N-methyl-D-aspartate Receptor Desensitization Research Articles

Theoretical calculation of NMDA receptor desensitization and intracellular calcium determination through simulation

One of the crucial processes in preventing cellular damage due to excitotoxicity involves the calcium-mediated desensitization of the N-methyl-D-aspartate (NMDA) receptor. The opening of NMDA channels results in the influx of Na+ and Ca2+ ions and efflux of K+ ions. This condition leads to an increase in intracellular calcium ([Ca2+]i), thereby diminishing the NMDA current and preventing damage induced by constant stimulation of the receptor by glutamate. In this study, a simulator based on a phenomenological mathematical model was developed, employing the Visual Basic 6.0 language for the Windows® environment. Input variables encompass intracellular calcium, peak currents (Ip), and steady-state currents (Iss). Input currents were derived from previously published electrophysiological recordings. The simulator adeptly reproduces the NMDA receptor desensitization phenomenon under varying experimental conditions and accurately calculates internal calcium with a 10% margin of error. It proves valuable in scenarios where simultaneous recordings of NMDA current and [Ca2+]i via fluorescence labeling techniques are impractical. In the realm of education, it enables students to conduct virtual experiments, providing an immersive introduction to the topic.

Chemical shift assignments of calmodulin bound to a cytosolic domain of GluN2A (residues 1004-1024) from the NMDA receptor.

N-methyl-D-aspartate receptors (NMDARs) consist of glycine-binding GluN1 and glutamate-binding GluN2 subunits that form tetrameric ion channels. NMDARs in the neuronal post-synaptic membrane are important for controlling neuroplasticity and synaptic transmission in the brain. Calmodulin (CaM) binds to the cytosolic C0 domains of both GluN1 (residues 841-865) and GluN2 (residues 1004-1024) that may play a role in the Ca2+-dependent desensitization of NMDAR channels. Mutations that disrupt Ca2+-dependent desensitization of NMDARs are linked to Alzheimer's disease, depression, stroke, epilepsy, and schizophrenia. NMR chemical shift assignments are reported here for Ca2+-saturated CaM bound to the GluN2A C0 domain of NMDAR (BMRB no. 51821).

Nutritional and Metabolic Factors, Ethanol and Cholesterol, Interact With Calcium-Dependent N-Methyl-D-Aspartate Receptor Inhibition by Tricyclic Antidepressants.

It is known that overexpression of N-methyl-D-aspartate receptors (NMDARs) contributes to central sensitization and development of neuropathic pain. Tricyclic antidepressants (TCAs), amitriptyline (ATL), and desipramine (DES) exhibit analgetic anti-NMDAR activity and are commonly utilized for pain therapy. This property is determined by their ability to enhance the calcium-dependent desensitization (CDD) of NMDARs. Coincidently ethanol and cholesterol, the ubiquitous food supplements, also modulate NMDAR CDD. The convergence of the effects of these compounds on a similar calcium-dependent process allows to assume their interaction on NMDARs. Since there is no information on whether ethanol supplementation and cholesterol deficit interfere with TCA inhibition of NMDARs at a cellular level, here we investigated this issue. Whole-cell NMDA-activated currents were recorded in rat cortical neurons of primary cultures to study how the IC50 values for TCA inhibition of NMDARs are influenced by ethanol and cholesterol extraction from the plasma membrane with methyl-β-cyclodextrin. Ethanol at 0.03% did not reliably affect the steady-state NMDA-activated currents. At this threshold concentration ethanol, however, increased IC50s for ATL and DES abolishing their calcium-dependent inhibition of NMDARs but did not change IC50 for clomipramine (CLO), which is calcium-independent. Whereas the ethanol effects on ATL-induced NMDAR inhibition reached a maximum at 2 mM external [Ca2+], for DES the maximum was achieved already at 1 mM external [Ca2+], that correlates with the manifestation of the calcium-dependent inhibition of NMDARs by these agents. Cholesterol depletion also increased IC50s for both ATL and DES abolishing the calcium-dependent inhibition of NMDARs. The restitution of cholesterol in the plasma membrane reversed the ATL IC50 back to the low values, by a restoration of calcium-dependence of ATL. These observations are consistent with the explanation that either 0.03% ethanol or cholesterol extraction may interrupt some intermediate step of CDD transduction or augment NMDAR CDD to the maximal level so that ATL and DES could not further enhance CDD. It is likely that anti-NMDAR action of ATL and DES against neuropathic pain could demonstrate peculiarities in therapeutic profiles during cholesterol decline in aging or medical treatments and ethanol supplementations even in quantities that are insufficient to cause the symptoms of intoxication.

