Effects of Ischaemia on Neurotransmitter Release from the Isolated Retina

Abstract: The effects of "ischaemia" (glucose-free Krebs-bicarbonate medium gassed with N₂/CO₂ on the release of glutamate and other major neurotransmitters in the retina were examined using the isolated rat and rabbit retina. Amino acid transmitters, acetylcholine, and dopamine were measured by HPLC. The release of glutamate, aspartate, GABA, and glycine from ischaemic retinas was more than doubled after 30 min, and after 90 min of ischaemia the release of amino acids was ∼ 15–20-fold that of control values. Ischaemia also produced large increases in the release of dopamine from both the rat and especially the rabbit retina. In contrast, the release of acetylcholine from the rat retina was significantly decreased by ischaemia, although the release of choline was increased. Because the ischaemia-induced release of glutamate, aspartate, and GABA from the rat retina was completely Ca independent, and exposure of the retina to high K (50 m M ) did not stimulate amino acid release, it is concluded that the mechanisms underlying the ischaemia-induced release do not involve an initial release of K or an influx of calcium.     

Elevation of the Extracellular Concentrations of Glutamate and Aspartate in Rat Hippocampus During Transient Cerebral Ischemia Monitored by Intracerebral Microdialysis

Rats were implanted with 0.3-mm-diameter dialysis tubing through the hippocampus and subsequently perfused with Ringer's solution at a flow rate of 2 microliter/min. Samples of the perfusate representing the extracellular fluid were collected over 5-min periods and subsequently analyzed for contents of the amino acids glutamate, aspartate, glutamine, taurine, alanine, and serine. Samples were collected before, during, and after a 10-min period of transient complete cerebral ischemia. The extracellular contents of glutamate and aspartate were increased, respectively, eight- and threefold during the ischemic period; the taurine concentration also was increased 2.6-fold. During the same period the extracellular content of glutamine was significantly decreased (to 68% of the control value), whereas the concentrations of alanine and serine did not change significantly during the ischemic period. The concentrations of gamma-aminobutyric acid (GABA) were too low to be measured reliably. It is suggested that the large increase in the content of extracellular glutamate and aspartate in the hippocampus induced by the ischemia may be one of the causal factors in the damage to certain neurons observed after ischemia.     

Dopaminergic Modulation of Glutamate Release in Striatum as Measured by Microdialysis

Glutamate and aspartate are the primary neurotransmitters of projections from motor and premotor cortices to the striatum. Release of glutamate may be modulated by dopamine receptors located on corticostriatal terminals. The present study used microdialysis to investigate the dopaminergic modulation of in vivo striatal glutamate and aspartate release in the striatum of awake-behaving rats. Local perfusion with a depolarizing concentration of K+ through a dialysis probe into the rat striatum produced a significant increase in the release of glutamate, aspartate, and taurine. The D2 agonist LY171555 blocked the K(+)-induced release of glutamate and aspartate, but not taurine, in a concentration-dependent manner. The D1 agonist SKF 38393 did not alter K(+)-induced release of glutamate and taurine, but did significantly decrease aspartate release. Neither agonist had any effect on basal amino acid release. The D2 antagonist (-)-sulpiride reversed the inhibitory effects of LY 171555 on K(+)-induced glutamate release. These results provide in vivo evidence for a functional interaction between dopamine, the D2 receptor, and striatal glutamate release.     

Excitatory Amino Acid-Induced Toxicity in Chick Retina: Amino Acid Release, Histology, and Effects of Chloride Channel Blockers

