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.     

High potassium-induced glutamate release in the cochlea: in vivo microdialysis study combined with on-line enzyme fluorometric detection of glutamate

The time course of changes in perilymphatic glutamate release and their Ca²⁺-dependency were studied in the guinea pig cochlea during high K⁺-evoked depolarization. The glutamate concentration was analyzed continuously by an enzyme-linked fluorometric assay combined with microdialysis. Two peaks of glutamate increase were found in response to perfusion for 10 min. In the absence of Ca²⁺, the first peak was diminished, whereas the inhibition of the second peak was minimal.     

Electrochemical detection of endogenous glutamate release from rat spinal cord organotypic slices as a real-time method to monitor excitotoxicity

Excessive release of glutamate is believed to be a major component of cell damage following excitotoxicity associated with acute spinal cord injury. Using an in vitro model of excitotoxicity evoked by kainate on rat organotypic spinal slice cultures, we investigated the timecourse and extent of endogenous glutamate release following 1h application of kainate using a commercially available biosensor placed on the ventral horn area of such slices. The release of glutamate peaked 7 min from the start of kainate (0.5 mM) application and then slowly declined to baseline prior to kainate washout. A lower concentration of kainate (0.1 mM) induced a smaller release that developed more slowly. At the end of each experiment, the number of pyknotic nuclei was counted to quantify cell death that was found to be about 10% of the total population with no significant neuronal loss. This finding accords with previous studies showing that, on the basis of neuronal counts at various times after kainate application, neuronal death was delayed. The present data demonstrate that a glutamate biosensor can be employed for real-time monitoring of endogenous glutamate release from an in vitro model of acute spinal cord injury applied to organotypic slices. This method can, therefore, be useful to study the cellular action of neuroprotective drugs targeting glutamate release mechanisms.     

Quantifying the effect of light activated outer and inner retinal inhibitory pathways on glutamate release from mixed bipolar cells.

Inhibition mediated by horizontal and amacrine cells in the outer and inner retina, respectively, are fundamental components of visual processing. Here, our purpose was to determine how these different inhibitory processes affect glutamate release from ON bipolar cells when the retina is stimulated with full‐field light of various intensities. Light‐evoked membrane potential changes (ΔV_m) were recorded directly from axon terminals of intact bipolar cells receiving mixed rod and cone inputs (Mbs) in slices of dark‐adapted goldfish retina. Inner and outer retinal inhibition to Mbs was blocked with bath applied picrotoxin (PTX) and NBQX, respectively. Then, control and pharmacologically modified light responses were injected into axotomized Mb terminals as command potentials to induce voltage‐gated Ca²⁺ influx (Q^Ca) and consequent glutamate release. Stimulus‐evoked glutamate release was quantified by the increase in membrane capacitance (ΔC_m). Increasing depolarization of Mb terminals upon removal of inner and outer retinal inhibition enhanced the ΔV_m/Q^Ca ratio equally at a given light intensity and inhibition did not alter the overall relation between Q^Ca and ΔC_m. However, relative to control, light responses recorded in the presence of PTX and PTX + NBQX increased ΔC_m unevenly across different stimulus intensities: at dim stimulus intensities predominantly the inner retinal GABAergic inhibition controlled release from Mbs, whereas the inner and outer retinal inhibition affected release equally in response to bright stimuli. Furthermore, our results suggest that non‐linear relationship between Q^Ca and glutamate release can influence the efficacy of inner and outer retinal inhibitory pathways to mediate Mb output at different light intensities.     

Changing sensitivity to cell death during development of retinal photoreceptors

Photoreceptor cell death occurs during both normal and pathological retinal development. We tested for selective induction and blockade of cell death in either retinal photoreceptors or their precursors. Organotypical retinal explants from rats at postnatal days 3-11 were treated in vitro for 24 hr with thapsigargin, okadaic acid, etoposide, anisomycin, or forskolin. Explant sections were examined for cell death, and identification of either photoreceptors or proliferating/immediate postmitotic cells followed imunohistochemistry for either rhodopsin or bromodeoxyuridine and proliferating cell nuclear antigen, respectively. Photoreceptor cell death was selectively induced by either thapsigargin or okadaic acid, whereas death of proliferating/immediate postmitotic cells was induced by etoposide. Prelabeling of proliferating precursors allowed direct demonstration of changing sensitivity of photoreceptors to various chemicals. Degeneration of both photoreceptors and proliferating/immediate postmitotic cells depended on protein synthesis. Increase of intracellular cyclic AMP blocked degeneration of postmitotic, but not of proliferating, photoreceptor precursors. The selective induction and blockade of cell death show that developing photoreceptors undergo progressive changes in mechanisms of programmed cell death associated with phenotypic differentiation.     

