Glu Receptor Agonists Research Articles

Hyperexcitability and brain morphological differences in mice lacking the cystine/glutamate antiporter, system xc.

System xc- (Sxc- ) is a heteromeric antiporter (L-cystine/L-glutamate exchanger) expressed predominately on astrocytes in the central nervous system. Its activity contributes importantly to the maintenance of the ambient extracellular glutamate levels, as well as, to cellular redox homeostasis. Since alterations in glutamate levels and redox modifications could cause structural changes, we analyzed gross regional morphology of thionin-stained brain sections and cellular and subcellular morphology of Golgi-Cox stained layer V pyramidal neurons in the primary motor cortex (PM1) of mice naturally null for SLC7A11 (SLC7A11sut/sut )-the gene that encodes the substrate specific light chain (xCT) for Sxc- . Intriguingly, in comparison to age- and sex-matched wild-type (SLC7A11+/+ ) littermate controls, we found morphologic changes-including increased dendritic complexity and mushroom spine area in males and reduced corpus callosum and soma size in females-that have previously been described, in each case, as morphological correlates of excitability. Consistent with this, we found that both male and female SLC7A11sut/sut mice had lower convulsive seizure thresholds and greater seizure severity than their sex-matched wild-type (SLC7A11+/+ ) littermates after acute challenge with two pharmacologically distinct chemoconvulsants: the Glu receptor agonist, kainic acid (KA), or the GABAA receptor antagonist, pentylenetetrazole (PTZ). These results suggest that the loss of Sxc- signaling in males and females perturbs excitatory/inhibitory (E/I) balance in vivo, potentially through its regulation of cellular and subcellular morphology.

Clustered burst firing in FMR1 premutation hippocampal neurons: amelioration with allopregnanolone

Premutation CGG repeat expansions (55-200 CGG repeats; preCGG) within the fragile X mental retardation 1 (FMR1) gene cause fragile X-associated tremor/ataxia syndrome (FXTAS). Defects in neuronal morphology and migration have been described in a preCGG mouse model. Mouse preCGG hippocampal neurons (170 CGG repeats) grown in vitro develop abnormal networks of clustered burst (CB) firing, as assessed by multielectrode array recordings and clustered patterns of spontaneous Ca(2+) oscillations, neither typical of wild-type (WT) neurons. PreCGG neurons have reduced expression of vesicular GABA and glutamate (Glu) transporters (VGAT and VGLUT1, respectively), and preCGG hippocampal astrocytes display a rightward shift on Glu uptake kinetics, compared with WT. These alterations in preCGG astrocytes and neurons are associated with 4- to 8-fold elevated Fmr1 mRNA and occur despite consistent expression of fragile X mental retardation protein levels at ∼50% of WT levels. Abnormal patterns of activity observed in preCGG neurons are pharmacologically mimicked in WT neurons by addition of Glu or the mGluR1/5 agonist, dihydroxyphenylglycine, to the medium, or by inhibition of astrocytic Glu uptake with dl-threo-β-benzyloxyaspartic acid, but not by the ionotropic Glu receptor agonists, α-2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid or N-methyl-d-aspartic acid. The mGluR1 (7-(hydroxyimino)cyclopropa [b]chromen-1a-carboxylate ethyl ester) or mGluR5 (2-methyl-6-(phenylethynyl)pyridine hydrochloride) antagonists reversed CB firing. Importantly, the acute addition of the neurosteroid allopregnanolone mitigated functional impairments observed in preCGG neurons in a reversible manner. These results demonstrate abnormal mGluR1/5 signaling in preCGG neurons, which is ameliorated by mGluR1/5 antagonists or augmentation of GABA(A) receptor signaling, and identify allopregnanolone as a candidate therapeutic lead.

