Neuronal N-methyl-D-aspartate Research Articles

Calpain‐mediated degradation of G‐substrate plays a critical role in retinal excitotoxicity for amacrine cells

The role of neuronal N-methyl-D-aspartate (NMDA) receptor-mediated intracellular signaling has been elucidated in both physiological and pathological conditions. However, the details of relative vulnerability for excitotoxicity remain unknown. Retinal excitotoxicity is involved in various diseases leading to irreversible blindness. Here, we used the visual system and explored the mechanistic details of the NMDA-elicited intracellular events, especially in the amacrine cells, which are the most vulnerable type of neuron in the retina. G-substrate, a specific substrate of cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase, is colocalized with amacrine cells and acts as an endogenous inhibitor of protein phosphatase. To elucidate how G-substrate was involved in NMDA-induced amacrine cell death, the immunohistochemical analysis with G-substrate antibody was performed following NMDA injury. In vivo, NMDA immediately decreased G-substrate immunoreactivity, and the suppression of calpain activation using ALLN or calpain III, an inhibitor of calpain, blocked this decrease. In vitro, degraded fragments of G-substrate were detected within 10 min after coincubation of G-substrate and calpain. Moreover, G-substrate knockout (G-substrate(-/-)) mice were more susceptible to NMDA injury than wild-type mice. ALLN did not have a neuroprotective effect in G-substrate(-/-) mice. These data strongly suggest that calpain-mediated loss of G-substrate represents an important mechanism contributing to NMDA-induced amacrine cell death.

N-Methyl-D-Aspartate (NMDA) Antagonists—S(+)-ketamine, Dextrorphan, and Dextromethorphan—Act as Calcium Antagonists on Bovine Cerebral Arteries

Ketamine, an intravenous anesthetic and a major drug of abuse, is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. Ketamine's enantiomer, S(+)-ketamine, acts stereoselectively on neuronal NMDA receptors. The purpose of this in vitro study was to compare the direct effects of S(+)-ketamine, 2 other noncompetitive NMDA receptor antagonists (dextrorphan and dextromethorphan), and the calcium entry blocker nimodipine on the cerebral vasculature, using bovine middle cerebral arteries as an experimental model. Arterial rings were mounted in isolated tissue chambers equipped with isometric tension transducers to obtain pharmacologic dose-response curves. In the absence of exogenous vasoconstrictors, the NMDA antagonists or nimodipine had negligible effects on cerebral arterial tone. When rings were preconstricted with either potassium or the stable thromboxane A2 mimetic U46619, the NMDA antagonists and nimodipine each produced dose-dependent relaxation. Prior endothelial stripping had no effect on subsequent drug-induced relaxation of K+-constricted rings. In Ca2+-deficient media containing either potassium or U46619, the NMDA antagonists and nimodipine each produced competitive inhibition of subsequent Cainduced constriction. In additional experiments, arterial strips were mounted in isolated tissue chambers to directly measure calcium uptake, using 45calcium (45Ca) as a radioactive tracer. The NMDA antagonists and nimodipine each blocked potassium-stimulated or U46619-stimulated Ca2+ uptake into arterial strips. These results indicate that S(+)-ketamine, dextrorphan, and dextromethorphan, like nimodipine, directly dilate cerebral arteries by acting as calcium antagonists; they all inhibit 45Ca uptake through both potential-operated (potassium) and receptor-operated (U46619) channels in cerebrovascular smooth muscle.

Noble Meets Nouveau

Noble Meets Nouveau

Astrocytic glutamate targets NMDA receptors

Astrocytic glutamate targets NMDA receptors

Neuron-derived D-Serine Release Provides a Novel Means to Activate N-Methyl-D-aspartate Receptors

D-serine is a coagonist of N-methyl-D-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of D-serine is carried out by serine racemase, which converts L- to D-serine. D-serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of D-serine. We now report significant amounts of serine racemase and D-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and D-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and D-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived D-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous D-serine by addition of the recombinant enzyme D-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal D-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked D-serine release. Release of D-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. D-serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of D-serine. Glutamate-induced neuronal D-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.