Acute excitotoxicity in embryonic chick retina and the ability of Cl- channel blockers to prevent toxicity were evaluated by measurement of endogenous amino acid release and histology. Treatment of retina with kainate, quisqualate, or N-methyl-D-aspartate resulted in a large dose-dependent release of gamma-aminobutyric acid and taurine, moderate release of glutamine and alanine, and no measurable release of glutamate or aspartate. Concentrations inducing maximal gamma-aminobutyric acid release were 50 microM quisquaalate, 100 microM kainate, and 100 microM N-methyl-D-aspartate. Treatment with 1 mM glutamate resulted in significant gamma-aminobutyric acid release, as well as an elevation in medium aspartate levels. Typical excitotoxic retinal lesions were produced by the agonists and, at the lower concentrations tested, revealed a regional sensitivity. There was a positive correlation between the amount of gamma-aminobutyric acid release and the extent of tissue swelling, suggesting that release may be secondary to toxic cellular events. Omission of Cl- completely blocked cytotoxic effects due to kainate or glutamate. Likewise, addition of the Cl-/bicarbonate anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate at 600 microM protected retina from cytotoxic damage from all excitotoxic analogs and restored amino acid levels to baseline values. Furosemide, which blocks Na+/K+/2Cl- cotransport, was only minimally effective in reducing amino acid release induced by the agonists. Consistent with the latter, histological examination showed the continued presence of the lesion but with general reduction of cellular edema. These results indicate that although influx of Cl- is a central component of the acute excitotoxic phenomenon, mechanisms other than passive Cl- flux may be involved.     

Exocytotic and Nonexocytotic Modes of Glutamate Release from Cultured Cerebellar Granule Cells During Chemical Ischaemia

Abstract: The mechanism of glutamate release from cultured cerebellar granule neurones in response to a chemical model of ischaemia (10 m M 2-deoxyglucose plus 1 m M sodium cyanide) was investigated. In the first 2 min of ischaemia, release of preloaded d -[³H]aspartate could be extensively attenuated by tetanus toxin and bafilomycin A₁ and was dependent on the activation of Ca²⁺ channels sensitive to the "Q" type Ca²⁺ channel antagonist, ω-conotoxin-MVIIC. During this period, ATP/ADP ratios fell rapidly. The extent of release in the first 2 min was comparable to that evoked by 2-min depolarization by 50 m M KCl. Free Ca²⁺ concentrations, determined in neurites and somata, did not increase until after 2 min. The neurite increase in cellular Ca²⁺ precedes that of the cell somata. Release of d -[³H]aspartate was partially inhibited by the NMDA receptor antagonist MK-801, which also delayed the increase in free Ca²⁺ concentration. Prolonging the period of ischaemia to 6 and 10 min produced no further increase in the apparently exocytotic component of release, but initiated an extensive nonexocytotic release of the amino acid. Studies with the synaptic vesicle membrane probe FM1-43 in which released amino acid was removed by superfusion indicated that Ca²⁺-dependent exocytosis was delayed in this system. It is concluded that chemical ischaemia initiates an initial exocytotic followed by nonexocytotic release and that the former is facilitated by NMDA receptor activation. These events occur in cells that are still able to exclude propidium iodide, indicating that cell death has not yet occurred.     

Effect of Reperfusion Following Cerebral Ischaemia on the Activity of the Mitochondrial Respiratory Chain in the Gerbil Brain

Abstract: The effect of reperfusion following 30 min of cerebral ischaemia on brain mitochondrial respiratory chain activity has been studied in the gerbil. The state 3 respiration rates with both FAD- and NAD-linked substrates were reduced after ischaemia. After 5 min of reperfusion, state 3 respiration with FAD-linked substrates was restored, but levels of NAD-linked substrates did not return to control values until 30 min of reperfusion. By 120 min of reperfusion state 3 respiration decreased relative to control values with all substrates studied. Measurement of the individual respiratory chain complexes showed that complex I, complex II–III, and complex V activities were reduced after ischaemia. By 5 min of reperfusion complex II–III activity was restored, but the activities of complexes I and V did not return to control values until 30 min of reperfusion. In contrast, complex IV activity was unaffected by ischaemia or 5 and 30 min of reperfusion but was significantly reduced after 120 min of reperfusion, possibly owing to free radical production and lipid peroxidation.     