Early Life Febrile Seizures Impair Hippocampal Synaptic Plasticity in Young Rats.

Febrile seizures (FSs) in early life are significant risk factors of neurological disorders and cognitive impairment in later life. However, existing data about the impact of FSs on the developing brain are conflicting. We aimed to investigate morphological and functional changes in the hippocampus of young rats exposed to hyperthermia-induced seizures at postnatal day 10. We found that FSs led to a slight morphological disturbance. The cell numbers decreased by 10% in the CA1 and hilus but did not reduce in the CA3 or dentate gyrus areas. In contrast, functional impairments were robust. Long-term potentiation (LTP) in CA3-CA1 synapses was strongly reduced, which we attribute to the insufficient activity of N-methyl-D-aspartate receptors (NMDARs). Using whole-cell recordings, we found higher desensitization of NMDAR currents in the FS group. Since the desensitization of NMDARs depends on subunit composition, we analyzed NMDAR current decays and gene expression of subunits, which revealed no differences between control and FS rats. We suggest that an increased desensitization is due to insufficient activation of the glycine site of NMDARs, as the application of D-serine, the glycine site agonist, allows the restoration of LTP to a control value. Our results reveal a new molecular mechanism of FS impact on the developing brain.

Desensitization of NMDA channels requires ligand binding to both GluN1 and GluN2 subunits to constrict the pore beside the activation gate.

N-methyl-D-aspartate (NMDA) receptor channels are activated by glutamate (or NMDA) and glycine. The channels also undergo desensitization, which denotes decreased channel availability, after prolonged exposure to the activating ligands. Glycine apparently has a paradoxical negative effect on desensitization, as the increase in ambient glycine in concentrations required for channel activation would increase sustained NMDA receptor currents. We hypothesized that this classical"glycine-dependent desensitization" could be glycine-dependentactivation in essence. By performing electrophysiological recordings and biophysical analyses with rat brain NMDA receptors heterogeneously expressed in Xenopus laevis oocytes, we characterizedthat the channel opened by "only" NMDA (in nominally glycine-free condition probably with the inevitable nanomolar glycine) would undergo a novel form of deactivation rather than desensitization, and is thus fully available for subsequent activation. Moreover, external tetrapentylammonium ions (TPentA), tetrabutylammonium ions, and tetrapropylammonium ions (TPA, in higher concentrations) block the pore and prohibit channel desensitization with a simple "foot-in-the-door" hindrance effect. TpentA and TPA have the same voltage dependence but show different flow dependence in binding affinity, revealing a common binding site at an electrical distance of ~0.7 from the outside yet differential involvement of the flux-coupling region in the external pore mouth. The smaller tetraethylammonium ion and the larger tetrahexylammonium and tetraheptylammonium ions may block the channel but could not affect desensitization. We conclude that NMDA receptor desensitization requires concomitant binding of both glycine and glutamate, and thus movement of both GluN1 and GluN2 subunits. Desensitization gateitself embodies a highly restricted pore reduction with a physical distance of ~4Å from the charged nitrogen atom of bound tetraalkylammonium ions, and is located very close to the activation gate in the bundle-crossing region in the externalpore vestibule.

Marine Mammals' NMDA Receptor Structure: Possible Adaptation to High Pressure Environment.