Purinergic Receptors Mediate Two Distinct Glutamate Release Pathways in Hippocampal Astrocytes

The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain.

Role of neuronal NR2B subunit-containing NMDA receptor-mediated Ca2+ influx and astrocytic activation in cultured mouse cortical neurons and astrocytes

The excitatory neurotransmitter glutamate has been shown to mediate such bidirectional communication between neurons and astrocytes. In the present study, we determined the role of N-methyl-D-aspartate (NMDA) receptors on glutamate-evoked Ca(2+) influx into neurons and astrocytes. Either a nonselective NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or selective NR2B subunit-containing NMDA receptor antagonists ifenprodil and (R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol (Ro25-6981) significantly inhibited the glutamate-evoked Ca(2+) influx into neurons, but not into astrocytes. Furthermore, we investigated whether NR2B subunit-containing NMDA receptor antagonists could suppress the astrocytic activation, as detected by glial fibrillary acidic protein (GFAP; as a specific marker of astrocyte)-like immunoreactivities in mouse cortical astrocytes. Here, we demonstrated that the increases in the level of GFAP-like immunoreactivities induced by glutamate were markedly suppressed by cotreatment with ifenprodil in cortical neuron/glia cocultures, but not in purified astrocytes. These results suggest that NR2B subunit-containing NMDA receptor plays a critical role in not only glutamate-evoked Ca(2+) influx into neurons, but also glutamate-induced astrocytic activation. Thus, glutamate-mediated pathway via NR2B subunit-containing NMDA receptor may, at least in part, contribute to neuron-to-astrocyte signaling.

Astrocyte Influences on Ischemic Neuronal Death

Glutamate excitotoxicity, oxidative stress, and acidosis are primary mediators of neuronal death during ischemia and reperfusion. Astrocytes influence these processes in several ways. Glutamate uptake by astrocytes normally prevents excitotoxic glutamate elevations in brain extracellular space, and this process appears to be a critical determinant of neuronal survival in the ischemic penumbra. Conversely, glutamate efflux from astrocytes by reversal of glutamate uptake, volume sensitive organic ion channels, and other routes may contribute to extracellular glutamate elevations. Glutamate activation of neuronal N-methyl-D-aspartate (NMDA) receptors is modulated by glycine and D-serine: both of these neuromodulators are transported by astrocytes, and D-serine production is localized exclusively to astrocytes. Astrocytes influence neuronal antioxidant status through release of ascorbate and uptake of its oxidized form, dehydroascorbate, and by indirectly supporting neuronal glutathione metabolism. In addition, glutathione in astrocytes can serve as a sink for nitric oxide and thereby reduce neuronal oxidant stress during ischemia. Astrocytes probably also influence neuronal survival in the post-ischemic period. Reactive astrocytes secrete nitric oxide, TNFalpha, matrix metalloproteinases, and other factors that can contribute to delayed neuronal death, and facilitate brain edema via aquaporin-4 channels localized to the astrocyte endfoot-endothelial interface. On the other hand erythropoietin, a paracrine messenger in brain, is produced by astrocytes and upregulated after ischemia. Erythropoietin stimulates the Janus kinase-2 (JAK-2) and nuclear factor-kappaB (NF-kB) signaling pathways in neurons to prevent programmed cell death after ischemic or excitotoxic stress. Astrocytes also secrete several angiogenic and neurotrophic factors that are important for vascular and neuronal regeneration after stroke.