On the Origin of Extracellular Glutamate Levels Monitored in the Basal Ganglia of the Rat by In Vivo Microdialysis

Abstract: Several putative neurotransmitters and metabolites were monitored simultaneously in the extracellular space of neostriatum, substantia nigra, and cortex and in subcutaneous tissue of the rat by in vivo microdialysis. Glutamate (Glu) and aspartate (Asp) were at submicromolar and γ-aminobutyric acid (GABA) was at nanomolar concentrations in all brain regions. The highest concentration of dopamine (DA) was in the neostriatum. Dynorphin B (Dyn B) was in the picomolar range in all brain regions. Although no GABA, DA, or Dyn B could be detected in subcutaneous tissue, Glu and Asp levels were ≈5 and ≈0.4 µM, respectively. Lactate and pyruvate concentrations were ≈200 and ≈10 µM in all regions. The following criteria were applied to ascertain the neuronal origin of substances quantified by microdialysis: sensitivity to (a) K⁺ depolarization, (b) Na⁺ channel blockade, (c) removal of extracellular Ca²⁺, and (d) depletion of presynaptic vesicles by local administration of α-latrotoxin. DA, Dyn B, and GABA largely satisfied all these criteria. In contrast, Glu and Asp levels were not greatly affected by K⁺ depolarization and were increased by perfusing with tetrodotoxin or with Ca²⁺-free medium, arguing against a neuronal origin. However, Glu and Asp, as well as DA and GABA, levels were decreased under both basal and K⁺-depolarizing conditions by α-latrotoxin. Because the effect of K⁺ depolarization on Glu and Asp could be masked by reuptake into nerve terminals and glial cells, the reuptake blocker dihydrokainic acid (DHKA) or l -trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was included in the microdialysis perfusion medium. The effect of K⁺ depolarization on Glu and Asp levels was increased by DHKA, but GABA levels were also affected. In contrast, PDC increased only Glu levels. It is concluded that there is a pool of releasable Glu and Asp in the rat brain. However, extracellular levels of amino acids monitored by in vivo microdialysis reflect the balance between neuronal release and reuptake into surrounding nerve terminals and glial elements.     

Stimulation-Induced Release of Coexistent Transmitters in the Prefrontal Cortex: An In Vivo Microdialysis Study of Dopamine and Neurotensin Release

Extracellular fluid levels of dopamine and neurotensin in the rat prefrontal cortex were measured using in vivo microdialysis. Electrical stimulation of the median forebrain bundle resulted in increased release of both dopamine and neurotensin from the prefrontal cortex. Thus, stimulation of neurons in which dopamine and neurotensin are colocalized can evoke the in vivo release of both substances.     

Origin of Ischemia-Induced Glutamate Efflux in the CA1 Field of the Gerbil Hippocampus: An In Vivo Brain Microdialysis Study

Abstract: In vivo brain microdialysis experiments were performed in the gerbil to evaluate the origin of accumulation of extracellular glutamate under transient ischemia. Microdialysis probes were positioned in the CA1 field of the hippocampus in which proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals had been induced by 5-min ischemia 10–14 days before the microdialysis experiment; in the white matter of the cerebral cortex, which contained few neurons, few presynaptic terminals, and many astrocytes; or in the histologically normal CA1 field of the hippocampus, and then 5- or 20-min ischemia was induced. When 5-min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter of the cerebral cortex, whereas a significant increase in glutamate (15-fold) was observed in the histologically normal CA1 field. When 20-min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter during the first 10 min after the onset of 20-min ischemia, but remarkable ischemia-induced increases in glutamate were observed during the last 10 min of 20-min ischemia in both areas. An excessive increase in glutamate (100-fold) was observed during 20-min ischemia in the normal CA1 field of the hippocampus. When a probe was positioned in the CA1 field of the hippocampus in which presynaptic terminals of Schaffer collaterals and commissural fibers had been eliminated by bilateral kainate injections into the lateral ventricles 4–7 days before the microdialysis experiment and then 5-min ischemia was induced, a significant increase in glutamate was observed during the last half of 5-min ischemia. These results suggest that the efflux of glutamate from astrocytes does not contribute to the large ischemia-induced glutamate accumulation in the CA1 field of the hippocampus during 5-min ischemia but contributes to the ischemia-induced increase in glutamate level during ischemia with a longer duration and that ischemia-induced efflux of glutamate in the CA1 field during 5-min ischemia originates mainly from neuronal elements: presynaptic terminals and postsynaptic neurons.     

