NMDA Receptor-mediated Ca2 Research Articles

Dopamine D1 receptors mediate CREB phosphorylation via phosphorylation of the NMDA receptor at Ser897-NR1.

Addictive drugs such as amphetamine and cocaine stimulate the dopaminergic system, activate dopamine receptors and induce gene expression throughout the striatum. The signal transduction pathway leading from dopamine receptor stimulation at the synapse to gene expression in the nucleus has not been fully elucidated. Here, we present evidence that D1 receptor stimulation leads to phosphorylation of the transcription factor Ca2+ and cyclic AMP response element binding protein (CREB) in the nucleus by means of NMDA receptor-mediated Ca2+ signaling. Stimulation of D1 receptors induces the phosphorylation of Ser897 on the NR1 subunit by protein kinase A (PKA). This phosphorylation event is crucial for D1 receptor-mediated CREB phosphorylation. Dopamine cannot induce CRE-mediated gene expression in neurons transfected with a phosphorylation-deficient NR1 construct. Moreover, stimulation of D1 receptors or increase in cyclic AMP levels leads to an increase in cytosolic Ca2+ in the presence of glutamate, but not in the absence of glutamate, indicating the ability of dopamine and cyclic AMP to facilitate NMDA channel activity. The recruitment of the NMDA receptor signal transduction pathway by D1 receptors may provide a general mechanism for gene regulation that is fundamental for mechanisms of drug addiction and long-term memory.

Glutamate-induced declustering of post-synaptic adaptor protein Cupidin (Homer 2/vesl-2) in cultured cerebellar granule cells.

Cupidin (Homer 2/vesl-2) is a post-synaptic adaptor protein that associates with glutamate receptor complexes and the actin cytoskeleton. We analyzed the developmental and activity-dependent localization of Cupidin in mouse cerebellar granule cells. Cupidin is predominantly localized to granule cell post-synapses connecting with mossy fiber terminals in developing post-natal cerebellum, but is diminished in adult cerebellum. In cultured granule cells 7 days in vitro, Cupidin was present as synaptic and extra-synaptic punctate clusters that largely co-localized with the actin-cytoskeletal binding partners F-actin and drebrin, as well as a post-synaptic scaffold protein PSD-95. Upon stimulation with glutamate, Cupidin clusters were rapidly dissociated without protein degradation, and by short-term but not sustained stimulation they were recovered after post-incubation without glutamate. The glutamate-induced declustering of Cupidin preceded that of F-actin and drebrin, was elicited by NMDA receptor-mediated Ca2+ influx, and was followed by a downstream pathway including MAPK/ERK and protein tyrosine kinase. Specific isoforms with post-translational modification were reduced depending on Ca2+-dependent protein phosphatase activity. In cultured hippocampal neurons, Homer family members Homer 1, Cupidin/Homer 2 and Homer 3 showed similar glutamate-induced declustering. We suggest that Cupidin acts as a mobile adaptor protein that changes the distribution states, clustered versus declustered, in response to synaptic activity.

Early postdenervation depolarization is controlled by acetylcholine and glutamate via nitric oxide regulation of the chloride transporter.

Resting non-quantal acetylcholine (ACh) and probably glutamate (Glu) release from nerve endings activates M1- and NMDA receptor-mediated Ca2+ entry into the sarcoplasm with following activation of NOS and production of NO. This is a trophic message from motoneurons, which keeps the Cl- transport inactive in the innervated sarcolemma. After denervation, the secretion of ACh and Glu at the neuromuscular junction is eliminated within 3-4 h and the production of NO in the sarcoplasm is lowered. As a result, the Cl- influx is probably activated by dephosphorylation of the Cl- transporter with subsequent elevation of intracellular Cl- concentration. The equilibrium Cl- potential becomes more positive and the muscle membrane becomes depolarized.

Corticosterone acutely prolonged N-methyl-d-aspartate receptor-mediated Ca2+ elevation in cultured rat hippocampal neurons.

This work reports the first demonstration that corticosterone (CORT) has a rapid and transient effect on NMDA receptor-mediated Ca2+ signaling in cultured rat hippocampal neurons. Using single cell Ca2+ imaging, CORT and agonists of glucocorticoid receptors were observed to modulate the NMDA receptor-mediated Ca2+ signals in a completely different fashion from pregnenolone sulfate. In the absence of steroids, 100 micro m NMDA induced a transient Ca2+ signal that lasted for 30-70 s in 86.1% of the neurons prepared from postnatal rats (3-5 days old). After pre-treatment with 0.1-100 micro m CORT for 10-20 min, NMDA induced extremely prolonged Ca2+ elevation. This prolonged Ca2+ elevation was terminated by the application of MK-801 and followed by washing out of CORT. The proportion of CORT-modulated neurons within the NMDA-responsive cells increased from 25.1 to 95.5% when the concentration of CORT was raised from 0.1 to 50 micro m. Substitution of BSA-conjugated CORT produced essentially the same results. When hippocampal neurons were preincubated with 10 micro m cortisol and 1 micro m dexamethasone for 20 min, a very prolonged Ca2+ elevation was also observed upon NMDA stimulation. The CORT-prolonged Ca2+ elevation caused a long-lasting depolarization of the mitochondrial membrane, as observed with rhodamine 123. In contrast, incubation with 100 micro m pregnenolone sulfate did not considerably alter the time duration of NMDA-induced transient Ca2+ elevation, but caused a significant increase in the peak amplitude of Ca2+ elevation in hippocampal neurons. These results imply that high levels of CORT induce a rapid and non-genomic prolongation of NMDA receptor-mediated Ca2+ elevation, probably via putative membrane surface receptors for CORT in the hippocampal neurons.