Molecular and pharmacological evidence for a facilitatory functional role of pre‐synaptic GLUK2/3 kainate receptors on GABA release in rat trigeminal caudal nucleus

BackgroundGamma-aminobutyric acid (GABA) and glutamate (GLU) are involved in nociceptive signals processing in the trigeminal system. In this study, we investigated the influence of excitatory transmission on GABA release in nerve terminals isolated from the rat trigeminal caudal nucleus (TCN).MethodsWe utilize biochemical (superfused synaptosomes loaded with [3H]GABA) and morphological (immunofluorescence experiments with specific antibody) techniques.ResultsOur results show that GLU potentiates the release of [3H]GABA evoked by 9, 15 and 30 mM [K+]e; 15 mM [K+]e-evoked [3H]GABA release was also reinforced by domoate and kainate (KA), two naturally occurring GLU-receptor agonists. The enhancement of 15 mM [K+]e-evoked [3H]GABA release produced by 100 μM KA was abolished by NBQX, a mixed AMPA/KA receptor antagonist, but was not affected by GYKI52466, a selective AMPA receptor antagonist. ATPA, a selective agonist for KA receptors containing the GLUK1 subunit, had no effect on depolarization-induced [3H]GABA release, and UBP310, which selectively antagonizes these same receptors, failed to reverse the KA-induced potentiation of 15 mM [K+]e-evoked [3H]GABA release. The KA-induced potentiation was also unaffected by concanavalin A (10 μM), a positive allosteric modulator of GLUK1- and GLUK2-containing KA receptors.Immunofluorescence experiments revealed that GABAergic nerve terminals in the TCN differentially expressed GLUK subunits, with GLUK2/3-positive terminals being twice more abundant than GLUK1-containing synaptosomes.ConclusionsThese findings indicate that pre-synaptic KA receptors facilitating GABA release from TCN nerve terminals mainly express GLUK2/GLUK3 subunits, supporting the notion that different types of KA receptors are involved in the various stages of pain transmission.

Gender‐dependent ATPA‐induced changes in long‐term potentiation in the rat lateral amygdala

There is increasing evidence that kainate receptors contribute to both postsynaptic and presynaptic signaling not only in the hippocampus but also in the amygdala. The present study demonstrates that low concentrations of the specific kainate GLU(K5) receptor agonist, ATPA, depressed baseline activity in the lateral nucleus of the rat amygdala (LA), induced by stimulation of external capsule fibers or by intranuclear stimulation in horizontal brain slices. ATPA reduced high-frequency-induced long-term potentiation (LTP) in males while it enhanced LTP in females during certain phases of the estrus cycle. In untreated slices from females, LA-LTP differed depending on the phase of the estrus cycle. In addition, we show for the first time that the p38 mitogen-activated protein (MAP) kinase inhibitor, SKF 86002, reduced LA-LTP. In males, the effects of ATPA and SKF 86002 were not additive. To the contrary, in females, the exposure to ATPA in control plus SKF 86002 increases LTP relative to control plus SKF 86002 alone. Thus, we demonstrate that the effectiveness of GLU(K5) stimulation on plasticity changes in the amygdala is gender-dependent and that the MAP kinase pathway might be involved in males.

Subthalamic Stimulation-Induced Forelimb Dyskinesias Are Linked to an Increase in Glutamate Levels in the Substantia Nigra Pars Reticulata

The neurobiological mechanisms by which high-frequency stimulation of the subthalamic nucleus (STN-HFS) alleviates the motor symptoms of Parkinson's disease (PD) remain unclear. In this study, we analyzed the effects of STN-HFS on motor behavior in intact or hemiparkinsonian rats (6-hydroxydopamine lesion of the substantia nigra pars compacta) and investigated the correlation between these effects and extracellular glutamate (Glu) and GABA levels, assessed by intracerebral microdialysis in the substantia nigra pars reticulata (SNr). STN-HFS at an intensity corresponding to the threshold inducing contralateral forelimb dyskinesia, increased Glu levels in the SNr of both intact and hemiparkinsonian rats. In contrast, STN-HFS at half this intensity did not affect Glu levels in the SNr in intact or hemiparkinsonian rats but increased GABA levels in hemiparkinsonian rats only. STN-HFS-induced forelimb dyskinesia was blocked by microinjection of the Glu receptor antagonist kynurenate into the SNr and facilitated by microinjection of a mixture of the Glu receptor agonists AMPA and NMDA into the SNr. These new neurochemical data suggest that STN-HFS-induced forelimb dyskinesia is mediated by glutamate, probably via the direct activation of STN axons, shedding light on the mechanisms of STN-HFS in PD.

