AMPA Receptor Stimulation Research Articles

Impaired Excitatory Transmission in the Striatum of Rats Chronically Intoxicated with Manganese

Chronic exposure to manganese (Mn) is known to produce a parkinsonian or dystonic state in humans caused by a rather selective involvement of the basal ganglia. Experimental observations suggest that secondary excitotoxic mechanisms play a crucial role in the development of Mn-induced neurodegeneration in the striatum, although the site of interference of Mn with glutamatergic transmission in this brain area is still unknown. To answer this question, in the present in vitro study, we investigated the physiological characteristics of striatal excitatory synaptic transmission in a rat model of Mn intoxication. We found that chronic Mn greatly increased both frequency and amplitude of spontaneous excitatory postsynaptic potentials, in the absence of appreciable changes of intrinsic membrane properties of striatal cells. The sensitivity of striatal neurons to glutamate AMPA and NMDA receptor stimulation was unaffected by Mn poisoning, as demonstrated by comparing the membrane responses produced in control and treated rats to the application of selective agonists of these receptors and to the direct activation of corticostriatal glutamatergic fibers. In addition, also paired-pulse facilitation was unaltered by Mn treatment, indicating that this toxin does not affect the pre- and postsynaptic mechanisms responsible for the appearance of this short-term form of synaptic plasticity at corticostriatal synapses. It is concluded, therefore, that hyperactivity of corticostriatal neurons, rather than increased postsynaptic sensitivity to glutamate, accounts for the abnormal excitation of striatal neurons in the course of Mn intoxication.

Inhibition of delayed rectifier K+ conductance in cultured rat cerebellar granule neurons by activation of calcium-permeable AMPA receptors.

Activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors in cerebellar granule cells during perforated-patch whole-cell recordings activated an inward current at negative voltages which was followed, after a delay, by the inhibition of an outward potassium current at voltages positive to -20 mV. The activated inward current was inwardly rectifying suggesting that the AMPA receptors were Ca2+-permeable. This was confirmed by direct measurements of intracellular calcium where Ca2+ rises were seen following AMPA receptor activation in Na+-free external solution. Ca2+ rises were equally large in the presence of 100 microM Cd2+ to block voltage-gated Ca2+ channels. Specific voltage-protocols, allowing selective activation of the delayed rectifier potassium current (KV) and the transient A current (KA), showed that kainate inhibited KV, but not to any great extent KA. The inhibition of KV was blocked by the AMPA receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and was no longer observed when the KV current was abolished with high concentrations of Ba2+. The responses to kainate were not altered by pre-treating the cells with pertussis toxin, suggesting that the AMPA receptor stimulation of the G-protein Gi cannot account for the effects observed. Replacing extracellular Na+ with choline did not alter the inhibition of KV by kainate, however, removing extracellular Ca2+ reduced the kainate response. The inhibition of KV by kainate was unaffected by the presence of 100 microM Cd2+. The guanylyl cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), did not alter kainate inhibition of KV. It is concluded that ion influx (particularly Ca2+ ions) through AMPA receptor channels following receptor activation leads to an inhibition of KV currents in cerebellar granule neurons.

Translocation of calbindin-D28k to the nucleus and the axon by stimulation of AMPA receptors in cortical neurons

Translocation of calbindin-D28k to the nucleus and the axon by stimulation of AMPA receptors in cortical neurons

Ca2+-Permeable AMPA Receptors Induce Phosphorylation of cAMP Response Element-Binding Protein through a Phosphatidylinositol 3-Kinase-Dependent Stimulation of the Mitogen-Activated Protein Kinase Signaling Cascade in Neurons

