified to have a significant role in the long-term damage to dopamine and serotonin terminals produced by methamphetamine (METH), but how ammonia contributes to this damage is unknown. Experiments were conducted to identify whether increases in brain ammonia affect METH-induced increases in glutamate and subsequent excitotoxicity. Increases in striatal glutamate were measured using in vivo microdialysis. To examine the role of ammonia in mediating changes in extracellular glutamate after METH exposure, lactulose was used to decrease plasma and brain ammonia. Lactulose is a non-absorbable disaccharide, which alters the intestinal lumen through multiple mechanisms that lead to the increased peripheral excretion of ammonia. METH caused a significant increase in extracellular glutamate that was prevented by lactulose. Lactulose had no effect on METH-induced hyperthermia. To determine if ammonia contributed to excitotoxicity, the effect of METH and lactulose treatment on calpain-mediated spectrin proteolysis was measured. METH significantly increased calpain-specific spectrin breakdown products, and this increase was prevented with lactulose treatment. To examine if ammonia-induced increases in extracellular glutamate were mediated by excitatory amino-acid transporters, the reverse dialysis of ammonia, the glutamate transporter inhibitor, DL-threo-β-benzyloxyaspartic acid (TBOA), or the combination of the two directly into the striatum of awake, freely moving rats was conducted. TBOA blocked the increases in extracellular glutamate produced by the reverse dialysis of ammonia. These findings demonstrate that ammonia mediates METH-induced increases in extracellular glutamate through an excitatory amino-acid transporter to cause excitotoxicity.

The antidepressant agomelatine inhibits stress-mediated changes in amino acid efflux in the rat hippocampus and amygdala

Agomelatine is a potent melatonergic (MT1 and MT2) receptor agonist and 5HT2C antagonist that is an effective antidepressant in animal models of depression and in patients suffering from depression. Our recent studies revealed that acute restraint stress increases extracellular levels of glutamate and GABA and that these increases in amino acid efflux are inhibited by some but not all antidepressants. In view of the increasing evidence supporting a role of amino acids in the pathology of depression, the current study examined whether acute stress-mediated changes in glutamate and GABA neurotransmission are modulated by agomelatine. In agreement with our previous work, acute stress increases extracellular glutamate levels in the basolateral nucleus of the amygdala (BLA). Similarly, acute stress increases glutamate efflux in the central nucleus of the amygdala (CeA). In the hippocampus, acute stress increases glutamate efflux and elicits an oscillatory pattern of GABA efflux. Agomelatine administration (40mg/kg ip) prior to acute stress inhibited stress-mediated increases in glutamate efflux in the hippocampus, BLA and CeA. These results demonstrate that acute agomelatine administration effectively inhibits acute stress-mediated changes in extracellular glutamate in the rat hippocampus and amygdala. While acute stress did not modulate GABA efflux in these regions, agomelatine treatment induced an overall reduction of GABA levels in the hippocampus. These data suggest that agomelatine modulates amino acid efflux in limbic structures implicated in major depressive disorder.

Rapid changes in extracellular glutamate induced by natural arousing stimuli and intravenous cocaine in the nucleus accumbens shell and core

Glutamate (Glu) is a major excitatory neurotransmitter, playing a crucial role in the functioning of the nucleus accumbens (NAc), a critical area implicated in somatosensory integration and regulation of motivated behavior. In this study, high-speed amperometry with enzyme-based biosensors was used in freely moving rats to examine changes in extracellular Glu in the NAc shell and core induced by a tone, tail pinch (TP), social interaction with a male conspecific (SI), and intravenous (iv) cocaine (1 mg/kg). To establish the contribution of Glu to electrochemical signal changes, similar recordings were conducted with null (Glu(0)) sensors, which were exposed to the same chemical and physical environment but were insensitive to Glu. TP, SI, and cocaine, but not a tone, induced relatively large and prolonged current increases detected by both Glu and Glu(0) sensors. However, current differentials revealed very rapid, much smaller, and transient increases in extracellular Glu levels, more predominantly in the NAc shell than core. In contrast to monophasic responses with natural stimuli, cocaine induced a biphasic Glu increase in the shell, with a transient peak during the injection and a slower postinjection peak. Therefore, Glu is phasically released in the NAc after exposure to natural arousing stimuli and cocaine; this release is rapid, stimulus dependent, and structure specific, suggesting its role in triggering neural and behavioral activation induced by these stimuli. This study also demonstrates the need for multiple in vitro and in vivo controls to reveal relatively small, highly phasic, and transient fluctuations in Glu levels occurring under behaviorally relevant conditions.