Abolition of chondral mineralization by group III metabotropic glutamate receptors expressed in rodent cartilage

1 Previous studies have demonstrated the functional expression by osteoblasts of glutamate (Glu) signaling machineries responsible for the stimulation of cell proliferation and differentiation in bone, while there is no information available on the expression of the Glu signaling system by cartilage to date. 2 In cultured mouse embryonic metatarsals isolated before vascularization, chondral mineralization was almost completely inhibited in the presence of the group III metabotropic Glu receptor (mGluR) agonist L-(1)-2-amino-4-phosphonobutyrate (L-AP4) in a manner sensitive to an antagonist, with the total length being unchanged. 3 A group II mGluR agonist was similarly more effective in inhibiting the mineralization than a group I mGluR agonist, while none of ionotropic GluR agonists drastically affected the mineralization. 4 Both histological and in situ hybridization analyses revealed that L-AP4 specifically inhibited chondral mineralization, without apoptotic cell death, in cultured metatarsals. 5 In addition to the constitutive expression of mRNA for particular mGluRs in both cultured mouse metatarsals and rat costal chondrocytes, L-AP4 significantly inhibited the accumulation of cyclic AMP by forskolin and parathyroid hormone in a manner sensitive to a group III mGluR antagonist in cultured chondrocytes. 6 Moreover, L-AP4 drastically inhibited the expression of osteopontin mRNA in both cultured metatarsals and chondrocytes. 7 These results suggest that Glu may at least in part play a role as a signal mediator in mechanisms associated with chondral mineralization through the group III mGluR subtype functionally expressed by chondrocytes in rodent cartilage.

Effects of glutamate transporter and receptor ligands on neuronal glutamate uptake

The excitatory amino acids (EAAs) transporters regulate the balance between physiological and pathological signaling over stimulation of the glutamatergic system pathway. The effect of transportable substrates and glutamate (Glu) receptor agonists on Glu uptake in neuronal cells was assessed at different conditions. Cells pre-incubated with Glu, l- or d-aspartate (Asp) and washed presented an inhibition on [ 3H]-Glu uptake and this effect was not mimicked by Glu receptors agonists. The effects of l- and d-Asp were not altered by the presence of N-methyl- d-aspartate (NMDA) receptor antagonists. Thus, the reduction on Glu uptake induced by EAAs is probably linked to the transporter activity. In contrast, the presence of NMDA or (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (SR-ACPD) during the pre-incubation and the [ 3H]-Glu uptake assay period increased Glu uptake, whilst kainic acid (KA) had no effect. The NMDA effect was not altered by its antagonists (±)-2-amino-5-phosphonopentanoic acid (AP-5) or dizocilpine (MK-801). The SR-ACPD effect was due to the activation of metabotropic Glu receptor, since it was abolished by its antagonist, l(±)-2-amino-3-phosphonopropionic acid ( l-AP3). Thus, the current studies suggest that the neuronal EAAs transporter is regulated in different manner by transportable substrates and Glu receptor agonists. The possible involvement of this modulation after certain neurotoxicity insults is discussed.

Glutamate modulation of human lymphocyte growth: in vitro studies

Peripheral blood mononuclear cell (PBMC) proliferation induced by phytohemagglutinin, or by anti-CD3 alone or plus anti-CD28 monoclonal antibodies (mAb) was inhibited by glutamate (Glu) in a concentration-dependent manner. This inhibition was not reproduced by selective ionotropic Glu receptor agonists, whereas it was potentiated by l-buthionine-( S, R)-sulfoximine, which depletes glutathione (GSH) stores, and counteracted by 2-mercaptoethanol, a preserver of cell thiols. The inhibitory effects of Glu were related to depletion of intracellular GSH stores, since it decreased GSH levels in a concentration-dependent manner. Furthermore, Glu modulated cytokine secretion by anti-CD3 mAb activated PBMC: it increased IFN-γ (+44.3 ± 8.2%) and IL-10 (+31.6 ± 9.7%) secretion, whereas that of IL-2, IL-4, IL-5, and TNF-α was not affected. These data suggest that high levels of Glu, which can be reached in damaged tissues, modulate lymphocyte responses to activating stimuli by favouring polarization of the T helper effector response.