Ca(2+)-permeable AMPA receptors may play a key role during developmental neuroplasticity, learning and memory, and neuronal loss in a number of neuropathologies. However, the intracellular signaling pathways used by AMPA receptors during such processes are not fully understood. The mitogen-activated protein kinase (MAPK) cascade is an attractive target because it has been shown to be involved in gene expression, synaptic plasticity, and neuronal stress. Using primary cultures of mouse striatal neurons and a phosphospecific MAPK antibody we addressed whether AMPA receptors can activate the MAPK cascade. We found that in the presence of cyclothiazide, AMPA caused a robust and direct (no involvement of NMDA receptors or L-type voltage-sensitive Ca(2+) channels) Ca(2+)-dependent activation of MAPK through MAPK kinase (MEK). This activation was blocked by GYKI 53655, a noncompetitive selective antagonist of AMPA receptors. Probing the mechanism of this activation revealed an essential role for phosphatidylinositol 3-kinase (PI 3-kinase) and the involvement of a pertussis toxin (PTX)-sensitive G-protein, a Src family protein tyrosine kinase, and Ca(2+)/calmodulin-dependent kinase II. Similarly, kainate activated MAPK in a PI 3-kinase-dependent manner. AMPA receptor-evoked neuronal death and arachidonic acid mobilization did not appear to involve signaling through the MAPK pathway. However, AMPA receptor stimulation led to a Ca(2+)-dependent phosphorylation of the nuclear transcription factor CREB, which could be prevented by inhibitors of MEK or PI 3-kinase. Our results indicate that Ca(2+)-permeable AMPA receptors transduce signals from the cell surface to the nucleus of neurons through a PI 3-kinase-dependent activation of MAPK. This novel pathway may play a pivotal role in regulating synaptic plasticity in the striatum.

Flow cytometric study of mitochondrial dysfunction after AMPA receptor activation.

The effect of AMPA-receptor stimulation on MMP and on the concentration of intracellular calcium ([Ca2+]i) was studied in dissociated CGC from rat pups, by flow cytometry. In the presence of cyclothiazide, AMPA induced a sodium-independent decrease in MMP up to 30.7+/-2.5%. This effect was antagonized by CNQX and NBQX. Mepacrine and dibucaine reversed the effect of AMPA on MMP, suggesting that it is mediated by a release of arachidonic acid. AMPA alone induced a slight (about 7%) increase in [Ca2+]i. In the presence of cyclothiazide, AMPA induced a concentration-dependent [Ca2+]i increase up to 29.10+/-2.10% that was not reversed by flunarizine. This increase was similar to that observed in a Na+-free medium, and was antagonized by CNQX and NBQX, but not by MK-801. Mitochondria play a key role in the modulation of [Ca2+]i since a significant [Ca2+]i increase was found in the presence of FCCP. On the other hand, the dantrolene-sensitive calcium pools do not participate in the [Ca2+]i increase induced by stimulation of AMPA receptors. It is concluded that when AMPA-receptor desensitization is blocked, a decrease in MMP and an increase in [Ca2+]i occurs, which could be additional events to potentiate neuronal cell death induced by glutamate.

Flow cytometric study of mitochondrial dysfunction after AMPA receptor activation

The effect of AMPA-receptor stimulation on MMP and on the concentration of intracellular calcium ([Ca2+]i) was studied in dissociated CGC from rat pups, by flow cytometry. In the presence of cyclothiazide, AMPA induced a sodium-independent decrease in MMP up to 30.7 ± 2.5%. This effect was antagonized by CNQX and NBQX. Mepacrine and dibucaine reversed the effect of AMPA on MMP, suggesting that it is mediated by a release of arachidonic acid. AMPA alone induced a slight (about 7%) increase in [Ca2+]i. In the presence of cyclothiazide, AMPA induced a concentration-dependent [Ca2+]i increase up to 29.10 ± 2.10% that was not reversed by flunarizine. This increase was similar to that observed in a Na+-free medium, and was antagonized by CNQX and NBQX, but not by MK-801. Mitochondria play a key role in the modulation of [Ca2+]i since a significant [Ca2+]i increase was found in the presence of FCCP. On the other hand, the dantrolene-sensitive calcium pools do not participate in the [Ca2+]i increase induced by stimulation of AMPA receptors. It is concluded that when AMPA-receptor desensitization is blocked, a decrease in MMP and an increase in [Ca2+]i occurs, which could be additional events to potentiate neuronal cell death induced by glutamate. J. Neurosci. Res. 52:684–690, 1998. © 1998 Wiley-Liss, Inc.