Interstitial 3-Methoxytyramine Reflects Striatal Dopamine Release: An In Vivo Microdialysis Study

Previous ex vivo studies have provided indirect evidence that the dopamine (DA) metabolite 3-methoxytyramine (3-MT) may be a useful index of DA release in vivo. In the present study, in vivo microdialysis was utilized to assess directly the relationship between extracellular DA and 3-MT in the striatum of rats following a variety of pharmacological manipulations. Apomorphine, a DA receptor agonist, produced a rapid, transient decrease in both DA and 3-MT. Conversely, the DA receptor antagonist haloperidol produced a concomitant increase in extracellular DA and 3-MT. Increases in DA and 3-MT were also noted following the administration of the DA uptake inhibitor, bupropion. Local application of tetrodotoxin resulted in the complete elimination of measurable amounts of DA and 3-MT in the dialysate, gamma-Butyrolactone also greatly decreased DA and 3-MT. Finally, d-amphetamine produced a large increase in DA and 3-MT in animals that had been treated previously with gamma-butyrolactone. The Pearson correlation coefficients for DA and 3-MT following these manipulations ranged from 0.87 to 0.97. These data indicate that interstitial 3-MT is an accurate index of DA release. However, when compared with previous ex vivo findings, the present results also suggest that changes in tissue concentrations of 3-MT may not reliably reflect DA release following certain pharmacological manipulations.     

Endogenous Amino Acid Release from Cultured Cerebellar Neuronal Cells: Effect of Tetanus Toxin on Glutamate Release

Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 mM K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, gamma-aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+-dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 micrograms/ml) for 24 h resulted in a total inhibition of the Ca2+-dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.     

In Vivo Measurement Using Microdialysis of the Release and Metabolism of 5-Hydroxytryptamine in Raphe Neurones Grafted to the Rat Hippocampus

The overflow and metabolism of serotonin (5-hydroxytryptamine; 5-HT) from transplants of embryonic medullary and mesencephalic raphe neurones in the previously 5-HT-denervated hippocampus have been analyzed in vivo using intracerebral dialysis. The average density of 5-HT-immunoreactive fibres in the grafted hippocampus was less than in nonlesioned hippocampus. Nonetheless, both basal and potassium-stimulated levels of 5-HT in the dialysates were restored to approximately normal after transplantation of medullary raphe cells, whereas mesencephalic implants resulted in over twice the 5-HT output observed in control hippocampus. However, 5-hydroxyindoleacetic acid (5-HIAA) overflow was increased only after grafting of mesencephalic raphe and then only to normal levels; medullary implants, by contrast, failed to enhance 5-HIAA output above that from lesion-only hippocampus. The evidence of a relative hyperactivity of the grafted neurones may explain the disproportionate improvements in various lesion-induced behavioural deficits after grafting of nervous tissue. In addition, differences in the presynaptic regulation of 5-HT release and metabolism are also apparent in the transplants; these variations are dependent on the precise origin of the serotoninergic cells.     

Extracellular Glutamate During Focal Cerebral Ischaemia in Rats: Time Course and Calcium Dependency