Structural basis for 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor activation

Abstract (S)-Glutamic acid (Glu), the major excitatory neurotransmitter in the central nervous system, operates through ionotropic as well as metabotropic receptors and is considered to be involved in a number of degenerative brain diseases that are currently without any satisfactory therapeutic treatment. Until recently, development of selective Glu receptor agonists had mainly been based on structural optimization of naturally occurring lead compounds structurally related to Glu. Crystallization of the agonist binding domain of the GluR2 subunit of the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of ionotropic Glu receptors (iGluRs) in the presence or absence of an agonist has provided important information about ligand-receptor interaction mechanisms. The availability of such binding domain crystal structures has formed the basis for rational design of ligands, especially for the AMPA subtypes of iGluRs.

Stereostructure–activity studies on agonists at the AMPA and kainate subtypes of ionotropic glutamate receptors

(S)-Glutamic acid (Glu), the major excitatory neurotransmitter in the central nervous system, operates through ionotropic as well as metabotropic receptors and is considered to be involved in certain neurological disorders and degenerative brain diseases that are currently without any satisfactory therapeutic treatment. Until recently, development of selective Glu receptor agonists had mainly been based on lead compounds, which were frequently naturally occurring excitants structurally related to Glu. These Glu receptor agonists generally contain heterocyclic acidic moieties, which has stimulated the use of bioisosteric replacement approaches for the design of subtype-selective agonists. Furthermore, most of these leads are conformationally restricted and stereochemically well-defined Glu analogs. Crystallization of the agonist binding domain of the GluR2 subunit of the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of ionotropic Glu receptors in the presence or absence of an agonist has provided important information about ligand-receptor interaction mechanisms. The availability of these binding domain crystal structures has formed the basis for rational design of ligands, especially for the AMPA and kainate subtypes of ionotropic Glu receptors. This mini-review will focus on structure-activity relationships on AMPA and kainate receptor agonists with special emphasis on stereochemical and three-dimensional aspects.

Kainate receptor-mediated apoptosis in primary cultures of cerebellar granule cells is attenuated by mitogen-activated protein and cyclin-dependent kinase inhibitors.

1. Previous studies have suggested that neuronal apoptosis is the result of an abortive attempt to re-enter the cell cycle, and more recently the cyclin-dependent (CDKs) and the mitogen-activated protein (MAP) kinases, two superfamilies of kinases that influence and control cell cycle progression, have been implicated in neuronal apoptosis. 2. Here, to examine whether CDK/MAPK related pathways are involved in excitotoxicity, we studied the actions of various kinase inhibitors on apoptosis induced by the ionotropic glutamate (Glu) receptor agonist, kainate (KA), in primary cultures of murine cerebellar granule cells (CGCs). 3. KA-mediated neurotoxicity was concentration-dependent, as determined by a cell viability assay monitoring the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and largely apoptotic in nature, as shown by morphological examination and labelling of DNA fragmentation in situ using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick-end labelling (TUNEL). 4. KA-mediated neurotoxicity and apoptosis was completely attenuated by the mixed CDK and MAP kinase inhibitor, olomoucine, in a concentration-dependent manner (50 - 600 microM), and partially by roscovitine (1 - 100 microM), a more selective CDK inihibitor. 5. The p38 MAP kinase inhibitor, SB203580 (1 - 100 microM), partially attenuated KA receptor-mediated apoptosis, as did the MAP kinase kinase inhibitors PD98509 (1 - 100 microM) and U0126 (1 - 100 microM). 6. These findings provide new evidence for a complex network of interacting pathways involving CDK/MAPK that control apoptosis downstream of KA receptor activation in excitotoxic neuronal cell death.