AMPA and NMDA receptor-dependent spinal LTP after nociceptive tetanic stimulation

LTP is often studied as a model of synaptic plasticity. Plasticity in pain control systems may involve mechanisms similar to those involved in learning. We recently reported LTP of both the A beta and C-fibre evoked responses of single dorsal horn neurons after a tetanic stimulation of the sciatic nerve, lasting for at least 6 h. In the present paper we show that identical stimulation induced LTP only of the C-fibre evoked response after blockade of muscular contractions during the tetanus. The C-fibre evoked response increased significantly less after pretreatment with either the AMPA antagonist NBQX or the NMDA antagonist D-AP5 (mean increase 33%) than in untreated animals (105%, p < 0.001), indicating that both AMPA and NMDA receptor stimulation are involved in the induction of LTP.

Evidence for a role of presynaptic AMPA receptors in the control of neuronal glutamate release in the rat forebrain

The role of presynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in controlling the neuronal release of excitatory amino acids has been investigated. Stimulation of presynaptic AMPA receptors by the endogenous agonist l-glutamate, or by ( R, S)-AMPA, dose-dependently enhanced the Ca 2+-dependent, tetrodotoxin-insensitive, electrically-stimulated release of [ 3 H ] d-aspartate from rat forebrain slices. This AMPA receptor-mediated response showed marked stereoselectivity with the activity residing solely in the ( S)-isomer. ( R)-AMPA was inactive in this respect. AMPA-evoked responses were significantly enhanced in the presence of the AMPA receptor desensitization inhibitor, cyclothiazide (10 μM). Moreover, responses to both AMPA and glutamate were inhibited by competitive (NBQX) and non-competitive (GYKI 52466) AMPA receptor-selective antagonists in a dose-dependent manner. These results provide strong support for the existence of presynaptic AMPA receptors acting to enhance the synaptic release of excitatory amino acids in the mammalian forebrain. Such a positive feedback system may play an important functional role in physiological (e.g. long-term potentiation) and/or pathological (e.g. epileptogenesis) processes in the mammalian central nervous system. AMPA-type autoreceptors may provide new targets for drug action.

Response to repetitive stimulation of AMPA receptors in patches excised from fields CA1 and CA3 of the hippocampus

Patches excised from fields CA1 and CA3 of the hippocampus were used to study AMPA receptor responses to repetitive application of brief pulses of agonist. AMPA receptors in field CA1 exhibited a larger degree of paired-pulse depression and a slower recovery rate from desensitization than those from CA3 patches. Responses became progressively smaller during a train of short pulses (4 at 100 Hz), an effect that was greater in CA1 than CA3 patches and that was blocked by a drug (cyclothiazide) that slows desensitization. Cyclothiazide also increased steady state currents during a long pulse of glutamate and had a lower EC 50 for CA3 than CA1 AMPA receptors. These results suggest (i) that differences in the relative balance of flip vs. flop subunits affect the kinetic and pharmacological properties of AMPA receptors in vivo, and (ii) that the rate of recovery from desensitization may influence responses to the bursts of afferent activity commonly used to induce long-term potentiation.

Properties of AMPA receptors expressed in rat cerebellar granule cell cultures: Ca2+ influx studies.

Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), in terms of AMPA-induced toxicity or 45Ca2+ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of 45Ca2+ accumulation. 45Ca2+ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall 45Ca2+ influx. These included Ca2+ uptake through NMDA-receptor channels, voltage-sensitive Ca2+ channels, and via Na+/Ca2+ exchange. However, nearly one-fifth of the total 45Ca2+ influx remained unaccounted for and this estimate was similar to 45Ca2+ influx observed under Na(+)-free conditions. This observation suggested that a significant proportion of the Ca2+ flux passes through the AMPA-receptor channel proper, a view supported by Co2+ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro.

P-171 Inhibition by ethanol of HGF-induced DNA synthesis and Ca 2+ mobilization in rat hepatocytes