Modulation of voltage‐ and ligand‐gated ion channels by neuronal P2Y receptors

AbstractThe present review summarizes data about the properties, distribution, and effects of neuronal P2Y receptors. Extracellular nucleotide receptors can be classified into two types belonging either to the P2X (ligand‐gated cationic channels) or the P2Y type (G protein‐coupled receptors). Neuronal P2Y receptors comprise five cloned and functionally defined classes of which P2Y1, P2Y6, P2Y11, P2Y12, and P2Y13 occur in the central nervous system. Various types of P2Y receptors stimulate inwardly rectifying potassium channels, positively or negatively modulate the high‐voltage activated Ca2+ current and increase the intracellular free Ca2+ concentration in neuronal preparations. Moreover, in a subpopulation of pyramidal cells of the rat prefrontal cortex (PFC), the P2Y2 receptor potentiates the function of a ligand‐gated cationic channel, the N‐methyl‐D‐aspartate (NMDA)‐type excitatory amino acid receptor. This effect is G protein‐mediated and utilizes the phospholipase C/inositol 1,4,5‐trisphosphate/Ca2+/calmodulin kinase II pathway. It appears that P2Y2 receptors situated at astrocytes release glutamate, which in turn modulates NMDA receptors of neighbouring neurons via stimulation of group I metabotropic glutamate receptors. In contrast, P2Y1 receptors inhibit the conductance of NMDA receptor‐channels in all PFC pyramidal cells investigated. This effect is fast in onset and does not depend on G protein activation. It is suggested that P2Y1 receptors alter the function of NMDA receptors by a direct protein–protein coupling in the membrane. Because ATP and dopamine are supposed to be coreleased from dopaminergic fibers onto layer V pyramidal cells, both neurotransmitters may interact with the neuronal NMDA receptor. Hence, D1 dopamine receptors and P2Y1/P2Y2 receptors may be involved in the fine tuning of higher order cognitive functions including learning and memory. Drug Dev. Res. 59:49–55, 2003. © 2003 Wiley‐Liss, Inc.

Spermine modulates neuronal excitability and NMDA receptors in juvenile gerbil auditory thalamus

Medial geniculate body (MGB) neurons process synaptic inputs from auditory cortex. Corticothalamic stimulation evokes glutamatergic excitatory postsynaptic potentials (EPSPs) that vary markedly in amplitude and duration during development. The EPSP decay phase is prolonged during second postnatal week but then shortens, significantly, until adulthood. The EPSP prolongation depends on spermine interactions with a polyamine-sensitive site on receptors for N-methyl- D-aspartate (NMDA). We examined effects of spermine application on EPSPs, firing modes, and membrane properties in gerbil MGB neurons during the P14 period of highest polyamine sensitivity. Spermine slowed EPSP decay and promoted firing on EPSPs, without changing passive membrane properties. Spermine increased membrane rectification on depolarization, which is mediated by tetrodotoxin (TTX)-sensitive, persistent Na + conductance. As a result, spermine lowered threshold and increased tonic firing evoked with current injection by up to ∼150%. These effects were concentration-dependent (ED 50=100 μM), reversible, and eliminated by NMDA receptor antagonist, 2-amino-5-phosphonovalerate (APV). In contrast, spermine increased d V/d t of the low threshold Ca 2+ spike (LTS) and burst firing, evoked from hyperpolarized potentials. LTS enhancement was greater at −55 mV than at hyperpolarized potentials and did not result from persistent Na + conductance or glutamate receptor mechanisms. In summary, spermine increased excitability by modulating NMDA receptors in juvenile gerbil neurons.

Interaction of dopamine D1 and NMDA receptors mediates acute clozapine potentiation of glutamate EPSPs in rat prefrontal cortex.