Divers that are exposed to high pressure (HP) above 1.1 MPa suffer from High Pressure Neurological Syndrome (HPNS), which is implicated with central nervous system (CNS) malfunction. Marine mammals performing extended and deep breath-hold dives are exposed to almost 20 MPa without apparent HPNS symptoms. N-methyl-D-aspartate receptor (NMDAR) has repeatedly been implicated as one of the major factors in CNS hyperexcitability as part of HPNS. Electrophysiological studies in rat brain slices at He HP showed a significant increase in the synaptic NMDAR response, followed by postsynaptic excitability changes. Molecular studies of Rattus norvegicus NMDARs have revealed that different subunit combinations of the NMDAR exhibit different, increased or decreased, current responses under He HP conditions. The purpose of the present research was to disclose if the breath-hold deep diving mammals exhibit NMDAR structural modifications related to HP. We used sequence alignment and homology structure modeling in order to compare deep diving marine mammals’ NMDARs to those of terrestrial mammals. We discovered that deep diving mammals have a special tertiary TMD structure of the GluN2A subunit that differs from that of the terrestrial mammals. In addition, the GluN2A subunit has a group of four conserved a.a. substitutions: V68L (N-terminal domain, NTD) and V440I (agonist-binding domain, ABD) are cetacean specific, E308D (N-terminal domain, NTD) and I816V (transmembrane domain, TMD) were also singularly found in some terrestrial mammals. Since I816V is localized in M4 α-helix region, which is critical for NMDAR activation and desensitization, we hypothesize that the presence of all 4 substitutions rather than a single one, is the combination that may enable HP tolerance. Furthermore, additional special substitutions that were found in the marine mammals’ NTD may affect the Zn2+ binding site, suggesting less or no voltage-independent inhibition by this ion. Our molecular studies of NMDARs containing the GluN2A subunit showed that HP removal of the Zn2+ voltage-independent inhibition could be the mechanism explaining its current increase at HP. Thus, this mechanism could play a crucial role in the CNS hyperexcitability at HP. Less or no voltage-independent Zn2+ inhibition, different conformations of the TMD, and special mutation in the M4 α-helix region of cetaceans’ NMDAR, may give them the advantage they need in order to perform such deep dives without CNS malfunction.

Downregulation of calcium-dependent NMDA receptor desensitization by sodium-calcium exchangers: a role of membrane cholesterol

BackgroundThe plasma membrane Na+/Ca2+-exchanger (NCX) has recently been shown to regulate Ca2+-dependent N-methyl-d-aspartate receptor (NMDAR) desensitization, suggesting a tight interaction of NCXs and NMDARs in lipid nanoclasters or “rafts”. To evaluate possible role of this interaction we studied effects of Li+ on NMDA-elicited whole-cell currents and Ca2+ responses of rat cortical neurons in vitro before and after cholesterol extraction by methyl-β-cyclodextrin (MβCD).ResultsSubstitution Li+ for Na+ in the external solution caused a concentration-dependent decrease of steady-state NMDAR currents from 440 ± 71 pA to 111 ± 29 pA in 140 mM Na+ and 140 mM Li+, respectively. The Li+ inhibition of NMDAR currents disappeared in the absence of Ca2+ in the external solution (Ca2+-free), suggesting that Li+ enhanced Ca2+-dependent NMDAR desensitization. Whereas the cholesterol extraction with MβCD induced a decrease of NMDAR currents to 136 ± 32 pA in 140 mM Na+ and 46 ± 15 pA in 140 mM Li+, the IC50 values for the Li+ inhibition were similar (about 44 mM Li+) before and after this procedure. In the Ca2+-free Na+ solution the steady-state NMDAR currents after the cholesterol extraction were 47 ± 6% of control values. Apparently this amplitude decrease was not Ca2+-dependent. In the Na+ solution containing 1 mM Ca2+ the Ca2+-dependent NMDAR desensitization was greater when cholesterol was extracted. Obviously, this procedure promoted its development. In agreement, Li+ and KB-R7943, an inhibitor of NCX, both considerably reduced NMDA-activated Ca2+ responses. The cholesterol extraction itself caused a decrease of NMDA-activated Ca2+ responses and, in addition, abolished the effects of Li+ and KB-R7943. The cholesterol loading into the plasma membrane caused a recovery of the KB-R7943 effects.ConclusionsTaken together our data suggest that NCXs downregulate the Ca2+-dependent NMDAR desensitization. Most likely, this is determined by a tight functional interaction of NCX and NMDAR molecules because of their co-localization in membrane lipid rafts. The destruction of these rafts is accompanied by an enhancement of NMDAR desensitization and a loss of NCX-selective agent effects on NMDARs.