Reduction in GluR2 subunit expression and subsequent increases in AMPA receptor mediated Ca2+ currents were postulated to exacerbate glutamate neurotoxicity following seizures or global ischemia. To directly test the effects of shifting the GluR1/GluR2 subunit ratio on excitotoxicity, GluR2 antisense deoxyoligonucleotides (AS-ODNs) were applied to dissociated hippocampal cultures for 1–8 days. The GluR1/GluR2 protein ratio was examined immunohistochemically and by Western blotting. [Ca2+]i concentrations were determined by ratiometric imaging of Fura 2-loaded cells. The cultures were exposed to glutamate, AMPA, NMDA or kainic acid (KA) 3 days after GluR2 knockdown and cell viability was determined 1 day later by MTT reduction assay or Trypan blue exclusion. Although GluR2 AS-ODNs increased the GluR1/GluR2 protein ratio in a time dependent manner, neurons and glia appeared healthy and MTT reduction values were similar to untreated and sense controls. Basal [Ca2+]i levels were unchanged but [Ca2+]i was selectively increased by agonist stimulation of AMPA receptors. Unexpectedly, delayed neurotoxicity was attenuated at saturating doses of glutamate while little difference in cell viability was observed at lower doses or with the other excitotoxins at any concentration. Therefore, there was a dissociation between rises in AMPA receptor-mediated Ca2+ influx and neurotoxicity despite marked decreases in GluR2 but not GluR1 immunoreactivity. It is proposed that a modification of AMPA receptor stochiometry that raises agonist-stimulated Ca2+ influx during an excitotoxic insult may have eventual neuroprotective effects.

The AMPA antagonist NBQX protects thalamic reticular neurons from degeneration following cardiac arrest in rats

Thalamic reticular (RT) neurons are selectively vulnerable to degeneration following global ischemia. The degenerative mechanism is thought to involve an excitotoxic component, mediated in part by sustained post-ischemia activation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) type excitatory amino acid (EAA) receptors. In order to test this hypothesis, the selective competitive AMPA type EAA antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxalinedione) was administered at 30 mg/kg to rats 1, 3, and 6 h after resuscitation from 10 min cardiac arrest. NBQX treatment resulted in a 2-fold increase of spared RT neurons, from a mean density of 3.6 ± 0.8 × 10 3 neurons/mm 3 in cardiac arrest cases to 7.4 ± 1.1 × 10 3 neurons/mm 3 in the NBQX treated group, which represents sparing of 41.7% of the normal population of RT neurons, and protection of 26.9% of vulnerable RT neurons. Neurons within the central core of the RT manifest both a higher degree of vulnerability to ischemic degeneration, >92% loss, and a higher sensitivity to sparing following NBQX administration, 460% increased sparing, than neuronal sub-populations in the medial or lateral 13 of the RT. Protection by post-arrest administration of NBQX suggests that sustained post-arrest stimulation of AMPA receptors is an important component in the process of ischemic degeneration of RT neurons.

Stimulation of glutamate receptors in the intermediate/caudal striatum induces contralateral turning

The aim of the present study was to investigate the role of striatal NMDA, kainate and AMPA receptors in the turning behaviour of rats. N-methyl- d-aspartate (NMDA, 500 ng/0.5 μl), kainic acid (50 ng/0.5 μl) or α-amino-3-hydroxy-5-methyl-4-isoxasole-propionic acid (AMPA, 1000 ng/0.5 μl), injected into the intermediate and caudal parts of the caudate-putamen, induced contralateral head turns and rotations. This effect was delayed or was not observed after administration of the compounds into the globus pallidus. The antagonist of non-NMDA receptors, 6,7-dinitroquinoxaline-2,3-dione (DNQX, 1000 ng/0.5 μl), antagonized the contralateral head turns and rotations induced by AMPA (1000 ng/0.5 μl) or kainic acid (50 ng/0.5 μl), and evoked per se (2000 ng/0.5 μl) the ipsilateral head turns and rotations. The NMDA receptor antagonist, (±)-2-amino-5-phosphonopentanoic acid (AP5, 1000 ng/0.5 μl), induced mainly ipsilateral head turns and rotations; when injected in a dose of 500 ng/0.5 μl, it inhibited the contralateral head turns and rotations after NMDA. The results seem to suggest that the contralateral head turns and rotations induced by stimulation of NMDA, AMPA and kainate receptors in the intermediate and caudal parts of the caudate-putamen may result from activation of the γ-aminobutyrate (GABA)-ergic strionigral pathway.