Differential mechanisms of glutamate-stimulated perturbations in the kinetics of c-fos mRNA induction are associated with maturation of cerebellar granule cells in primary culture.

In further exploring proposals for the measurement of early gene (c-fos mRNA) levels as a predictive index for in vitro excitotoxicity, this study, using immature (2 days in vitro) cultures of mouse cerebellar granule cells as an experimental model system, was undertaken to determine the effect of glutamate (Glu) i) in stimulating increases in intracellular free-calcium ([Ca2+]), ii) on cell viability and iii) on induction of steady-state c-fos mRNA levels. In parallel experiments the action of agents (viz. 55 mM KCl and the calcium ionophore, A23187) that mediate Ca2+ entry into cells via different routes was also evaluated. Glu was unable to induce excitotoxicity in granule cells at this stage of development in culture, but did stimulate a concentration-dependent and marked increase in [Ca2+], levels while also mediating a dramatic concentration-dependent perturbation in the kinetics of c-fos mRNA induction that appeared to arise solely from NMDA receptor-mediated Ca2+ influx. The results are presented in comparison to the actions of KCI and A23187 and considered in relation to earlier studies undertaken using mature (7 days in vitro) cultures of cerebellar granule cells.

Brain tissue responses to ischemia.

Brain tissue responses to ischemia.

Mechanisms of [2,3-Butanedione Bis(N4-Dimethylthiosemicarbazone)]zinc (Zn-ATSM2)-Induced Protection of Cultured Hippocampal Neurons Against N-Methyl-D-Aspartate Receptor-Mediated Glutamate Cytotoxicity

Hyperexcitation of glutamatergic neurons may play a key role in ischemia-related neurodegeneration. Recent studies have suggested that the zinc ion (Zn2+), which is present in the central nervous system, has a modulatory role in glutamatergic neuron activity. Zinc ions block glutamate-induced depolarizing currents and neuronal damage by binding with zinc sites on the NMDA subtypes. Therefore, we examined the usefulness of zinc as a therapeutic agent for the prevention of ischemic neuronal damage in the brain. In our previous study, 2,3-butanedione bis(N4-dimethylthiosemicarbazonato) zinc complex (Zn-ATSM2), with high brain uptake, showed significant neuroprotective effects against cerebral ischemia in rats when administered systemically. In this study, to elucidate the mechanism of the neuroprotective effect of Zn-ATSM2, we first examined its in vitro protective effects against glutamate-, NMDA- and kainite-induced neurotoxicity in primary cultures of hippocampal neurons. Zn-ATSM2 elicited protective effects against this glutamate- and NMDA-induced neurotoxicity, but did not affect kainite-induced cytotoxicity. In addition, we studied the effects of Zn-ATSM2 on influx of Ca2+, which undergoes modification subsequent to NMDA activation. Zn-ATSM2 significantly decreased glutamate-induced 45Ca2+ uptake. Thus, Zn-ATSM2 protected against glutamate-induced neurotoxicity and its protective effect was, at least in part, due to the blockage of NMDA receptor-mediated Ca2+ influx.

The return of the spike: postsynaptic action potentials and the induction of LTP and LTD.

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Peroxynitrite and nitric oxide donors induce neuronal apoptosis by eliciting autocrine excitotoxicity.

Endogenous generation of nitric oxide and its congeners, including peroxynitrite (ONOO-), has been implicated in the mechanism of neuron loss in neurodegenerative diseases. Accordingly, nitric oxide donors and ONOO- can elicit both apoptosis and necrosis in neuron cultures. Here we show that nitric oxide donors and ONOO- are each able to trigger apoptosis of mouse cerebellar granule cells by an excitotoxic mechanism requiring exocytosis and NMDA receptor-mediated intracellular Ca2+ overload. This conclusion is supported by the following findings. Apoptosis was induced by various nitric oxide donors or by direct addition of ONOO- to differentiated cerebellar granule cell cultures that were sensitive to NMDA toxicity, but not in cerebellar granule cells that did not display NMDA-induced cell death (i.e. early days in culture) or in various glial cell populations. Donors of ONOO- or nitric oxide stimulated a sustained increase in intracellular Ca2+, which was prevented by inhibitors of NMDA receptors, such as MK-801 and 5-phospho-aminovaleric acid, or by dampening neuronal electrical activity with high concentrations of extracellular Mg2+. Moreover, these treatments and the exposure of cerebellar granule cells in nominally Ca2+-free media prevented apoptotic cell death. Both the intracellular Ca2+ increase and apoptosis elicited by ONOO- or the nitric oxide donors were prevented by blocking exocytosis with tetanus toxin or botulinum neurotoxin C.