Abstract: The time course of changes in extracellular glutamic acid levels and their Ca²⁺ dependency were studied in the rat striatum during focal cerebral ischaemia, using microdialysis. Ischaemia-induced changes were compared with those produced by high K⁺-evoked local depolarization. To optimize time resolution, glutamate was analysed continuously as the dialysate emerged from the microdialysis probe by either enzyme fluorimetry or biosensor. The Ca²⁺ dependency of glutamate changes was examined by perfusing the probe with Ca²⁺-free medium. With normal artificial CSF, ischaemia produced a biphasic increase in extracellular glutamate, which started from the onset of ischaemia. During the first phase lasting ～10 min, dialysate glutamate level increased from 5.8 ± 0.9 µM· min^?1 to 35.8 ± 6.2 µM where it stabilized for ～3 min. During the second phase dialysate glutamate increased progressively to its maximum (82 ± 8 µM), reached after 55 min of ischaemia, where it remained for as long as it was recorded (3 h). The overall changes in extracellular glutamate were similar when Ca²⁺ was omitted from the perfusion medium, except that the first phase was no longer detectable and, early in ischaemia, extracellular glutamate increased at a significantly slower rate than in the control group (2.2 ± 1 µM· min^?1; p < 0.05). On the basis of these data, we propose that most of the glutamate released in the extracellular space in severe ischaemia is of metabolic origin, probably originating from both neurons and glia, and caused by altered glutamate uptake mechanisms. Comparison with high K⁺-induced glutamate release did not suggest that glutamate “exocytosis,” early after middle cerebral artery occlusion, was markedly limited by deficient ATP levels.     

Extracellular Dopamine and Serotonin in the Rat Striatum During Transient Ischaemia of Different Severities: A Microdialysis Study

Abstract: Generalised neurotransmitter overflow into the extracellular space on cerebral ischaemia has been widely reported and implicated in events leading to subsequent neu-ronal death. As little is known about the effect of depth of ischaemia on these changes, we have subjected anaesthetised rats to a sequence of four challenges [high K⁺ stimulus, moderate (penumbral) ischaemia, severe ischaemia, cardiac arrest] and have concurrently monitored both electrophysio-logical parameters and changes in extracellular dopamine, serotonin, and their metabolites in the striatum. Oi'particu-lar relevance to human stroke therapy was penumbral ischaemia, where ionic homeostasis was maintained even though electrical function was lost. All challenges increased extracellular monoamines, although levels were significantly greater when ischaemia was severe enough to produce sustained anoxic depolarisation. Baseline levels were rapidly restored during recovery phases. Acidic monoamine metabolites decreased significantly during each insult, returning to basal levels during reperfusion after moderate ischaemia, and to significantly higher levels after severe ischaemia. Results indicate that sustained anoxic depolarisation may be a critical factor in determining outcome after ischaemia, being associated with significantly greater release of monoamines, and impairment of electrical function recovery.     

Septal and Hippocampal Glutamate Receptors Modulate the Output of Acetylcholine in Hippocampus: A Microdialysis Study

Abstract: In the present study, glutamate receptor agonists and antagonists were administered by retrograde microdialysis into either the medial septum/vertical limb of the diagonal band (MS/vDB), or hippocampus, and the output of acetylcholine (ACh) was measured in the hippocampus by using intracerebral microdialysis. Perfusion with N -methyl- d -aspartate (NMDA) and ( S )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the MS/vDB caused an increase in ACh output in the hippocampus. This increase was completely blocked by coadministration of their respective antagonists d (−)-2-amino-5-phosphonopentanoic acid ( d -AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Perfusion in the MS/vDB with kainic acid also caused an increase in ACh output, but coadministration of CNQX attenuated the increase only partially. Perfusion with d -AP5 or CNQX alone in the septal probe did not affect ACh output from the hippocampus. In contrast to the results of septal administration of NMDA and AMPA, local perfusion with the same drugs in the hippocampus caused a decrease in ACh output. Whereas the results of septal administration of drugs indicate that septal cholinergic neurons probably receive excitatory glutamatergic innervation, the decrease in ACh output caused by administration of NMDA and AMPA in the hippocampus is poorly understood.     