Neurogranin in the nucleus accumbens regulates NMDA receptor tolerance and motivation for ethanol seeking

Dysfunction of N-methyl-d-aspartate receptor (NMDAR) signaling in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng), a calmodulin-binding protein, is exclusively expressed in the post-synapse, and mediates NMDAR driven synaptic plasticity by regulating the calcium-calmodulin (Ca2+-CaM) pathway. To study the functional role of Ng in AUD, we administrated behavior tests including Pavlovian instrument transfer (PIT), operant conditioning, and rotarod test using Ng null mice (Ng–/– mice). We used adeno-associated virus (AAV)-mediated Ng expression and pharmacological manipulation to validate behavioral responses in Ng–/– mice. The results from our multidisciplinary approaches demonstrated that deficit of Ng increases tolerance to NMDAR inhibition and elicit faster cue reactivity during PIT without changes in ethanol reward. Operant conditioning results demonstrated that Ng–/– mice self-administered significantly more ethanol and displayed reduced sensitivity to aversive motivation. We identified that ethanol exposure decreases mGluR5 (metabotropic glutamate receptor 5) expression in the NAc of Ng–/– mice and pharmacological inhibition of mGluR5 reverses NMDAR desensitization in Ng–/– mice. Together these findings specifically suggest that accumbal Ng plays an essential role in the counterbalance between NMDAR and mGluR5 signaling; which alters NMDAR resistance, and thereby altering aversive motivation for ethanol and may ultimately contribute to susceptibility for alcohol addiction.

CaMKII-mediated phosphorylation of GluN2B regulates recombinant NMDA receptor currents in a chloride-dependent manner.

Some forms of long-term synaptic plasticity require docking of Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) to residues 1290-1309 within the intracellular C-terminal tail of the N-methyl-d-aspartate (NMDA) receptor GluN2B subunit. The phosphorylation of Ser1303 within this region destabilizes CaMKII binding. Interestingly, Ser1303 is a substrate for CaMKII itself, as well as PKC and DAPK1, but these kinases have been reported to have contradictory effects on the activity of GluN2B-containing NMDA receptors. Here, we re-assessed the effect of CaMKII on NMDA receptor desensitization in heterologous cells, as measured by the ratio of steady-state to peak currents induced during 3s agonist applications. CaMKIIα co-expression or infusion of constitutively active CaMKII limits the extent of desensitization and preserves current amplitude with repeated stimulation of recombinant GluN1A/GluN2B when examined using low intracellular chloride (Cl-) levels, characteristic of neurons beyond the first postnatal week. In contrast, CaMKIIα enhances the acute rate and extent of desensitization when intracellular Cl- concentrations are high. The apparent dependence of CaMKIIα effects on NMDA receptor desensitization on Cl- concentrations is consistent with the presence of a Ca2+-activated Cl- conductance endogenous to HEK 293 cells, which was confirmed by photolysis of caged-Ca2+. However, Ca2+-activated Cl- conductances are unaffected by CaMKIIα expression, indicating that CaMKII affects agonist-induced whole cell currents via modulation of the NMDA receptor. In support of this idea, CaMKIIα modulation of GluN2B-NMDA receptors is abrogated by the phospho-null mutation of Ser1303 in GluN2B to alanine and occluded by phospho-mimetic mutation of Ser1303 to aspartate regardless of intracellular Cl- concentration. Thus, CaMKII-mediated phosphorylation of GluN2B-containing NMDA receptors reduces desensitization at physiological (low) intracellular Cl-, perhaps serving as a feed-forward mechanism to sustain NMDA-mediated Ca2+ entry and continued CaMKII activation during learning and memory.