Short- and long-term changes in extracellular glutamate and acetylcholine concentrations in the rat hippocampus following hypoxia

Hypoxia at birth is a major source of brain damage and it is associated with serious neurological sequelae in survivors. Alterations in the extracellular turnover of glutamate (Glu) and acetylcholine (ACh), two neurotransmitters that are essential for normal hippocampal function and learning and memory processes, may contribute to some of the neurological effects of perinatal hypoxia. We set out to determine the immediate and long-lasting effects of hypoxia on the turnover of these neurotransmitters by using microdialysis to measure the extracellular concentration of Glu and ACh in hippocampus, when hypoxia was induced in rats at postnatal day (PD) 7, and again at PD30. In PD7 rats, hypoxia induced an increase in extracellular Glu concentrations that lasted for up to 2.5h and a decrease in extracellular ACh concentrations over this period. By contrast, perinatal hypoxia attenuated Glu release in asphyxiated rats, inducing a decrease in basal Glu levels when these animals reached PD30. Unlike Glu, the basal ACh levels in these animals were greater than in controls at PD30, although ACh release was stimulated less strongly than in control animals. These results provide the first evidence of the initial and long term consequences of the hypoxia on Glu and ACh turnover in the brain, demonstrating that hypoxia produces significant alterations in hippocampal neurochemistry and physiology.

Central interleukin-10 attenuated lipopolysaccharide-induced changes in core temperature and hypothalamic glutamate, hydroxyl radicals and prostaglandin-E2

It has been documented that intravenous lipopolysaccharide (LPS) in rabbits causes fever accompanied by increased levels of extracellular glutamate, hydroxyl radicals, and prostaglandin E2 (PGE2) in the hypothalamus and circulating tumor necrosis factor-alpha (TNF-α). This investigation was to determine whether central interleukin-10 (IL-10) exerted its antipyresis by reducing changes in circulating TNF-α and extracellular glutamate, hydroxyl radicals and PGE2 in the hypothalamus. The microdialysis probes were stereotaxically and chronically implanted into the preoptic anterior hypothalamus of rabbit brain for determinating extracellular glutamate, hydroxyl radicals, and PGE2 in situ. It was found that systemically injected LPS (2μg/kg, intravenously) increased the levels of core temperature, and extracellular glutamate, hydroxyl radicals, and PGE2 in the hypothalamus accompanied by increased plasma levels of TNF-α. Pretreatment with IL-10 (10–100ng, intracerebroventricularly) 1h before intravenous LPS significantly reduced the LPS-induced changes in extracellular glutamate, hydroxyl radicals, and PGE2 in the hypothalamus and fever, but not the increased levels of TNF-α in rabbits. These findings suggested that directly injected IL-10 into the lateral cerebral ventricle 1h before intravenous LPS exerted its antipyresis by inhibiting the changes in extracellular glutamate, hydroxyl radicals and PGE2 in the hypothalamus during LPS fever in rabbits.

Chronic electrographic seizure reduces glutamine and elevates glutamate in the extracellular fluid of rat brain