(3)H](2S,4R)-4-Methylglutamate: a novel ligand for the characterization of glutamate transporters.

[(3)H](2S,4R)-4-Methylglutamate ([(3)H]4MG), used previously as a ligand for low-affinity kainate receptors, was employed to establish a binding assay for glutamate transporters (GluTs), as 4MG has also been shown to have affinity for the glial GluTs, GLT1 and GLAST. In rat brain membrane homogenates in the presence of Na(+) ions at 4 degrees C, specific binding of [(3)H]4MG was rapid and saturable (t(1/2) approximately 15 min), representing > 90% of total binding. Dissociation of [(3)H]4MG occurred in a biphasic manner, however, saturation studies and Scatchard analysis indicated a single site of binding (n(H) = 0.85) and a K(d) of 6.2 +/- 0.8 microM with a B(max) of 111.8 +/- 23.8 pmol/mg protein. Specific binding of [(3)H]4MG was Na(+)-dependent and inhibited by K(+) and HCO(3-). Pharmacological inhibition with compounds acting at GluTs revealed that Glu, D- and L-aspartate, L-serine-O-sulfate and Ltrans-pyrrolidine-2,4-dicarboxylate fully displaced specific binding. Drugs having preferential affinity for GLT1, kainate, dihydrokainate and Lthreo-3-methylglutamate, all inhibited approximately 40% of specific binding. The inhibition pattern of L-serine-O-sulfate in the presence of a saturating concentration of dihydrokainate was suggestive of [(3)H]4MG also labelling GLAST. 6-Cyano-7-nitroquinoxaline, a kainate receptor antagonist, and a range of Glu receptor agonists and antagonists failed to significantly inhibit [(3)H]4MG binding. The pharmacological profile of binding of [(3)H]4MG resembled that found for [(3)H]D-aspartate, a ligand specific for GluTs, reinforcing the hypothesis that [(3)H]4MG was labelling GluTs in this assay. Together, these data illustrate the development of an efficient, economic binding assay that is suitable for the characterization of different subtypes of GLuTs.

Evidence for neuronal origin and metabotropic receptor-mediated regulation of extracellular glutamate and aspartate in rat striatum in vivo following electrical stimulation of the prefrontal cortex.

Extracellular levels of glutamate (Glu) and aspartate (Asp) were measured at 5-s intervals in the striatum of chloral hydrate-anesthetized rats by using microdialysis coupled to an automated assay system based on capillary electrophoresis with laser-induced fluorescence. Application of a single 10-s train of depolarizing pulses to the prefrontal cortex caused a rapid increase in Glu and Asp concentrations (200-300% of basal value), which returned to basal level within 60 s. The stimulated rise in Glu and Asp concentrations was blocked completely by 2 microM tetrodotoxin or depletion of extracellular Ca2+, suggesting a neuronal origin of the Glu and Asp. Infusion of the Glu transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (200 microM) increased resting Glu and Asp levels by 300-500% without altering electrically stimulated changes in Glu and Asp concentration. Stimulated Glu and Asp concentration changes were suppressed by 91 and 73%, respectively, by the metabotropic Glu receptor agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylate (200 microM). This effect was blocked by the metabotropic Glu receptor antagonist (RS)-alpha-methylcarboxyphenylglycine (MCPG; 200 microM). MCPG alone produced no effect on electrically stimulated changes in Glu and Asp levels; however, in the presence of L-trans-pyrrolidine-2,4-dicarboxylic acid, MCPG produced a five- to sixfold increase in stimulated overflow. Based on these results, it is concluded that release of Glu and Asp from corticostriatal neurons can be inhibited by activation of metabotropic Glu autoreceptors, which may be an important determinant of excitatory transmission at striatal synapses.