Homocysteine reduces NMDAR desensitization and differentially modulates peak amplitude of NMDAR currents, depending on GluN2 subunit composition

N-methyl-d-aspartate receptors (NMDARs) have been linked to schizophrenia because agents that bind the receptor, like ketamine and phencyclidine, are capable of inducing schizophrenia-like symptoms. Here we show that the amino acid homocysteine (HCY), which is increased in the blood of schizophrenia patients, reduces desensitization of NMDARs in cultured mouse neurons, human embryonic kidney cells transfected with GluN1 + GluN2A, GluN2B, or GluN2D subunits, and hippocampal slices. HCY also alters the peak amplitude of NMDAR currents, depending on the GluN2 subunit the receptor contains; GluN1 + GluN2A-containing NMDARs show an increase in peak amplitude when exposed to HCY, while GluN1 + GluN2B-containing NMDARs show a decrease in peak amplitude. Both peak amplitude and desensitization effects of HCY can be occluded by saturating the NMDAR with glycine. Since glycine concentrations are not saturating in the brain, HCY could play an NMDAR-modulating role in the nervous system. We also show that HCY shares characteristics with glutamate and suggest that HCY affects both the agonist and co-agonist site of the NMDAR.

Substrate-selective and Calcium-independent Activation of CaMKII by α-Actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.

NMDA Receptor Desensitization Regulated by Direct Binding to PDZ1-2 Domains of PSD-95

Regulation of N-methyl-d-aspartate receptor (NMDAR) activity by desensitization is important in physiological and pathological states; NMDAR desensitization contributes in shaping synaptic responses and may be protective by limiting calcium influx during sustained glutamate insults. We previously reported that glycine-independent desensitization decreases during hippocampal neuronal development, correlating with NMDAR synaptic localization and association with postsynaptic density 95 (PSD-95). PSD-95/Discs large/zona occludens (PDZ)-1,2 domains of PSD-95 bind to the C-terminus of NMDAR NR2 subunits. The role of PSD-95 in anchoring signaling proteins near NMDARs is well documented. To determine if PSD-95-induced changes in NMDAR desensitization occur because of direct binding to NR2 or due to recruitment of regulatory proteins, we tested the effects of various PSD-95 constructs on NMDAR currents in human embryonic kidney 293 (HEK293) cells and neurons. In HEK cells, wild-type PSD-95 significantly reduced wild-type NMDAR desensitization without altering currents of NMDARs containing NR2A-S1462A, a mutation that abolishes PSD-95 binding. The PSD-95 N-terminus truncated after the PDZ1-2 domains was sufficient for this effect in neurons with low endogenous PSD-95 levels; in NMDAR-expressing HEK cells, the effect persisted when PSD-95 multimerization was eliminated. Moreover other PSD-95 family members with highly homologous PDZ1-2 domains significantly reduced NMDAR desensitization. In mature neurons, disruption of PSD-95/NMDAR interaction through protein kinase C (PKC) activation increased desensitization to levels found in immature neurons, and this effect was not due to PKC direct regulation of NMDAR activity. We conclude that direct binding of PSD-95 increases stability of NMDAR responses to agonist exposure in neuronal and nonneuronal cells.