Effects of spontaneous seizures on extracellular glutamate and glutamine were studied in the kainate-induced rat model of epilepsy in the chronic phase. Extracellular fluid from the CA1–CA3 regions of the hippocampus was collected with a 2-mm microdialysis probe every 2min for 5h. EEG seizures with no or mild behavioral components caused 2- to 6-fold elevation of extracellular glutamate. Concomitantly, extracellular glutamine decreased at t=5h to 48% of the initial value (n=6). The changes in extracellular glutamate and glutamine correlated with the frequency and magnitude of seizure activity. In contrast, no change in either metabolite was observed in kainate-injected rats that did not undergo seizure during microdialysis (n=6). In hippocampal tissue (9.4±1.1mg) that contained the region sampled by microdialysis and the site of kainate injection, intracellular glutamine concentration was significantly reduced in the seizure group, compared to that in no-seizure group. The observed elevation of extracellular glutamate strongly suggests that neurotransmitter glutamate was released at a rate faster than the rate of its uptake into glia, possibly due to down-regulation of the transporter. This reduces the availability of substrate glutamate for glutamine synthesis, as corroborated by the observed reduction of intracellular glutamine. This is likely to reduce the rate of glutamine efflux from glia and result in the observed decrease of extracellular glutamine. There remains an intriguing possibility that seizure-induced decrease of extracellular glutamine also reflects its increased uptake into neurons to replenish neurotransmitter glutamate during enhanced epileptiform activity.

Rapid microelectrode measurements and the origin and regulation of extracellular glutamate in rat prefrontal cortex

Glutamate in the prefrontal cortex (PFC) plays a significant role in several mental illnesses, including schizophrenia, addiction and anxiety. Previous studies on PFC glutamate-mediated function have used techniques that raise questions on the neuronal versus astrocytic origin of glutamate. The present studies used enzyme-based microelectrode arrays to monitor second-by-second resting glutamate levels in the PFC of awake rats. Locally applied drugs were employed in an attempt to discriminate between the neuronal or glial components of the resting glutamate signal. Local application of tetrodotoxin (sodium channel blocker), produced a significant (∼ 40%) decline in resting glutamate levels. In addition significant reductions in extracellular glutamate were seen with locally applied ω-conotoxin (MVIIC; ∼ 50%; calcium channel blocker), and the mGluR(2/3) agonist, LY379268 (∼ 20%), and a significant increase with the mGluR(2/3) antagonist LY341495 (∼ 40%), effects all consistent with a large neuronal contribution to the resting glutamate levels. Local administration of D,L-threo-β-benzyloxyaspartate (glutamate transporter inhibitor) produced an ∼ 120% increase in extracellular glutamate levels, supporting that excitatory amino acid transporters, which are largely located on glia, modulate clearance of extracellular glutamate. Interestingly, local application of (S)-4-carboxyphenylglycine (cystine/glutamate antiporter inhibitor), produced small, non-significant bi-phasic changes in extracellular glutamate versus vehicle control. Finally, pre-administration of tetrodotoxin completely blocked the glutamate response to tail pinch stress. Taken together, these results support that PFC resting glutamate levels in rats as measured by the microelectrode array technology are at least 40-50% derived from neurons. Furthermore, these data support that the impulse flow-dependent glutamate release from a physiologically -evoked event is entirely neuronally derived.

Alcohol‐seeking behavior is associated with increased glutamate transmission in basolateral amygdala and nucleus accumbens as measured by glutamate‐oxidase‐coated biosensors

Relapse is one of the most problematic aspects in the treatment of alcoholism and is often triggered by alcohol-associated environmental cues. Evidence indicates that glutamate neurotransmission plays a critical role in cue-induced relapse-like behavior, as inhibition of glutamate neurotransmission can prevent reinstatement of alcohol-seeking behavior. However, few studies have examined specific changes in extracellular glutamate levels in discrete brain regions produced by exposure to alcohol-associated cues. The purpose of this study was to use glutamate oxidase (GluOx)-coated biosensors to monitor changes in extracellular glutamate in specific brain regions during cue-induced reinstatement of alcohol-seeking behavior. Male Wistar rats were implanted with indwelling jugular vein catheters and intracerebral guide cannula aimed at the basolateral amygdala (BLA) or nucleus accumbens (NAc) core, and then trained to self-administer alcohol intravenously. A separate group of animals were trained to self-administer food pellets. Each reinforcer was accompanied by the presentation of a light/tone stimulus. Following stabilization of responding for alcohol or food reinforcement, and subsequent extinction training, animals were implanted with pre-calibrated biosensors and then underwent a 1-hour cue-induced reinstatement testing period. As determined by GluOx-coated biosensors, extracellular levels of glutamate were increased in the BLA and NAc core during cue-induced reinstatement of alcohol-seeking behavior. The cumulative change in extracellular glutamate in both regions was significantly greater for cue-induced reinstatement of alcohol-seeking behavior versus that of food-seeking behavior. These results indicate that increases in glutamate transmission in the BLA and NAc core may be a neurochemical substrate of cue-evoked alcohol-seeking behavior.