Effect of Ischemia on the In Vivo Release of Striatal Dopamine, Glutamate, and γ-Aminobutyric Acid Studied by Intracerebral Microdialysis

We have previously described a marked attenuation of postischemic striatal neuronal death by prior substantia nigra (SN) lesioning. The present study was carried out to evaluate whether the protective effect of the lesion involves changes in the degree of local cerebral blood flow (ICBF) reduction, energy metabolite depletion, or alterations in the extracellular release of striatal dopamine (DA), glutamate (Glu), or gamma-aminobutyric acid (GABA). Control and SN-lesioned rats were subjected to 20 min of forebrain ischemia by four-vessel occlusion combined with systemic hypotension. Levels of ICBF, as measured by the autoradiographic method, and energy metabolites were uniformly reduced in both the ipsi- and contralateral striata at the end of the ischemic period, a finding implying that the lesion did not affect the severity of the ischemic insult itself. Extracellular neurotransmitter levels were measured by microdialysis; the perfusate was collected before, during, and after ischemia. An approximately 500-fold increase in DA content, a 7-fold increase in Glu content, and a 5-fold increase in GABA content were observed during ischemia in nonlesioned animals. These levels gradually returned to baseline by 30 min of reperfusion. In SN-lesioned rats, the release of DA was completely prevented, the release of GABA was not affected, and the release of Glu was partially attenuated. However, excessive extracellular Glu concentrations were still attained, which are potentially toxic. This, taken together with the previous neuropathological findings, suggests that excessive release of DA is important for the development of ischemic cell damage in the striatum.     

Determination of Acetylcholine Release in the Striatum of Anesthetized Rats Using In Vivo Microdialysis and a Radioimmunoassay

A vertical-type in vivo microdialysis probe and a sensitive, specific radioimmunoassay (RIA) were used to study the mechanism of acetylcholine (ACh) release in the striatum of anesthetized rats. Without the use of physostigmine, a cholinesterase inhibitor, our RIA could still detect the amount of ACh present in the perfusate (5.6 +/- 0.6 fmol/min, n = 16). Tetrodotoxin (1 microM) produced a significant decrease in the amount of ACh collected in the perfusate, suggesting that basal ACh determined under the present experimental conditions was related to cholinergic neural activity. Atropine (0.1-1 microM) applied topically via the dialysis probe did not affect the amount of ACh recovered in the perfusate in the absence of physostigmine. Addition of physostigmine (10 microM) to the perfusion fluid produced about a 100-fold increase in the amount of ACh collected. In the presence of physostigmine, topical administration of atropine and pirenzepine (0.01-1 microM) through a dialysis probe produced a further three- to fourfold increase in ACh output, whereas a slight increase was produced by AF-DX 116 at the highest concentration (1 microM). These results indicate that presynaptic modulation of ACh release in the striatum does not occur under basal conditions, and that presynaptic M1 muscarinic receptors are involved in the modulation of ACh release when the ACh concentration is raised under certain conditions.     

N-methyl-d-Aspartic Acid Differentially Regulates Extracellular Dopamine, GABA, and Glutamate Levels in the Dorsolateral Neostriatum of the Halothane-Anesthetized Rat: An In Vivo Microdialysis Study

Abstract: The effects of local perfusion with the glutamate receptor agonist NMDA and the noncompetitive NMDA receptor antagonist dizolcipine (MK-801) on extracellular dopamine (DA), GABA, and glutamate (Glu) levels in the dorsolateral striatum were monitored using in vivo microdialysis in the halothane-anesthetized rat. In addition, the sensitivity of both the basal and NMDA-induced increases in levels of these neurotransmitter substances to perfusion with tetrodotoxin (TTX; 10^?5 M) and a low Ca²⁺ concentration (0.1 mM) was studied. The results show that the local perfusion (10 min) with both the 10^?3 and 10^?4 M dose of NMDA increased striatal DA and GABA outflow, whereas only the (10^?3 M) dose of NMDA was associated with a small and delayed increase in extracellular Glu levels. The NMDA-induced effects were dose-dependently counteracted by simultaneous perfusion with MK-801 (10^?6 and 10^?5 M). Both the basal and NMDA (10^?3 M)-induced increase in extracellular striatal DA content was reduced in the presence of TTX and a low Ca²⁺ concentration, whereas both basal and NMDA-stimulated GABA levels were unaffected by these treatments. Both the basal and NMDA-stimulated Glu levels were enhanced following TTX treatment, whereas perfusion with a low Ca²⁺ concentration reduced basal Glu levels and enhanced and prolonged the NMDA-induced stimulation. These data support the view that NMDA receptor stimulation plays a role in the regulation of extracellular DA, GABA, and Glu levels in the dorsolateral neostriatum and provide evidence for a differential effect of NMDA receptor stimulation on these three striatal neurotransmitter systems, possibly reflecting direct and indirect actions mediated via striatal NMDA receptors.     