Non-opioid antitussives inhibit endogenous glutamate release from rabbit hippocampal slices.

The non-opioid antitussive drugs, dextromethorphan, caramiphen and carbetapentane, are also anticonvulsant. The effects of these antitussives on potassium-stimulated release of endogenous amino acids from rabbit hippocampal slices was tested. All 3 drugs significantly reduced the release of glutamate, with carbetapentane being the most potent (IC50 approximately 40 microM). We suggest that the anticonvulsant action may be due to their ability to decrease glutamate release.     

Endogenous glutamate stimulates release of alpha-melanocyte-stimulating hormone from the rat hypothalamus.

Release of alpha-melanocyte-stimulating hormone (alpha-MSH) and glutamic acid was quantified from superfused slices of rat hypothalamus. Application of L-glutamic acid 10(-4) M failed to evoke release of alpha-MSH but, in the presence of 10(-4) M dihydrokainic acid (DHK) an inhibitor of glutamate uptake systems, caused significant stimulation of release. DHK caused gradual and sustained increases in both alpha-MSH and glutamate release. That in alpha-MSH was blocked by 10(-4) M DL-2-amino-5-phosphopentanoic acid, a competitive N-methyl-D-aspartic acid (NMDA)-type glutamate receptor antagonist. We conclude that hypothalamic glutamate is subject to rapid uptake through mechanisms blocked by DHK and that alpha-MSH release is stimulated by endogenous and exogenous glutamate through NMDA-type receptors.     

Deficient NMDA-mediated glutamate release from synaptosomes of schizophrenics

Previous studies from our laboratory indicated that the veratridine-induced release of glutamate and GABA from synaptosomes derived from brains of schizophrenics was decreased. In the present study, synaptosomes were prepared from frozen brain samples from schizophrenics and from controls. Stimulation by 10 μmol/L 2-amino-3-hydroxy-5-methoxylisoxazole-4-propionic acid (AMPA) produced equal glutamate release from both groups. Release induced by either 10 μmol/L kainic acid (KA) or n-methyl-d-asparate (NMDA) was reduced significantly in the preparations derived from schizophrenics. Similarly, the amount of GABA released by 50 μmol/L glutamate was also reduced in the schizophrenic-derived synaptosomes. However, in membranes derived from the crude synaptosomal pellet, no differences between the controls and schizophrenics were observed in measures of total glutamate binding or its displacement by NMDA. The data demonstrate a deficiency in NMDA (and possibly KA) receptor functioning in schizophrenics and support the “second-generation” theories of schizophrenia as a glutamatergic deficiency disorder.     

Carrier-mediated release of GABA from retinal horizontal cells

H1 horizontal cells in goldfish retina are probably GABAergic and receive excitatory synaptic input from red cones. This input should affect the synaptic release of GABA from H1 cells. We studied the uptake and release of [3H]GABA from the isolated goldfish retina by use of autoradiography. When retinas were incubated in the light for 15 min in 0.72 microM [3H]GABA, heavy label was found over the somata (HS) and axon terminals (HAT) of H1 horizontal cells, and over pyriform amacrine cell bodies and their processes in sublamina b of the IPL. Postincubation of retinas, preloaded with [3H]GABA, in 0.5-10 mM L-glutamate or 0.1-10 mM L-aspartate, resulted in a dose-dependent and selective loss of [3H]GABA from HS and very little loss from HAT. This loss was not due to an efflux of metabolites of [3H]GABA or to any calcium-dependent vesicular release of [3H]GABA from HS. The glutamate-evoked release of [3H]GABA by H1 cells was sodium dependent, sensitive to substitution of lithium for sodium, and inhibited by nipecotic acid. In addition, [3H]GABA was released from HS by 0.1 mM ouabain but not by 50 mM potassium chloride. Our results suggest that the chemically evoked release of [3H]GABA from HS is mediated by a sodium-dependent transport carrier which may be responsible for the high affinity uptake of [3H]GABA by H1 cells as well. Since synaptic vesicles are not found at presumed synaptic release sites in H1 cells, we suggest that the GABA which is released synaptically from H1 cells may derive from a cytoplasmic pool of GABA and is released by means of a transport carrier. This carrier appears to depend primarily on the sodium concentration gradient across the H1 cell membrane rather than on the membrane potential of the H1 cell for its action. The relevance of the carrier-mediated release of GABA from HS in regard to the synaptic function of H1 cells is discussed.     