Molecular Determinants of Glycine-Independent Desensitization of NR1/NR2A Receptors

Glycine-independent desensitization is thought to be an important regulatory mechanism for the function of N-methyl-D-aspartate (NMDA) receptors. Previous studies have suggested that the molecular determinants for glycine-independent desensitization are located at two distinct domains of NR2A, i.e., the amino-terminal domain (ATD) and the pre-M1 domain. Since the glycine-independent desensitization described in these earlier studies was a mixture of glycine-independent desensitization and zinc-dependent apparent desensitization, the exact role of these two domains in glycine-independent desensitization remains in question. In the present study, we show that deletion of the ATD of NR2A or mutating the pre-M1 region of NR2A causes no detectable changes in the degree or the time constant of glycine-independent desensitization. Therefore, the ATD and the pre-M1 domain of NR2A play no significant role in glycine-independent desensitization of NR1/NR2A receptors. On the other hand, several residues in the lurcher motif of either NR1 or NR2A are critical for the glycine-independent desensitization of NR1/NR2A receptors. In addition to NR1a(A653T) and NR2A(A651T), NR1a(T648C), NR1a(A649C), NR2A(T646C), and NR2A(A647C) show significantly reduced glycine-independent desensitization. Since all these mutations also alter the proton sensitivity or deactivation time constants of NR1/NR2A receptors, our data suggest that the channel gating and desensitization of NMDA receptors share common molecular determinants and that the lurcher motif of NR1 and NR2A is critical for both processes.

The function of the amino terminal domain in NMDA receptor modulation

N-methyl- d-aspartate (NMDA) receptors are ligand-gated channels important in neurotransmission which are activated by the combined presence of glutamate and glycine. They are comprised of four subunits that form a dimer of dimers. The activity of NMDA receptors is modulated by a variety of endogenous ligands such as zinc ions, phenylethanolamines, polyamines and protons. Findings show that the binding sites for these modulators are found in the amino terminal domain of such receptors, but different modulators appear to affect different subunits. However, despite the enormous efforts expended in mutagenesis and patch clamp experiments on NMDA receptors, the exact assembly of these subunits and the effects of the modulatory species are not well understood. We have modelled dimers of the amino terminal domains of these receptors based on their homology with the extracellular dimer of a metabotropic glutamate receptor. Conserved cysteine residues, which have been highlighted as important in previous work, are shown to form a disulphide bridge, stabilizing a four-helix bundle between subunits. This establishes a hinge in the receptor. The model also highlights a zinc binding site in the binding crevice of the NR2a subunit of the receptor that stabilizes the open state of the amino terminal domain. The similar effect of ifenprodil is thus explained by its stabilization of the open state of the amino terminal domain (ATD). The presence of three histidine residues in the zinc site is used to explain the pH dependence of zinc inhibition. Previous work has also implicated certain residues in spermine stimulation of such receptors. The homology model shows that this site is found at the inter-subunit boundary of the dimer. This predicts a binding site between subunits, a result not calculable by the homology modelling of single subunits done previously. Finally, these results are drawn together to yield a consistent picture of NMDA receptor activation and desensitization. An understanding of how these receptors work and how they can be modulated is an important step toward rational drug design.

Calcineurin acts via the C-terminus of NR2A to modulate desensitization of NMDA receptors

Phosphatase IIb (calcineurin, CaN) can reduce N-methyl- D-aspartate (NMDA) synaptic responses by enhancing glycine-independent desensitization. We examined the action of CaN on desensitization in recombinant NMDA receptors comprised of NMDA receptor 1 (NR1) and NR2A subunits. The C-terminus of NR2A, but not NR1, was critical for modulation of desensitization by CaN. Alanine-scanning mutagenesis indicated that serines 900 and 929 in NR2A altered desensitization, as did inhibition of tyrosine phosphatases. Our data suggest that dephosphorylation-dependent regulation of the C-terminus of NR2A increases desensitization of NMDA receptors, providing an additional mechanism for modulation of synaptic signals.

NMDA receptor-dependent periodic oscillations in cultured spinal cord networks.