Homocysteate-Evoked Release of Acetylcholine from the Rabbit Retina

Abstract: The cholinergic amacrine cells of the rabbit retina can be labeled with [³H]choline and the activity of the cholinergic population monitored by following the release of [³H]acetylcholine. It has been proposed that l -homocysteate may be the main endogenous transmitter released onto cholinergic amacrine cells by bipolar cells. Therefore, we have examined the effects of the isomers of homocysteate on the release of [³H]acetylcholine. In magnesium-free medium, d -homocysteate was slightly more potent than the l -isomer. The addition of magnesium, which blocks responses mediated by NMDA receptors, preferentially reduced but did not eliminate, the response to l -homocysteate. 2-Amino-7-phosphonoheptanoate, a potent NMDA antagonist, preferentially blocked l -homocysteate evoked responses. 6,7-Dinitroquinoxaline-2,3-dione, a potent kainate antagonist, preferentially blocked d -homocysteate-evoked responses. Therefore, in the rabbit retina, l -homocysteate is an NMDA-preferring agonist, whereas d -homocysteate is a kainate-preferring agonist. In addition, we found that l -homocysteate can activate the physiologically activated kainate receptor but only when used in millimolar concentrations and under conditions that minimize NMDA-receptor activation. However, the low potency of l -homocysteate combined with low affinity for the glutamate transporter, lack of immunocytochemical localization in bipolar cells, and low retinal content place serious limitations on the role of l -homocysteate at the bipolar-to-cholinergic amacrine cell synapse.     

Effect of the Non-NMDA Receptor Antagonist GYKI 52466 on the Microdialysate and Tissue Concentrations of Amino Acids Following Transient Forebrain Ischaemia

Abstract: The effect of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) on ischaemia-induced changes in the microdialysate and tissue concentrations of glutamate, aspartate, and γ-aminobutyric acid (GABA) was studied in rats. Twenty minutes of four-vessel occlusion resulted in a transient increase in microdialysate levels of glutamate, aspartate, and GABA in striatum, cortex, and hippocampus. Administration of GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min intravenously starting 20 min before onset of ischaemia) inhibited ischaemia-induced increases in microdialysate glutamate and GABA in striatum without affecting the increases in hippocampus or cortex. Twenty minutes of four-vessel occlusion resulted in immediate small decreases and larger delayed (72 h) decreases in tissue levels of glutamate and aspartate. Transient increases in tissue levels of GABA were shown in all three structures at the end of the ischaemic period. At 72 h, after the ischaemic period, significantly reduced GABA levels were observed in striatum and hippocampus. GYKI 52466, given under identical conditions as above, augmented the ischaemia-induced decrease in striatal tissue levels of glutamate and aspartate, without significantly affecting the decreases in hippocampus and cortex. Twenty minutes of ischaemia resulted in a large increase in microdialysate dopamine in striatum. GYKI 52466 failed to inhibit this increase. Kainic acid (500 μM infused through the probe for 20 min) caused increases in microdialysate glutamate and aspartate in the striatum. GYKI 52466 (10 mg/ kg bolus + 10 mg/kg/60 min) completely inhibited the kainic acid-induced glutamate release. In conclusion, the action of the non-NMDA antagonist, GYKI 52466, in the striatum is different from that in the cortex and hippocampus. The inhibition by GYKI 52466 of ischaemia-induced and kainate-induced increases in microdialysate glutamate concentration in the striatum may be related to the neuroprotection provided by GYKI 52466 in this region.