Methamphetamine neurotoxicity and striatal glutamate release: comparison to 3,4-methylenedioxymethamphetamine.

The effect of repeated administration of either methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA) or vehicle on the extracellular concentrations of glutamate (GLU), aspartate, taurine, dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), was studied in awake, freely moving rats using in vivo microdialysis. MA (7.5 mg/kg, i.p.) administered every 2 h for a total of 3 injections, increased the extracellular concentration of GLU in the anteromedial striatum. By contrast, neither vehicle nor MDMA (9.2 and 13.8 mg/kg) increased GLU efflux following repeated administration. Both MA and MDMA increased the extracellular concentration of DA in the striatum. However, the cumulative increase in DA was significantly greater in the MDMA treated animals as compared to the MA group. The concentrations of DA, serotonin (5-HT) and their metabolites were determined in the striatum 7 days following the repeated administration of MA, MDMA and vehicle. MA, but not MDMA or vehicle, decreased the concentration of DA in the striatum. Conversely, MDMA (13.8 mg/kg) decreased the concentration of 5-HT, whereas MA, MDMA (9.2 mg/kg) and vehicle had no effect on striatal 5-HT content. These data are suggestive that the long-term (7 day) DA neurotoxicity produced by the repeated administration of MA is mediated, in part, by a delayed increase in extracellular concentrations of GLU. In contrast, repeated administration of MDMA, at a dose which produced a long-term (7 day) depletion of striatal 5-HT content, had no effect on GLU efflux in the striatum.     

Sick photoreceptors attract activated microglia from the ganglion cell layer: a model to study the inflammatory cascades in rats with inherited retinal dystrophy.

Understanding of neuron-glial interactions in neurodegenerative diseases remains limited, but is of crucial importance for unravelling the etiology of such disorders both in humans and in animals. The present work employed a new, function-dependent technique for examining the role of microglia in rats afflicted with inherited retinal photoreceptor degeneration (strain: royal college of surgeons, RCS). In this rat strain, which served as a surrogate for human inherited retinal photoreceptor dystrophy, the optic nerve was cut and the ganglion cells were retrogradely labelled with the fluorescent dye 4Di-10ASP. The experiment was performed under three different conditions: (1) at the 50th day of postnatal age (P50) when there is ongoing degeneration of photoreceptor cells, (2) at P110 when most photoreceptors were degenerated and (3) at P50 in non-dystrophic rats of the Sprague-Dawley strain. After axotomy-induced ganglion cell death and labelling of activated microglia by phagocytosis of the ganglion cell debris, this study monitored whether the labelled and therefore identifiable microglial cells within the severed ganglion cell layer (GCL) are prompted to migrate and to participate in phagocytosis of debris produced within the endogenously degenerating photoreceptor cell layer (PRL). Massive migration of microglial cells from the GCL to the PRL occurred in dystrophic animals with optic nerve transection at P50. Double-labelling of microglial cells with the fluorescent dye ingested within the GCL and with lipofuscin ingested within the PRL indicated the ability of these cells to perform double-phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific interaction of glutamate with membranes from cultured retinal pigment epithelium