The atypical antipsychotic drug clozapine effectively alleviates both negative and positive symptoms of schizophrenia via unclear cellular mechanisms. Clozapine may modulate both glutamatergic and dopaminergic transmission in the prefrontal cortex (PFC) to achieve part of its therapeutic actions. Using whole cell patch-clamp techniques, current-clamp recordings in layers V-VI pyramidal neurons from rat PFC slices showed that stimulation of local afferents (in 2 microM bicuculline) evoked mixed [AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors] glutamate receptor-mediated excitatory postsynaptic potentials (EPSPs). Clozapine (1 microM) potentiated polysynaptically mediated evoked EPSPs (V(Hold) = -65 mV), or reversed EPSPs (rEPSP, V(Hold) = +20 mV) for >30 min. The potentiated EPSPs or rEPSPs were attenuated by elevating [Ca(2+)](O) (7 mM), by application of NMDA receptor antagonist 2-amino5-phosphonovaleric acid (50 microM), or by pretreatment with dopamine D1/D5 receptor antagonist SCH23390 (1 microM) but could be further enhanced by a dopamine reuptake inhibitor bupropion (1 microM). Clozapine had no significant effect on pharmacologically isolated evoked NMDA-rEPSP or AMPA-rEPSPs but increased spontaneous EPSPs without changing the steady-state resting membrane potential. Under voltage clamp, clozapine (1 microM) enhanced the frequency, and the number of low-amplitude (5-10 pA) AMPA receptor-mediated spontaneous EPSCs, while there was no such changes with the mini-EPSCs (in 1 microM TTX). Taken together these data suggest that acute clozapine can increase spike-dependent presynaptic release of glutamate and dopamine. The glutamate stimulates distal dendritic AMPA receptors to increase spontaneous EPSCs and enabled a voltage-dependent activation of neuronal NMDA receptors. The dopamine released stimulates postsynaptic D1 receptor to modulate a lasting potentiation of the NMDA receptor component of the glutamatergic synaptic responses in the PFC neuronal network. This sequence of early synaptic events induced by acute clozapine may comprise part of the activity that leads to later cognitive improvement in schizophrenia.

The NMDA type glutamate receptors expressed by primary rat osteoblasts have the same electrophysiological characteristics as neuronal receptors.

Cells of mammalian bone express glutamate receptors. Functional N-methyl-D-aspartate (NMDA) receptors have been demonstrated in human, osteoblastic MG-63 cells, but currents in these cells, unlike those of mammalian neurons, are blocked by Mg(2+) in a voltage-insensitive manner. Differences between the characteristics of NMDA currents in bone cells and in neurons may reflect molecular variation of the receptors or associated molecules, with implications for the role(s) of glutamate in these different tissues and for targeting of ligands/antagonists. To determine whether NMDA receptors in primary bone cells are functional, and whether the currents carried by these receptors resemble those of MG-63 cells or those of mammalian neurons, we have applied the whole cell patch clamp technique to primary cultures of rat osteoblasts. In 0-Mg(2+) saline, 25% of cells showed a slowly developing inward current in response to bath perfusion with 1 mM or 100 microM NMDA. Antibodies against NMDA receptors stained approximately 26% of cells. When NMDA was applied by rapid superfusion, kinetics of the currents were similar to those of neuronal NMDA currents, reaching a peak within 20-30 ms. 1 mM Mg(2+) reduced current amplitude at negative holding potentials and caused the I-V relationship of the currents to adopt a 'J' shape rather than the linear relationship seen in the absence of added Mg(2+). Co-application of glycine (20 microM) with NMDA increased current amplitude by only 18%, suggesting that glycine is released from cells within the cultures. Currents were blocked by (+)-MK-801 and DL-2-amino-5-phosphonovaleric acid. Fluorimetric monitoring of [Ca(2+)](i) using fura-2 showed that, in Mg(2+)-free medium, NMDA caused a sustained rise in [Ca(2+)](i) that could be reversed by subsequent application of MK-801. We conclude that rat femoral osteoblasts express functional NMDA receptors and that these receptors differ from those previously identified in MG-63 cells. NMDA receptors of primary osteoblasts show a 'classical' voltage-sensitive Mg(2+) block, similar to that seen in neuronal NMDA receptors, and will therefore function as detectors of coincident receptor activation and membrane depolarization.