Cultured spinal cord networks grown on microelectrode arrays display complex patterns of spontaneous burst and spike activity. During disinhibition with bicuculline and strychnine, synchronized burst patterns routinely emerge. However, the variability of both intra- and interculture burst periods and durations are typically large under these conditions. As a further step in simplification of synaptic interactions, we blocked excitatory AMPA synapses with 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulphonamide (NBQX), resulting in network activity mediated through the N-methyl-D-aspartate (NMDA) receptor (NMDA(ONLY)). This activity was APV sensitive. The oscillation under NMDA(ONLY) conditions at 37 degrees C was characterized by a period of 2.9 +/- 0.3 s (16 separate cultures). More than 98% of all neurons recorded participated in this highly rhythmic activity. The temporal coefficients of variation, reflecting the rhythmic nature of the oscillation, were 3.7, 4.7, and 4.9% for burst rate, burst duration, and interburst interval, respectively [mean coefficients of variation (CVs) for 16 cultures]. The oscillation persisted for at least 12 h without change (maximum observation time). Once established, it was not perturbed by agents that block mGlu receptors, GABA(B) receptors, cholinergic receptors, purinergic receptors, tachykinin receptors, serotonin (5-HT) receptors, dopamine receptors, electrical synapses, burst afterhyperpolarization, NMDA receptor desensitization, or the hyperpolarization-activated current. However, the oscillation was destroyed by bath application of NMDA (20-50 microM). These results suggest a presynaptic mechanism underlying this periodic rhythm that is solely dependent on the NMDA synapse. When the AMPA/kainate synapse was the sole driving force (n = 6), the resulting burst patterns showed much higher variability and did not develop the highly periodic, synchronized nature of the NMDA(ONLY) activity. Network size or age did not appear to influence the reliability of expression of the NMDA(ONLY) activity pattern. For this reason, we suggest that the NMDA(ONLY) condition unmasks a fundamental rhythmogenic mechanism of possible functional importance during periods of NMDA receptor-dominated activity, such as embryonic and early postnatal development.

Competitive inhibition of NMDA receptor-mediated currents by extracellular calcium chelators.

Calcium chelators have been widely used in electrophysiological recordings of N-methyl-D-aspartate (NMDA) receptor-mediated currents, as well as in studies of excitotoxicity. Intracellularly applied calcium chelators are known to inhibit, at least in part, such calcium-dependent processes as calmodulin-dependent inactivation, calcineurin-dependent desensitization, and rundown of NMDA receptors. On the other hand, the functional consequences and potential nonspecific effects of extracellularly applied chelators have not been extensively investigated. In whole-cell patch-clamp recordings from human embryonic kidney (HEK) 293 cells transiently transfected with recombinant NMDA receptors, we found that addition of calcium chelators such as EGTA shifted the glutamate dose-response curve to the right, from an EC(50) for NR1A/NR2A of 8 microM in 1.8 mM Ca(2+) to approximately 24 microM in a solution containing nominal 0 Ca(2+)/5 mM EGTA and further to approximately 80 microM in 20 mM EGTA. A similar shift in glutamate dose-response was observed for NR1A/NR2B currents. This dose-response shift was not due to a decrease in extracellular Ca(2+) concentration because there was no change in the glutamate EC(50) at Ca(2+) concentrations ranging from 10 mM to nominal 0/200 microM EGTA. Moreover, addition of 5 mM EGTA fully chelated with 6.8 mM Ca(2+) did not produce any shift in the glutamate dose-response curve. We propose that calcium chelators, containing four free carboxyl moieties, competitively inhibit glutamate binding to NMDA receptors.

Histamine slows the onset of desensitization of rat cortical NMDA receptors.