Excitatory amino acids (EAA) have been shown to induce phagocytosis in retinal pigment epithelial (RPE) cells. In order to explore if this action is receptor-mediated, we have identified and characterized receptors for L-glutamate through the binding of [³H]L-glutamate to membranes from chick RPE cells in primary culture. Specific binding was found saturable, with K_B = 333nM and B_max = 3.2 pmol/mg protein in frozen/thawed membranes. Na⁺-independent binding was present in cultures of 16 and 25 days in vitro, and was not affected by temperature. Pharmacological profile of analogues of EAA at different receptor types suggests the presence of a metabotropic type receptor (L-glutamate > S-2-amino-3-phosphonopropionate > 2-amino-4-phosphonobutyrate = trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate > quisqualate). Excitatory amino acid analogues acting at the NMDA-receptor also displaced bound L-glutamate, and a noticeable stimulation of specific binding of this ligand by glycine was shown; this effect was mimicked by D-serine and 1-hydroxy-3-aminopyrrolidone-2 (HA-966) but not by 7-chlorokynurenate, and was not inhibited by strychnine. Since taurine and GABA also increased specific binding, it is likely that modulation of EAA receptors in RPE differs from that in neurons. © 1993 Wiley-Liss, Inc.     

Rapid desensitization converts prolonged glutamate release into a transient EPSC at ribbon synapses between retinal bipolar and amacrine cells

The mechanisms underlying the conversion of prolonged glutamate release from ribbon synapses in bipolar cells to sustained and transient excitatory postsynaptic responses in identified retinal amacrine cells were studied in tiger salamander (Ambystoma tigrinum) retina. A retina slice preparation with whole cell patch recording techniques under voltage- and current-clamp conditions was used to assay the electrical properties of bipolar and amacrine cells. Amacrine cells were categorized into two basic forms: (i) transient amacrine cells that respond to a step of light with a burst of spikes only at the transitions of the step; and (ii) sustained amacrine cells that respond with continuous spiking during the entire light step. The two cell types each had a characteristic morphology: transient amacrine cells possessed wide dendritic fields (χ = 375 μm), while sustained cells had much more narrowly confined dendritic fields (χ = 85 μm). Whole cell voltage-gated currents of the transient and sustained cell types were not significantly different. Both cell types had spikes that were sensitive to tetradotoxin (TTX, 0.3 μm ) with voltage deflections of up to 100 mV. Light-evoked excitatory synaptic currents relaxed rapidly in transient neurons (τ_1/2 = 100 ms) and more slowly in sustained neurons (τ_1/2 = 1.2 s). EPSCs in both cells reversed near 0 mV. Rapid application of glutamate or kainate elicited rapidly desensitizing ionic currents (τ_1/2 = 85 ms) followed by a slowly desensitizing component. Cyclothiazide, a drug that eliminates rapid desensitization, lengthened the time course of the glutamate gated current from τ_1/2 = 85 ms to about 3 s, and the relaxation kinetics of the glutamatergic EPSC from τ_1/2 = 85 ms to about 1.0 s. These data suggest that a key determinant in forming transient versus sustained responses in amacrine cells of vertebrate retina is the differences in their excitatory, glutamatergic synaptic inputs, and that rapid desensitization of glutamate receptors plays a role in converting the presynaptic signal associated with sustained glutamate release into a postsynaptic, transient signal at the ribbon synapse.     

Glutaminase inhibition and the release of neurotransmitter glutamate from synaptosomes

Cerebrocortical synaptosomes were incubated with glutamine together with 6-diazo-5-oxo-L-norleucine (DON) and NH4Cl (which are known to inhibit phosphate-stimulated glutaminase) in order to assess the effect of such inhibition on the pool sizes and extent of evoked release of endogenous amino acids, particularly glutamate. DON (5 mM) inhibited glutaminase by 73-89% and NH4Cl (1-4 mM) inhibited the enzyme by 45-53% under the conditions employed. NH4Cl (4 mM) incubated with synaptosomes for 30 min reduced pool sizes of aspartate and glutamate by 28% and inhibited release of glutamate by 55% compared to control release. DON caused a decrease in both the pool size of glutamate (22%) and the extent of veratrine-evoked release of amino acids (21%).     