Nitric oxide-mediated Ca ++-influx in neuronal nuclei and cortical synaptosomes of normoxic and hypoxic newborn piglets

Previous studies have shown that hypoxia results in the generation of nitric oxide (NO) free radicals in the cerebral cortex of newborn animals. The present study tested the hypothesis that NO increases Ca ++-influx in neuronal nuclei as well as N-methyl- d aspartate (NMDA) receptor-mediated Ca ++-influx in cortical synaptosomes of newborn piglets. Studies were performed in five normoxic (Nx) and 6 hypoxic (Hx) newborn piglets. Cerebral tissue hypoxia was documented by determining the levels of ATP and phosphocreatine (PCr). 45Ca ++ -influx was determined in the presence of sodium nitroprusside (SNP, 10 μM), a NO donor, and peroxynitrite (10 μM). In the Hx group, ATP levels decreased to 1.40±0.69 vs 4.27±0.80 μmoles/g brain in the Nx group ( P<0.05). Similarly, PCr levels decreased to 0.91±0.57 vs 3.40±0.99 μmoles/g brain ( P<0.001). Nuclear 45Ca ++-influx increased from 3.57±1.46 pmoles/mg protein in Nx nuclei to 8.64±3.50 in Hx nuclei ( P<0.05). SNP increased neuronal nuclear Ca ++ influx in the Nx from 3.57±1.46 to 5.47±2.52 pmoles/mg protein ( P<0.05) but did not affect Ca ++ influx in the Hx group (8.64±3.50 vs. 10.17±4.00 pmoles/mg protein). The level of Ca ++ influx in the presence of SNP in Nx nuclei was similar to that seen in Hx nuclei alone. Peroxynitrite did not affect nuclear Ca ++-influx in either Nx or Hx group. Synaptosomal Ca ++-influx in the presence of glu+gly was 40±11 pmoles/mg protein in the Nx group and 80±16 pmoles/mg protein in the Hx group ( P<0.05). Both SNP and peroxynitrite increased Ca ++ influx in Nx and Hx synaptosomes. These results show that hypoxia results in increased nuclear and synaptosomal Ca ++-influx. Further, the data demonstrate that NO increases intranuclear as well as intrasynaptosomal Ca ++-influx and suggest that during hypoxia, the increase in intranuclear and intraynaptosomal Ca ++ is NO-mediated. We propose that NO-mediated modification (by nitrosylation/nitration) of nuclear membrane high affinity Ca ++-ATPase and neuronal membrane NMDA receptor, resulting in increased intranuclear and intracellular Ca ++ influx, are potential NO-mediated mechanisms of Hx neuronal injury.

NMDA-responsible, APV-insensitive receptor in cultured human astrocytes

The present study was conducted to assess N-methyl-D-aspartate (NMDA)-responsible receptors in cultured human astrocytes by monitoring whole-cell membrane currents. NMDA generated currents, that are potentiated by glycine and blocked by Mg 2+, with the current/voltage relation showing a reversal potential of ± 0 mV. The currents were not inhibited by either the selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV), or the non-selective ionotropic glutamate receptor antagonist, kynurenic acid. The currents were inhibited only by 19% in Ca 2+-free extracellular solution. Furthermore, GDPβS, a broad G-protein inhibitor, inhibited NMDA-induced currents to 82% of original levels. The results of the present study thus suggest that an NMDA-responsible, APV-insensitive receptor with low Ca 2+ permeability, distinct from the neuronal NMDA receptors, is expressed in human astrocytes and that the receptor is regulated in part by an unknown G-protein-linked receptor.

A brain slice model for in vitro analyses of astrocytic gap junction and connexin43 regulation: actions of ischemia, glutamate and elevated potassium