Selective stimulation of excitatory amino acid receptor subtypes and the survival of granule cells in culture: effect of quisqualate and AMPA

Differentiating granule cells develop survival requirements in vitro which can be met by treatment with high K + or excitatory amino acids. Promotion of cell survival by N -methyl- d-aspartate (NMDA) or kainate has already been established and here we report that treatment of the cells with α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) or quisqualate (QA) also leads to cell rescue. In comparison with the effect of NMDA, the influence of AMPA/QA is small, resulting in a 20–30% increase in cell survival, with a peak at a very narrow concentration range (0.5–2.0 μM QA and 5–10 μM AMPA). The effect is exclusive to AMPA receptor stimulation, since stimulation of metabotropic glutamate receptors with (1 S3 R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) has no effect. Furthermore, AMPA/QA rescue of cells is blocked by ionotropic non-NMDA receptor antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline (NBQX). In addition, both nifedipine and dizolcipline (MK-801) interfered with the cell survival promoting effect of AMPA, suggesting that the influence of AMPA is mediated via calcium influx involving both depolarization-activated voltage sensitive calcium channels and NMDA receptors stimulated as a result of AMPA-induced release of glutamate. Possible reasons for the small cell survival promoting effect of AMPA/QA compared with the influence of high K + or NMDA are discussed.

Persistent AMPA receptor stimulation alters [Ca 2+] i homeostasis in cultures of embryonic dopaminergic neurons

The effect of the ionotropic glutamate receptor agonist, AMPA, on intracellular Ca 2+ concentrations ([Ca 2+] i) was studied in dopaminergic neurons present in primary cultures of ventral tegmental mesencephalon of 14 day rat embryos. Exposure of cells to 10 μM AMPA for 1 min increased [Ca 2+] i by 2–3 fold in dopaminergic and other neurons and this response was obliterated within 5 min by superfusion with AMPA-free incubation buffer. In dopaminergic neurons, 1 min or 5 min exposure to 50 μM AMPA increased [Ca 2+] i 3 to 5 times over control values. This rise in [Ca 2+] 1 persisted even after a 20 min superfusion with AMPA-free media, whereas, [Ca 2+] i in non-dopaminergic neurons was reversed to control values during this time. Preincubation (2 min) of cultured cells with NBQX or the L-type channel blocker, nifedipine, but not with MK-801 blunted the rise of [Ca 2+] i in dopaminergic and other neurons. Pretreatment with 2 μM NBQX shifted the dose response curve for AMPA to the right without changing the basal [Ca 2+] i. The presence of 10 μM dantrolene, a blocker of Ca 2+ release from intracellular stores, did not alter the initial rise of [Ca 2+] i elicited by 50 μM AMPA, but prevented the destabilization of Ca 2+ homeostasis by facilitating the recovery to normal of basal [Ca 2+] i. Exposure to 50 μM AMPA (5 min) caused an irreversible increase of [Ca 2+] i in dopaminergic neurons and cell death was manifested by propidium iodide uptake 6–7 h after AMPA exposure. The present results show that in dopaminergic neurons the increase of [Ca 2+] i elicited by activation of AMPA receptors is mediated by sustained Ca 2+ flux through voltage-gated channels and through a Ca 2+-dependent Ca 2+ release from intracellular stores. Protracted stimulation of AMPA and kainate receptors caused death of dopaminergic neurons before that of other neurons present in the same culture. The increased vulnerability of dopaminergic neurons to AMPA may represent a mechanism of pathological relevance during ontogenesis.

Regulation of GABAA and AMPA receptors by agonist and depolarizing stimulation requires phosphatase or kinase activity

Using a live in vitro slice preparation of adult rat neocortex we have examined the role of phosphorylation and dephosphorylation in the regulation of GABAA and AMPA receptors. Alkaline phosphatase increased binding levels for both receptors while protein kinase A had the opposite effect. For both receptor populations, phosphatase effects were blocked by sodium beta-D-glycerol phosphate and sodium vanadate (phosphatase inhibitors) while kinase actions were blocked by a protein kinase inhibitor. Increases in cell depolarizations by veratridine led to an increase in labelled GABAA receptors, but to decreases in labelled AMPA receptors. Increases in binding were differentially blocked by the two phosphatase inhibitors, sodium beta-D-glycerol phosphate and sodium vanadate while decreases in binding were blocked by protein kinase inhibitor. Agonist stimulation of GABAA and AMPA receptors led to a decrease in receptor binding which could be blocked in both cases by protein kinase inhibitor. These data show that certain cortical excitatory and inhibitory amino acid receptors can be regulated by the action of phosphorylating/dephosphorylating enzymes and further suggest that the regulation induced by cell depolarization and agonist stimulation is based on similar mechanisms.