Mitochondria accumulate Ca2+ following intense glutamate stimulation of cultured rat forebrain neurones.

1. In cultures of rat forebrain neurones, mitochondria buffer glutamate-induced, NMDA receptor-mediated Ca2+ influx. Here, we have used the fluorescent calcium indicator, indo-1 AM to record [Ca2+]i from single cells. We varied either the glutamate concentration or the duration of exposure to investigate the cellular mechanisms recruited to buffer [Ca2+]i within different stimulation protocols. 2. For a 15 s stimulus, the recovery time doubled as the glutamate concentration was raised from 3 to 300 microM. Changing the duration of exposure from 15 s to 5 min increased the recovery time tenfold even when the glutamate concentration was held at 3 microM. 3. We used a selective inhibitor of the mitochondrial Na(+)-Ca2+ exchange, CGP-37157. When applied immediately after a 15 s, 100 microM glutamate challenge, CGP-37157 consistently caused a rapid fall in [Ca2+]i followed by a slow rise after the drug was washed out. A similar pattern was seen with the 5 min, 3 microM glutamate stimulus. The effects of CGP-37157 are consistent with the release of substantial mitochondrial Ca2+ stores during recovery from an intense glutamate stimulus. 4. These studies suggest that mitochondria become progressively more important for buffering glutamate-induced Ca2+ loads as the stimulus intensity increases. The recovery of [Ca2+]i to baseline following glutamate removal is critically regulated by the release of Ca2+ from mitochondrial stores via mitochondrial Na(+)-Ca2+ exchange. The data highlight a previously under-appreciated role for [Na+]i in the regulation of [Ca2+]i in central neurones.

Cerebral protection by adenosine.

Delayed selective nerve cell death as seen in the hippocampal CA1 area of gerbils after transient forebrain ischemia goes along with neuronal hyperactivity and an early demonstrable accumulation of calcium in circumscribed groups of nerve cells. Application of NMDA receptor-blockers such as MK 801 prevents neuronal damage. This suggests the involvement of NMDA receptors which are operated by glutamate and known to mediate a special Ca2+ influx required also for establishing sustained enhancement of synaptic efficacy. Thus, the excessive postischemic accumulation of calcium, thought to be instrumental in the generation of nerve cell death, seems to result from turning on a dangerous, but primarily physiological mechanism which ran out of control. We studied the endogenous control mechanisms by which the firing pattern of nerve cells and the initiation of NMDA receptor-mediated neuronal Ca2+ influx are controlled focusing in particular on the role of adenosine. This nucleoside is released from nerve- and glial cells in larger amounts after ischemia. It counteracts at increased extracellular concentration the generation of burst discharges, an effect which is ascribed to a modulation of the dendritic membrane properties. Removal of a possible action of endogenous adenosine by receptor antagonists such as theophylline was found to enhance postischemic nerve cell death. This together with other reported experimental evidences points to a protective action of this nucleoside in the brain. The presumed mechanisms by which this effect is achieved were studied in a rat hippocampal slice using ion selective electrodes.(ABSTRACT TRUNCATED AT 250 WORDS)

Redox modulation of NMDA receptor-mediated Ca2+ flux in mammalian central neurons

NMDA receptor-mediated Ca2+ flux was studied in cultured rat retinal ganglion cells and neocortical neurons. Intracellular free calcium ([Ca2+]i was measured with fura-2 fluorescence imaging. Baseline [Ca2+]i was 59 +/- 5 nM. In low [Mg2+]o, 200 microM NMDA reversibly increased [Ca2+]i to 421 +/- 70 nM. This rise in [Ca2+]i was blocked by the NMDA antagonists APV (200 microM) or [Mg2+]o (1 mM), but only slightly inhibited by the non-NMDA antagonist CNQX (10 microM). Chemical reduction with dithiothreitol (DTT) had no effect on resting [Ca2+]i. However, DTT increased the NMDA-induced rise in [Ca2+]i approximately 1.6-fold; the oxidizing agent dithiobisnitrobenzoic acid (DTNB) reversed this effect. In patch-clamp experiments, DTT increased NMDA-activated whole-cell conductance approximately 1.7-fold in low and high [Ca2+]o. The Ca2+/Na+ permeability ratio of approximately 7 for NMDA channels remained unaltered by chemical reduction. Thus, redox modulation of the NMDA receptor/channel complex results in a dramatic alteration in current magnitude but no change in ionic permeabilities.