Brain slices prepared from adult rats and maintained for up to 3 h in vitro were used to investigate the effects of pharmacological treatments on the phosphorylation state, immunolabelling characteristics and ultrastructural localization of astrocytic gap junctions and connexin43 (Cx43). Slices deprived of glucose/oxygen to mimic ischemia or those exposed to 1 mM glutamate for 1 h exhibited Cx43 dephosphorylation, epitope masking and gap junction internalization as revealed by Western blotting and Cx43 immunolocalization with various antibodies. Treatment with 15 mM K+ caused Cx43 dephosphorylation without junction internalization. The effects of glutamate and K+ were completely blocked by the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist 2-amino-5-phosphonovalerate (APV), which acts largely on neuronal NMDA receptors, suggesting neuronal mediation of glial gap junction responses to these treatments. Astrocytes contained a dephosphorylated form of Cx43 with a typical migration profile at 41 kDa as well as novel, apparently dephosphorylated or partially phosphorylated, forms migrating at 43 kDa. These results indicate that slices prepared from adult brain can serve as a convenient model to investigate the molecular basis and receptor-mediated mechanisms underlying astrocytic Cx43 responses that have been observed in vivo following cerebral ischemia or neural activation. These processes can be related in part to neuronal regulation of astrocytic gap junctional coupling state, which is also amenable to analysis in brain slices.

Spermine and Arcaine Block and Permeate N-Methyl- d-Aspartate Receptor Channels

Polyamines such as spermine are thought to be endogenous regulators of NMDA ( N-methyl- d-aspartate)-type glutamate receptors. Polyamine block of NMDA receptors was studied in excised outside-out patches from rat hippocampal neurons and Xenopus oocytes expressing recombinant receptors. Extracellular spermine and arcaine reduced NMDA single-channel conductance in a voltage-dependent manner, with partial relief of block evident at large inside negative membrane potentials. Reducing extracellular Na + concentration increased the apparent affinities for spermine and arcaine, indicating strong interaction between spermine and permeant ions. Internal spermine also blocked NMDA channels in a voltage-dependent manner, with relief of block evident at large inside positive potentials. The Woodhull model of channel block by an impermeant ion adequately described the actions of external spermine from −60 to +60 mV, but failed for more negative potentials. Eyring rate theory for a permeable blocker with two barriers and one binding site adequately described the voltage-dependent block and relief from block by both external and internal spermine over the range of −120 to +60 mV. These findings indicate that polyamines block and permeate neuronal NMDA receptor channels from the extracellular and intracellular sides, although sensitivity to internal spermine is probably too low to be physiologically relevant.

Neuroactive compounds produced by bacteria from the marine sponge Halichondria panicea: activation of the neuronal NMDA receptor

Previous studies revealed that the marine sponge Halichondria panicea habors symbiotic- and commensalic bacteria ( Althoff et al., 1998. Marine Biol. 130, 529–536). In the present study the hypothesis was tested whether some of those bacteria synthesize neuroactive compounds. For the first time the effect of bacterial bioactive compounds on the neuronal ionotropic glutamate receptors [iGluR], subtype N-methyl- d-aspartate (NMDA) receptor, was checked. In cortical neurons from rats as cell system the supernatant of two bacterial cultures isolated from H. panicea proved to agonize the NMDA receptor. The response of the NMDA receptor to the bioactive compounds was determined by measuring the intracellular Ca 2+ level. The supernatants of cultures 697 and 698 were found to upregulate the intracellular Ca 2+ level. To validate the specificity of the effects, inhibition studies with Memantine and d-AP5 were performed. The two bacteria were identified by polymerase chain reaction-amplification of the 16S rDNA genes and subsequent sequencing; they displayed highest identity to Antarcticum vesiculatum and to Psychroserpens burtonensis, respectively. Based on these data first experimental evidence is presented indicating that bacteria associated with sponges display neuroactivity by agonizing the NMDA receptor.

Calmodulin Mediates Calcium-Dependent Inactivation of N-Methyl-D-Aspartate Receptors

Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors activates signal transduction pathways critical for many forms of synaptic plasticity in the brain. NMDA receptor–mediated Ca2+ influx also downregulates the gating of NMDA channels through a process called Ca2+-dependent inactivation (CDI). Recent studies have demonstrated that the calcium binding protein calmodulin directly interacts with NMDA receptors, suggesting that calmodulin may play a role in CDI. We report here that the mutation of a specific calmodulin binding site in the C0 region of the NR1 subunit of the NMDA receptor blocks CDI. Moreover, intracellular infusion of a calmodulin inhibitory peptide markedly reduces CDI of both recombinant and neuronal NMDA receptors. Furthermore, this inactivating effect of calmodulin can be prevented by coexpressing a region of the cytoskeletal protein α-actinin2 known to interact with the C0 region of NR1. Taken together, these results demonstrate that the binding of Ca2+/calmodulin to NR1 mediates CDI of the NMDA receptor and suggest that inactivation occurs via Ca2+/calmodulin-dependent release of the receptor complex from the neuronal cytoskeleton.