Evaluation of glutamate as a hippocampal neurotransmitter: Glutamate uptake and release from synaptosomes

The uptake and release of glutamic acid by synaptosomal preparations from rat hippocampus was studied. Glutamate uptake showed a low and high affinity component. High affinity glutamate uptake was highly depedent upon Na⁺, competitively inhibited by aspartic acid and was somewhat more active in fractions containing synaptosomes than in microsomal or nuclear fractions. Different hippocampal subfields showed similar kinetic properties for glutamate accumulation.The release of endogenous and exogenously loaded glutamate was studied and compared to GABA, a known neurotransmitter in the hippocampus. There were two types of K⁺-evoked release from crude synaptosomal fractions for both amino acids. One was Ca²⁺-dependent and the other was not. Purified synaptosomal fractions showed an enrichment of the Ca²⁺-depedent, K⁺-stimulated release over crude synaptosomal fractions, whereas Ca²⁺-independet, K⁺-stimulated efflux was markedly reduced in purified synaptosomes. Efflux from microsomes was K⁺-stimulated and showed no dependence on external Ca²⁺, even in the presence of A23187, the Ca²⁺ ionophore. Qualitatively, the characteristics of GABA and glutamate efflux were identical for both Ca²⁺-dependent and -independent release.Our results show that glutamate release originates primarily from synaptic endings, and that this release meets the characteristics of well-known stimulus secretion coupling processes seen for another transmitter in the hippocampus as well as many other transmitters in a wide variety of systems. These data strengthen our previous suggestion that glutamate may be a neurotransmitter in the rat hippocampus.     

A micromethod to measure platelet aggregation and atp release by impedance.

A standard electrode has been modified in order to detect platelet aggregation by impedance in small volumes of whole blood. The modified electrode was constructed to fit into a standard 0.5-ml cuvette normally employed for detecting platelet aggregation in platelet-rich plasma (PRP) on a Chrono-log Aggregometer (Haverford, PA). Whole blood samples were mixed 1:1 with saline and total volumes of 1 ml, 500 microl and 300 microl were tested. ATP release was recorded concurrently with aggregation by measuring the luminescence from the firefly luciferin-luciferase reaction. The modified electrode could detect aggregation in the 300- and 500-microl samples with the same efficiency as the 1-ml samples. ATP release in the 300- and 500-microl samples yielded equivalent levels of release as detected with the 1-ml samples. This electrode is well suited for evaluation of platelet function in research and clinical blood samples where only small volumes of blood are available.     

Calcium-dependent release of N-acetylaspartylglutamate from retinal neurons upon depolarization

N-Acetylaspartylglutamate (NAAG) is present in high concentrations specifically in the nervous system. Its neuronal distribution, presence in synaptic vesicles and its excitatory actions support the hypothesis that this dipeptide participates in communication between neurons. Following the incorporation of [3H]glutamate by frog retinal cells in vivo, the release of radiolabeled glutamate, GABA and NAAG was studied during acute incubation of the retina in vitro. Release of the radiolabeled amino acids and dipeptide was stimulated by elevated extracellular potassium. The release required the presence of extracellular calcium. These data are the first which demonstrate the release of NAAG following biosynthesis from a radiolabeled precursor and are consistent with synaptic release of this dipeptide.     

Functional remodeling of glutamate receptors by inner retinal neurons occurs from an early stage of retinal degeneration

Retinitis pigmentosa reflects a family of diseases that result in retinal photoreceptor death and functional blindness. The natural course of retinal changes secondary to photoreceptor degeneration involves anatomical remodeling (cell process alterations and soma displacement) and neurochemical remodeling. Anatomical remodeling predominantly occurs late in the disease process and cannot explain the significant visual deficits that occur very early in the disease process. Neurochemical remodeling includes modified glutamate receptor disposition and altered responses secondary to functional activation of glutamate receptors. We investigated the neurochemical remodeling of retinal neurons in the rd/rd (rd1) mouse retina by tracking the functional activation of glutamate receptors with a cation probe, agmatine. We provide evidence that bipolar cells and amacrine cells undergo selective remodeling of glutamate receptors during the early phases of retinal degeneration. These early neurochemical changes in the rd/rd mouse retina include the expression of aberrant functional ionotropic glutamate receptors on the cone ON bipolar cells from postnatal day 15 (P15), poor functional activation of metabotropic glutamate receptors on both rod and cone ON bipolar cells throughout development/degeneration, and poor functional activation of N‐methyl‐D‐aspartate receptors on amacrine cells from P15. Our results suggest that major neurochemical remodeling occurs prior to anatomical remodeling, and likely accounts for the early visual deficits in the rd/rd mouse retina. J. Comp. Neurol. 514:473–491, 2009. © 2009 Wiley‐Liss, Inc.