Real-time glutamate measurements in the putamen of awake rhesus monkeys using an enzyme-based human microelectrode array prototype

Commonly used for research studies in the central nervous system, microdialysis has revealed a link between dysregulation of the excitatory neurotransmitter glutamate and ischemia and seizure, however limitations like slow temporal resolution have stalled the advancement of microdialysis as a diagnostic tool. We have developed and extensively characterized an enzyme-based microelectrode array technology for second-by-second in vivo amperometric measurements of glutamate in the mammalian CNS. The current studies demonstrated the ability of a human microelectrode array prototype (Spencer-Gerhardt-2 (SG-2)) to measure tonic and phasic glutamate neurotransmission in the putamen of unanesthetized non-human primates. We also showed that the SG-2 remains functional following sterilization. Ability to monitor dynamic changes in glutamate in real-time may assist the development of clinical algorithms to potentially alert care-providers prior to onset of overt ischemia or seizure, or provide neurosurgeons with second-by-second measurements of rapid changes in extracellular glutamate which could help guide surgical procedures or aid in interventional strategies.

Facilitation of zinc influx via AMPA/kainate receptor activation in the hippocampus

Glutamate and zinc is co-released by excitation of hippocampal mossy fibers and both concentrations are increased in the extracellular compartment. In a novel environment, however, extracellular zinc is persistently decreased in spite of the increase in extracellular glutamate. The mechanism of the decrease in extracellular zinc was studied in the present paper. In rats subjected to the novelty stress under hippocampal perfusion, the differential changes in extracellular glutamate and zinc were abolished in the presence of 1 μM tetrodotoxin (TTX), a sodium channel blocker, which reduced exploratory behavior. When the hippocampus was perfused with corticosterone (50 ng/ml), extracellular zinc was increased. These results suggest that glutamatergic neuron activation elicited by novelty stress is involved in the decrease in extracellular zinc and that glucocorticoid is not a trigger for its decrease. The differential changes in extracellular glutamate and zinc was induced by electrical stimulation to analyze the decrease in extracellular zinc; the differential changes were elicited by delivery of tetanic stimuli (100 Hz for 1 s, 5 min intervals, three times) to the hippocampus instead of the novelty stress, as reported previously. The changes elicited by tetanic stimulation were abolished in the presence of 10 μM CNQX, an AMPA/kainate receptor antagonist. In a hippocampal slice double-labeled with zinc and calcium indicators, furthermore, CNQX inhibited the increase in intracellular zinc levels in mossy fiber synapses after delivery of tetanic stimuli (100 Hz for 5 s) to dentate granule cells. The in vivo and in vitro experiments suggest that AMPA/kainate receptor activation is involved in zinc influx into hippocampal cells, followed by the decrease in extracellular zinc. It is likely that zinc influx is persistently facilitated via stress-induced glutamatergic neuron activation.

Effects of Retigabine on the Neurodegeneration and Extracellular Glutamate Changes Induced by 4-Aminopyridine in Rat Hippocampus In Vivo

We have previously shown that microdialysis perfusion of the K+ channel blocker 4-aminopyridine (4-AP) in rat hippocampus induces convulsions and neurodegeneration, due to the stimulation of glutamate release from synaptic terminals. Retigabine is an opener of the KCNQ2/Q3-type K+ channel that possesses antiepileptic action and may be neuroprotective, and we have therefore studied its effect on the hyperexcitation, the neuronal damage and the changes in extracellular glutamate induced by 4-AP. Retigabine and 4-AP were co-administered by microdialysis in the hippocampus of anesthetized rats, with simultaneous recording of the EEG, and the extracellular concentration of glutamate was measured in the microdialysis fractions. In 70-80% of the rats tested retigabine reduced the 4-AP-induced stimulation of glutamate release and prevented the neuronal damage observed at 24 h in the CA1 hippocampal region. However, retigabine did not block the EEG epileptic discharges and their duration was reduced in only 20-25% of the tested animals. We conclude that the neuroprotective action of retigabine is probably due to the blockade of the 4-AP-induced stimulation of glutamate release. This inhibition, however, was not sufficient to block the epileptic activity.