The effects of histamine on N-methyl-d-aspartate (NMDA) receptor-mediated ion current in cultured rat cortical neurones have been investigated by superfusion of voltage-clamped (-80 mV) cells with 100 mM NMDA and with various concentrations of histamine. In a subpopulation of the neurones tested (30/53), histamine (10-100 mM) enhanced the peak amplitude, and slowed down the onset of desensitization of the NMDA-induced ion currents, both in a concentration-dependent manner. No effects of histamine on the kinetics of recovery from desensitization were detected. In other neurones (21/53), histamine had no effect on the NMDA-induced currents and in two of the neurones tested histamine potentiated NMDA-induced currents without a detectable effect on desensitization kinetics. The results indicate that in a subpopulation of rat cortical neurones histamine potentiates NMDA-induced ion currents by slowing the kinetics of onset of desensitization.

Modulation of excitatory synaptic transmission by low concentrations of glutamate in cultured rat hippocampal neurons.

1. The effects of low micromolar concentrations of glutamate on fast excitatory synaptic responses were studied in microcultures of postnatal rat hippocampal neurons using whole-cell patch clamp recordings. 2. Glutamate depressed the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor component of excitatory autaptic currents (EACs) with an EC50 of 3.8 microM. 3. Both pre- and postsynaptic effects contributed to the depression of AMPA receptor-mediated EACs. Cyclothiazide and wheatgerm agglutinin, agents which inhibit AMPA receptor desensitization, partially reversed the depression produced by glutamate, as did pertussis toxin, an agent that blocks presynaptic inhibition mediated by metabotropic glutamate receptors. 4. In neurons in which both the AMPA and N-methyl-D-aspartate (NMDA) receptor components of EACs were examined, low concentrations of glutamate depressed the NMDA component of EACs to a greater extent. The EC50 for inhibiting the NMDA component was 1.3 microM. 5. Calcium-dependent desensitization of postsynaptic NMDA receptors contributed to the depression of NMDA receptor-mediated synaptic responses. Both depolarization of postsynaptic neurons to +70 mV to decrease Ca2+ influx via NMDA channels and inclusion of high concentrations of a calcium chelator in recording pipettes decreased the depression of NMDA receptor-mediated EACs. 6. Threo-3-hydroxy-aspartate (THA), an inhibitor of glutamate transport, depressed EACs by about 10% and increased the degree of depression produced by 2.5 microM glutamate, suggesting that glutamate transport in microcultures helps to control ambient glutamate levels. 7. Because the normal extracellular concentration of glutamate is about 1 microM, these results suggest that the ambient glutamate level is an important determinant of synaptic efficacy. Relatively small changes in extracellular glutamate can alter fast excitatory synaptic transmission by both presynaptic and postsynaptic mechanisms.

G‐Protein Modulation of Glycine‐resistant NMDA Receptor Desensitization in Rat Cultured Hippocampal Neurons

Activation of N-methyl-D-aspartate (NMDA) subtype glutamate receptors increases the excitability of most neurons within the CNS. A common feature of ionotropic glutamate receptors is their ability to undergo desensitization. In the present experiments we have examined the role of guanine nucleotide-binding proteins (G-proteins) in the regulation of NMDA receptor desensitization. Repeated NMDA receptor activation with 2 mM extracellular Ca2+ increased the degree of glycine-resistant NMDA receptor desensitization of subsequent responses to NMDA recorded in the presence of 0.2 mM Ca2+. The recovery of glycine-resistant NMDA receptor desensitization after repeated NMDA receptor activation in the presence of 2 mM Ca2+ was significantly reduced in neurons intracellularly dialysed with guanosine-5'-O-(3-thiotriphosphate), guanosine-5'-triphosphate or AlCl3 and CsF, compounds known to activate G-proteins. Intracellular dialysis with guanosine-5'-O-(2-thiodiphosphate), adenosine triphosphate, adenosine triphosphate, or adenosine-5'-O-(3-thiotriphosphate) was ineffective. The calcium permeability of NMDA receptor-channels was not altered by intracellular dialysis with GTP gamma S. This suggests that modulation of NMDA receptor desensitization by G-proteins represents a novel mechanism for regulation of glutamate-gated ion channel activity.