In vivo and in vitro studies of glycine- and glutamate-evoked acetylcholinesterase release from spinal motor neurones: implications for amyotrophic lateral sclerosis/motor neurone disease pathogenesis

To investigate the spinal cellular structures and molecular mechanisms involved in acetylcholinesterase (AChE) release evoked by both glycine (GLY) and glutamate (GLU)—responses that might play a role in chronic neurotoxicity—we analysed AChE histochemistry and histology upon systemic administration of aspartate (ASP), and conducted in vitro experiments in synaptosomes and slices prepared from mouse spinal ventral horns. Upon superfusion and incubation exposure of these preparations to GLY- and GLU-receptor agonists, we assayed both tissue content and release of AChE, butyrylcholinesterase and lactic dehydrogenase. Histochemical reduction of motor neurone (MN) AChE, calcium dependency, decreases in intracellular AChE and the ratio amongst molecular forms released, suggest that both synaptosomal GLY-evoked AChE release (GLY-EAR) and GLU-receptor-elicited AChE release (GEAR) have release sites located at MN presynaptic terminals. These responses exhibited remarkable postnatal regulation. GEAR seems to be mediated through α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors after the fourth postnatal week and through both NMDA and non-NMDA receptors at earlier stages. Sustained rises of extracellular AChE might link acute excitotoxic injury with several long-lasting pathways leading to chronic neurotoxicity, since AChE molecular properties include: (1) the ability to block cholinergic mechanisms that protect MN against overactivity; (2) activation of ATP-dependent potassium channels; (3) promotion of neurite and axon outgrowth; and possibly (4) stimulation of brain macrophage migration and activation.

Nitric oxide involvement in regulating the dopamine transport in the striatal region of rat brain

Spontaneous [ 3H]dopamine ([ 3H]DA) overflow was measured from striatal slices in the presence of different glutamate (Glu) receptor agonists such as N-methyl- d-aspartate (NMDA), kainate (KA) and quisqualate (QA) and their corresponding antagonists, Dizocilpine maleate (MK-801), d-γ-glutamylaminomethanesulfonic acid (GAMS) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. [ 3H]DA uptake and release in the presence of l-Arginine ( l-Arg) and N G-nitro-arginine ( l- N-Arg), an inhibitor of nitric oxide (NO) synthesis were also evaluated. l- N-Arg alone or combined with l-Arg significantly reduced [ 3H]DA uptake at 10 and 100 μM from 33% to 44% from striatal slices. Whereas, in brain synaptosomal fractions l-Arg induced a biphasic effect on that [ 3H]DA uptake in a dose dependent manner, and l- N-Arg showed an absolute inhibition in 80–90% of this [ 3H]DA uptake at 1–500 μM. The amino acids, lysine, valine and histidine (100 μM) had a little effect inhibitory on [ 3H]DA uptake from synaptosomal fractions. Glu agonists, NMDA (10 μM) and KA (10 μM) importantly increased the spontaneous [ 3H]DA overflow, which was blocked by MK-801 (10 μM) and GAMS (10 μM), respectively. QA had no effect on [ 3H]DA release. l-Arg (10–200 μM) potentiated the spontaneous [ 3H]DA overflow in a dose dependent fashion from striatal slices, being reverted by 10 μM l- N-Arg alone or in combination with all other compounds; whereas, lysine, histidine and valine did not modify that spontaneous [ 3H]DA overflow. Results support the hypothesis related to the participation of NO on DA transport possibly synthesized at the dopaminergic (DAergic) terminals in the striatum; also that l-Arg concentration may determine alternative mechanisms to regulate the DAergic activity at the striatum.

Selected P2 purinoceptor modulators prevent glutamate-evoked cytotoxicity in cultured cerebellar granule neurons.