Apomorphine-induced alterations in striatal and substantia nigra pars reticulata glutamate following unilateral loss of striatal dopamine

We have reported time-dependent changes in extracellular glutamate within the striatum at 1 and 3 months following a unilateral lesion of the nigrostriatal pathway using the neurotoxin, 6-hydroxydopamine (6-OHDA) (Meshul, C.K., Emre, N., Nakamura, C.M., Allen, C., Donohue, M.K., Buckman, J.F., 1999. Time-dependent changes in striatal glutamate synapses following a 6-hydroxydopamine lesion. Neurosci. 88, 1–16.). The aim of the present study was to determine the effects of such a lesion on glutamate within the substantia nigra pars reticulata (SN-PR) and the effect of subchronic administration of the dopamine D-1/D-2 agonist, apomorphine, on extracellular glutamate within both the striatum and the SN-PR using in vivo microdialysis. One month after the lesion, there is an increase in extracellular glutamate within the striatum and apomorphine treatment leads to a further increase. Within the SN-PR, a loss of striatal dopamine leads to a decrease in extracellular glutamate, while apomorphine treatment leads to a further decrease in nigral glutamate. Three months after a 6-OHDA lesion, there is a decrease in extracellular striatal glutamate, with apomorphine administration leading to essentially no further change in glutamate. The loss of striatal dopamine increased extracellular glutamate within the SN-PR while apomorphine administration resulted in a decrease in extracellular glutamate back to the value observed in the control group. The data suggests that the increase in striatal glutamate 1 month following a 6-OHDA lesion alone or following subchronic apomorphine is consistent with the hypothesis that a decrease in glutamate within the SN-PR leads to activation of the thalamo–cortico–striatal pathway. The decrease in striatal glutamate 3 months after a nigrostriatal lesion is also consistent with the observed increase in extracellular glutamate within the SN-PR, thus leading to a decrease in output of the thalamo–cortico–striatal pathway.

Time-dependent changes in extracellular glutamate in the rat dorsolateral striatum following a single cocaine injection

Acute cocaine administration has been shown to alter dorsal striatal plasticity [Proc Natl Acad Sci USA 87 (1990) 6912; Brain Res Bull 30 (1993) 173] and produce long-term neurochemical changes [Pharmacol Biochem Behav 27 (1987) 533]. To date, the effects of acute cocaine on extracellular glutamate and nerve terminal glutamate immunolabeling in the rat dorsolateral striatum have not been reported. To investigate cocaine-induced changes in extracellular glutamate, in vivo microdialysis was carried out in the dorsolateral striatum of rats 1–14 days after receiving a single injection of either vehicle or 15mg/kg cocaine. There was an increase in the group injected with cocaine 1 day prior to measuring extracellular glutamate as compared with the control group. The group injected with cocaine 3 days prior to the microdialysis session had decreased extracellular glutamate levels. Furthermore, extracellular glutamate remained attenuated 14 days after acute cocaine treatment. Striatal glutamate decreased in the cocaine-treated rats after calcium removal, suggesting that cocaine-induced changes in extracellular glutamate were partially calcium-dependent. The density of nerve terminal glutamate immunolabeling was measured using immunogold electron microscopy in the contralateral striatum of the same rats that had been acutely treated with cocaine or vehicle. There were no changes in the density of glutamate immunolabeling within identified nerve terminals making an asymmetrical (excitatory) synaptic contact 1, 2, 3, or 14 days after acute cocaine exposure as compared with the control groups. Hence, these alterations in extracellular glutamate did not result from changes in glutamate immunolabeling within the synaptic vesicle pool. In addition, no changes in glutamate immunolabeling were found in rats that received cocaine 2 h previously or were withdrawn after 1 week of cocaine administration. The results demonstrate that a single injection of cocaine produces biphasic, time-dependent changes in extracellular glutamate in the rat dorsolateral striatum.