Human hippocampal AMPA and NMDA mRNA levels in temporal lobe epilepsy patients.

This study was designed to determine whether hippocampal neuronal AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and NMDA (N-methyl-D-aspartate) mRNA levels were differentially increased in temporal lobe epilepsy patients compared with those measured in control tissue from non-seizure autopsies. Hippocampi from hippocampal sclerosis patients (n = 28) and temporal mass lesion cases (n = 12) were compared with those from the autopsies (n = 4), and studied for AMPA GluR1-3 and NMDAR1-2 mRNAs using semi-quantitative in situ hybridization, along with fascia dentata and Ammon's horn neuron densities. Compared with the autopsies, and without correction for neuron counts, the mass lesion cases with neuron densities similar to autopsies showed: (i) significantly increased NMDAR2 hybridization densities for fascia dentata granule cells; (ii) increased AMPA GluR3 mRNA densities for Ammon's horn pyramids; and (iii) similar or numerically increased mRNAs for all other subunits and hippocampal subfields. Compared with the autopsies, hippocampal sclerosis cases with decreased neuron densities showed: (i) significantly decreased AMPA GluR1-2 and NMDAR1-2 hybridization densities for Ammon's horn pyramids and (ii) similar or numerically decreased mRNAs for all other subunits and subfields. However, correcting for changes in neuron densities showed that hippocampal sclerosis patients had increased AMPA and NMDA mRNA levels per neuron compared with autopsies, and in the CA2 resistant sector GluR2 mRNA levels were numerically greater than autopsies and mass lesion cases. Furthermore, relative to autopsies both sclerosis and mass lesion hippocampi showed that, in the stratum granulosum, the greatest mRNA increases were in AMPA GluR1 and NMDAR2 compared with the other mRNAs. In chronic temporal lobe seizure patients these results indicate that mass lesion and sclerosis cases show differential increases in hippocampal AMPA and NMDA mRNA levels per neuron compared with autopsies, especially for AMPA GluR1 and NMDAR2 in fascia dentata granule cells. These findings support the hypothesis that temporal lobe seizures are associated with increased ionotropic glutamate receptor mRNA levels and alterations in receptor subunit composition that probably contribute to neuronal hyperexcitability, synchronization and seizure generation.

Recovery from NMDA-induced intracellular acidification is delayed and dependent on extracellular bicarbonate.

A 30-s exposure to N-methyl-D-aspartate (NMDA) produced a dose-dependent and long-lasting (10-20 min) reduction in intracellular pH in cultured cortical neurons, detected by the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. This intracellular acidification could be blocked by addition of the NMDA antagonist, D-(-)-2-amino-5-phosphonovalerate, or by removal of extracellular Ca2+. Removal of extracellular HCO3- markedly impaired recovery from NMDA-induced intracellular acidification. Recovery was also impaired when 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, inhibitors of HCO3- transport, were added to the cultures immediately after NMDA exposure. In contrast, the Na+/H+ exchange blocker, 5-(N-ethyl-N-isopropyl)amiloride, did not affect pH recovery. Removal of extracellular Cl- partially prevented pH recovery after NMDA stimulation. In addition, extracellular HCO3- increased intracellular Na+ after NMDA exposure, consistent with HCO3- activation of a Na(+)-dependent exchanger. These results demonstrate that stimulation of cortical neuronal NMDA receptors is followed by long-lasting intracellular acidification and that the presence of extracellular HCO3- is important in the subsequent recovery of normal intracellular pH, likely acting at least in part via the Na(+)-dependent Cl-/HCO3- exchanger.