Primary cultures of granule neurons derived from cerebella of postnatal rats are endowed with Glu receptors. Glu receptor agonists exert a trophic influence on differentiating granule cells but, with maturation, the cells become vulnerable to excitatory amino acids. Here we show that the P2 purinoceptor antagonist basilen blue abolishes in rat cerebellar granule neurons the cytotoxic action of glutamate with an IC50 in the 10-20 microM range. Within the same concentrations, basilen blue inhibits binding of [3H] ATP to cerebellar granule cells, glutamate-evoked release (but not uptake) of [3H] D-aspartate and Ca2+ uptake. Furthermore, the extracellular phosphorylation of a major 45-kDa endogenous ecto-protein substrate of cerebellar granule neurons is inhibited with an IC50 of about 1 microM. Similar effects are elicited by 5-adenylylimidodiphosphate, a P2 purinoceptor agonist, when supplied to the neurons for 8 days previously to the addition of glutamate. Our data point to the use of P2 purinoceptor modulators as novel elements for understanding and controlling glutamate-mediated excitatory neurotoxicity and neurotransmission. We suggest a possible involvement of P2 purinoceptors in these actions.

Selected P2 purinoceptor modulators prevent glutamate‐evoked cytotoxicity in cultured cerebellar granule neurons

Primary cultures of granule neurons derived from cerebella of postnatal rats are endowed with Glu receptors. Glu receptor agonists exert a trophic influence on differentiating granule cells but, with maturation, the cells become vulnerable to excitatory amino acids. Here we show that the P2 purinoceptor antagonist basilen blue abolishes in rat cerebellar granule neurons the cytotoxic action of glutamate with an IC50 in the 10–20 μM range. Within the same concentrations, basilen blue inhibits binding of [3H]ATP to cerebellar granule cells, glutamate-evoked release (but not uptake) of [3H] D-aspartate and Ca2+ uptake. Furthermore, the extracellular phosphorylation of a major 45-kDa endogenous ecto-protein substrate of cerebellar granule neurons is inhibited with an IC50 of about 1 μM. Similar effects are elicited by 5-adenylylimidodiphosphate, a P2 purinoceptor agonist, when supplied to the neurons for 8 days previously to the addition of glutamate. Our data point to the use of P2 purinoceptor modulators as novel elements for understanding and controlling glutamate-mediated excitatory neurotoxicity and neurotransmission. We suggest a possible involvement of P2 purinoceptors in these actions. © 1996 Wiley-Liss, Inc.

Adrenergic calcium signaling in astrocyte networks within the hippocampal slice

Norepinephrine (NE) and glutamate (Glu) initiate intracellular calcium ([Ca2+]i) transients, oscillations, and intracellular [Ca2+]i waves in cultured astrocytes. To further elucidate the significance of NE- and Glu-evoked astrocytic [Ca2+]i signaling to neuron-astrocyte communication in the mature CNS, [Ca2+]i of astrocyte networks within hippocampal slices (P21-42) was measured during bath application of NE and Glu receptor agonists. Astrocytes in stratum radiatum were identified by highly negative membrane potentials (75 +/- 3 mV), absence of action potentials, and dye coupling following intracellular injection of the [Ca2+]-sensitive dye calcium orange. NE (2-100 microM) evoked [Ca2+]i increases (7 of 8 slices, 24 of 24 cells in responding slices) characterized by an initial rise, 20-50 sec to peak, followed by a slower return to baseline (over approximately 8 min). The alpha 1-agonist phenylephrine (PE) (10-100 microM) evoked complex [Ca2+]i signals (22 of 26 slices, 90 of 90 cells in responding slices) composed of both a prolonged component (5.1 +/- 1.8 min), synchronized in neighboring cells, and multiple, mainly asynchronous [Ca2+]i spikes (25.0 +/- 11.6 sec). PE responses were completely blocked by the alpha 1-antagonist prazosin (200 nM, n = 4 slices), but not by the alpha 2-antagonist yohimbine (n = 3 slices). The alpha 2-agonist clonidine (10-100 microM) did not increase [Ca2+]i (n = 4 slices). alpha 1-mediated [Ca2+]i transients were observed after removal of extracellular [Ca2+]o (n = 8 of 9 slices), indicating PE-induced Ca2+ release from intracellular stores. Adrenergic responses were mediated by alpha 1-receptors localized to astrocytes because PE and NE increased [Ca2+]i of acutely isolated hippocampal astrocytes. Glu (0.75-2.0 mM) did not increase astrocytic [Ca2+]i in slices (0 of 7), even in the presence of the Glu uptake inhibitor L-trans-pyrrollidine-2,4-dicarboxylic acid (PDC) (0 of 5 slices), or in acutely isolated astrocytes (0 of 7 cells). The metabotropic agonist t-ACPD (30 or 50 microM) did not increase astrocytic [Ca2+]i in hippocampal slices (0 of 5), while kainate (200 microM or 1 mM) induced brief (1-2 min) [Ca2+]i increases only rarely (2 of 8 applications in 6 slices). These results support a primary role of NE release and alpha 1-adrenoceptor stimulation in neuron-astrocyte communication in the mature CNS.