Extracellular glutamate changes in rat striatum during ischemia determined by a novel dialysis electrode and conventional microdialysis

Our newly developed method using a dialysis electrode has made it possible to perform real time monitoring of extracellular glutamate concentration ([Glu]e) utilizing the oxygen-independent reaction with glutamate oxidase and ferrocene. In this study, we therefore, investigated [Glu]e changes during brain ischemia using both the conventional microdialysis method and the dialysis electrode method. A comparison between our newly developed dialysis electrode and conventional microdialysis methods provided the following results. When the conventional microdialysis method was employed: (1) the elevation of [Glu]e during complete global ischemia was delayed; and (2) the elevation of concentration and reuptake of glutamate were delayed during 10-min transient ischemia, and the elevation of [Glu]e reached a maximum later using conventional microdialysis than using our dialysis electrode. (3) The biphasic [Glu]e elevation of glutamate concentration detected using the dialysis electrode method was not observed using the conventional microdialysis method. It was additionally investigated why the conventional microdialysis method provides inferior time resolution. In this study, we also demonstrated with the chromatographic SMART™ procedure coupled to UV detection that biogenic substances, i.e. low molecular weight proteins and peptides, are released during ischemic injury, and they may cause a delay in the time resolution in the microdialysis method.

Excitotoxicity in neurological disorders — the glutamate paradox

Beneficial effects of glutamate-receptor antagonists in models of neurological disorders are often used to support the notion that endogenous excitotoxicity (i.e. resulting from extracellular accumulation of endogenous glutamate) is a major contributor to neuronal death associated with these conditions. However, this interpretation conflicts with a number of robust and important experimental evidence. Here, emphasis is placed on two key elements: (i) very high extracellular levels of glutamate must be reached to initiate neuronal death, far above those measured in models of neurological disorders; and (ii) changes in extracellular glutamate as measured by microdialysis are not related to changes in the synaptic cleft, i.e. the compartment where neurotransmitter glutamate interacts with its receptors.It has become clear that the diversity and complexity of glutamate-mediated processes allow for a wide range of potential abnormalities (e.g. loss of selectivity of glutamate-operated ion channels, abnormal modulation of glutamate receptors). In addition, as neuronal death subsequent to ischemia and other insults is likely to result from multifactorial processes that may be inter-related, inhibition of glutamate-mediated synaptic transmission may be neuroprotective by increasing the resistance of neurons to other deleterious mechanisms (e.g. inadequate energy supply) that are not directly related to glutamatergic transmission.

Effects of nitric oxide synthase inhibition on extracellular glutamate and cerebral blood flow during forebrain ischemia-reperfusion in rat in vivo.

To evaluate factors involved in global forebrain ischemia-reperfusion, the effects of the systemically administered NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), on changes in extracellular glutamate and cerebral blood flow (CBF) were studied during the early period of global forebrain ischemia-reperfusion, simultaneously measuring the glutamate released in the rat forebrain cortex and cortical CBF. After injection of saline or L-NAME, forebrain ischemia-reperfusion was performed by bilateral carotid artery occlusion with controlled hemorrhagic hypotension (30 mmHg) for 10 min and reperfusion for 60 min. The microdialysis electrode and laser Doppler flowmetry were used for real-time monitoring of glutamate and CBF, respectively. During ischemia, glutamate increased linearly to over 100 muM and remained elevated 30 min after reperfusion in L-NAME-treated rats. In L-NAME-treated rats, CBF also remained significantly lower than baseline for 30-60 min after reperfusion, and glutamate was higher than in saline-treated rats throughout the experiment. A remarkable linear increase in glutamate release was observed during ischemia. L-NAME did not prevent this dramatic glutamate accumulation, and moreover, its level increased during reperfusion. The decrease in CBF response after reperfusion might be a factor of the elevated glutamate after reperfusion due to a decrease in reuptake of glutamate.