Decrease and increase of responses to glutamate receptor agonists in RNA-injected Xenopus oocytes by the epileptogenic agent pentylenetetrazol: Dependence on the agonist concentration

The effects of the epileptogenic agent pentylenetetrazol (PTZ) on current responses to glutamate (Glu) and to the Glu receptor agonists N- methyl- d-aspartate (NMDA), kainate (KA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualate (QA) were studied on RNA-injected Xenopus oocytes. PTZ antagonized the reactions to Glu as well as to NMDA, KA, AMPA and QA. With increasing concentration of AMPA, however, the depressive effect of PTZ turned to an augmentation. The complete change from a decreasing to an increasing effect of PTZ with elevated agonist concentration was unique for current responses by AMPA.

Growth conditions differentially modulate the vulnerability of developing cerebellar granule cells to excitatory amino acids

The survival of immature nerve cells in a cerebellar culture, predominantly excitatory granule cells, is known to be promoted by chronic exposure to high K + (> 20 mM) or glutamate (Glu) receptor agonists. These treatments are believed to mimic the in vivo effect of the incoming glutamatergic afferents, the mossy fibres. Here we report that with maturation the cells become vulnerable to excitatory amino acids (EAAs) and that the characteristics of EAA sensitivity are dependent on the environmental influences being either “trophic” (25 mM K + or 140 μM NMDA, K25 or K10 + NMDA) or “non-trophic” (10 mM K +, K10). Toxicity was assayed routenely at 9 days in vitro (DIV) after 24 h exposure to EAAs. Under all the tested conditions, the effect of Glu was mediated exclusively through NMDA receptors. However, the efficacy and potency of Glu were high in K25- and K10 + NMDA-grown cells compared with K10-grown cells. Growth conditions had the same influence on NMDA as on Glu-induced toxicity, but with the following special features: (1) in comparison with K25 cells, the potency of NMDA was significantly lower in K10 + NMDA cells. The K10 + NMDA cultures behaved as if they were completely insensitive to the NMDA which is present in their growth medium. (2) The K10-grown cells were not vulnerable to NMDA, unless the cell membrane was depolarised by shifting the cells into K25 medium. The efficacy of NMDA became then similar to that in K25 cultures, although the potency was about 7-fold less. Thus NMDA receptors can be activated by the depolarisation of K10 cells, implying the operation of Mg 2+ blockade of the channel at normal resting membrane potential. Although non-NMDA receptors did not seem to be involved in Glu toxicity, cells were vulnerable to kainate, which killed significantly more cells than Glu (about 80% vs 70%). This was partly due to the resistance of GABAergic interneurons present in the cultures to Glu- or NMDA-induced toxicity. In contrast to the effects of Glu or NMDA, KA vulnerability was lower in cells grown in K25 or K40 than K10 medium (rank order K10 > K25 > K40). Under our experimental conditions, cultured cells were resistant to AMPA, quisqualate and the selective metabotropic Glu receptor agonist 1 S,3 R-ACPD. Collectively, the observations indicated that EAA sensitivity of cultured cerebellar interneurons is significantly and differentially influenced by environmental factors, believed to mimic in vivo trophic influences on these cells.