N-methyl-D-aspartate Depolarization Research Articles

Analyses of rapid estrogen actions on rat ventromedial hypothalamic neurons

Rapid estrogen actions are widely diverse across many cell types. We conducted a series of electrophysiological studies on single rat hypothalamic neurons and found that estradiol (E2) could rapidly and independently potentiate neuronal excitation/depolarizations induced by histamine (HA) and N-Methyl-d-Aspartate (NMDA). Now, the present whole-cell patch study was designed to determine whether E2 potentiates HA and NMDA depolarizations – mediated by distinctly different types of receptors – by the same or by different mechanisms. For this, the actions of HA, NMDA, as well as E2, were investigated first using various ion channel blockers and then by analyzing and comparing their channel activating characteristics. Results indicate that: first, both HA and NMDA depolarize neurons by inhibiting K+ currents. Second, E2 potentiates both HA and NMDA depolarizations by enhancing the inhibition of K+ currents, an inhibition caused by the two transmitters. Third, E2 employs the very same mechanism, the enhancement of K+ current inhibition, thus to rapidly potentiate HA and NMDA depolarizations. These data are of behavioral importance, since the rapid E2 potentiation of depolarization synergizes with nuclear genomic actions of E2 to facilitate lordosis behavior, the primary female-typical reproductive behavior.

5-Hydroxytryptamine 5HT2C Receptors Form a Protein Complex with N-Methyl-d-aspartate GluN2A Subunits and Activate Phosphorylation of Src Protein to Modulate Motoneuronal Depolarization

N-Methyl-D-aspartate (NMDA)-gated ion channels are known to play a critical role in motoneuron depolarization, but the molecular mechanisms modulating NMDA activation in the spinal cord are not well understood. This study demonstrates that activated 5HT2C receptors enhance NMDA depolarizations recorded electrophysiologically from motoneurons. Pharmacological studies indicate involvement of Src tyrosine kinase mediates 5HT2C facilitation of NMDA. RT-PCR analysis revealed edited forms of 5HT2C were present in mammalian spinal cord, indicating the availability of G-protein-independent isoforms. Spinal cord neurons treated with the 5HT2C agonist MK 212 showed increased Src(Tyr-416) phosphorylation in a dose-dependent manner thus verifying that Src is activated after treatment. In addition, 5HT2C antagonists and tyrosine kinase inhibitors blocked 5HT2C-mediated Src(Tyr-416) phosphorylation and also enhanced NMDA-induced motoneuron depolarization. Co-immunoprecipitation of synaptosomal fractions showed that GluN2A, 5HT2C receptors, and Src tyrosine kinase form protein associations in synaptosomes. Moreover, immunohistochemical analysis demonstrated GluN2A and 5HT2C receptors co-localize on the processes of spinal neurons. These findings reveal that a distinct multiprotein complex links 5-hydroxytryptamine-activated intracellular signaling events with NMDA-mediated functional activity.

Instrumental role of Na+ in NMDA excitotoxicity in glucose-deprived and depolarized cerebellar granule cells.

In glucose-deprived cerebellar granule cells, substitution of extracellular Na+ with Li+ or Cs+ prevented N-methyl-D-aspartate (NMDA)-induced excitotoxicity. NMDA stimulated 45Ca2+ accumulation and ATP depletion in a Na-dependent manner, and caused neuronal death, even if applied while Na,K-ATPase was inhibited by 1 mM ouabain. The cells treated with NMDA in the presence of ouabain accumulated sizable 45Ca2+ load but most of them failed to elevate cytosolic [Ca2+] upon mitochondrial depolarization. Na/Ca exchange inhibitor, KB-R7943, inhibited Na-dependent and NMDA-induced 45Ca2+ accumulation but only if Na,K-ATPase activity was compromised by ouabain. In cells energized by glucose and exposed to NMDA without ouabain, KB-R7943 reduced NMDA-elicited ionic currents by 19% but failed to inhibit 45Ca2+ accumulation. It appears that a large part of NMDA-induced Ca2+ influx in depolarized and glucose-deprived cells is mediated by reverse Na/Ca exchange. A high level of reverse Na/Ca exchange operation is maintained by a sustained Na+ influx via NMDA channels and depolarization of the plasma membrane. In cells energized by glucose, however, most Ca2+ enters directly via NMDA channels because Na,K-ATPase regenerating Na+ and K+ concentration gradients prevents Na/Ca exchange reversal. Since under these conditions Na/Ca exchange extrudes Ca2+, its inhibition destabilizes Ca2+ homeostasis.

Role of muscarinic receptors, G-proteins, and intracellular messengers in muscarinic modulation of NMDA receptor-mediated synaptic transmission.

Previously, we reported that activation of muscarinic receptors modulates N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission in auditory neocortex [Aramakis et al. (1997a) Exp Brain Res 113:484-496]. Here, we describe the muscarinic subtypes responsible for these modulatory effects, and a role for G-proteins and intracellular messengers. The muscarinic agonist oxotremorine-M (oxo-M), at 25-100 microM, produced a long-lasting enhancement of NMDA-induced membrane depolarizations. We examined the postsynaptic G-protein dependence of the modulatory effects of oxo-M with the use of the G-protein activator GTP gamma S and the nonhydrolyzable GDP analog GDP beta S. Intracellular infusion of GTP gamma S mimicked the facilitating actions of oxo-M. After obtaining the whole-cell recording configuration, there was a gradual, time-dependent increase of the NMDA receptor-mediated slow-EPSP, and of iontophoretic NMDA-induced membrane depolarizations. In contrast, intracellular infusion of either GDP beta S or the IP3 receptor antagonist heparin prevented oxo-M mediated enhancement of NMDA depolarizations. The muscarinic receptor involved in enhancement of NMDA iontophoretic responses is likely the M1 receptor, because the increase was prevented by pirenzepine, but not the M2 antagonists methoctramine or AF-DX 116. Oxo-M also reduced the amplitude of the pharmacologically isolated slow-EPSP, and this effect was blocked by M2 antagonists. Thus, muscarinic-mediated enhancement of NMDA responses involves activation of M1 receptors, leading to the engagement of a postsynaptic G-protein and subsequent IP3 receptor activity.

Effects of hypomagnesia on histamine H1 receptor-mediated facilitation of NMDA responses.

1. The ability of histamine to facilitate the N-methyl-D-aspartate (NMDA) induced depolarization of cortical projection neurones was examined by use of grease-gap recording. 2. Histamine (1 to 15 microM) reversibly facilitated the NMDA-induced depolarization yielding a bellshaped concentration-response relationship. The peak enhancement was 167% above the control at 10 microM histamine. Desensitization was present in 4 out of 5 slices on second exposure 40 min following the first exposure. 3. Histamine did not alter the depolarization induced by 10 microM kainate. 4. The histamine-induced facilitation persisted in the presence of tetrodotoxin, but was reduced in a concentration-dependent manner by diphenhydramine (IC50 = 7.6 nM). Cyproheptadine (10 nM) also reduced the facilitation, whereas ranitidine (200 nM) and thioperamide (10 nM) were ineffective in this regard. 5. Histamine (10 microM) facilitated the NMDA (25 microM)-induced depolarization in nominally Mg(2+)-free medium. The magnitude of the facilitation was smaller than that observed in Mg(2+)-containing medium (17% above the control) and desensitization was not observed. This facilitation was not reduced by cyproheptadine (10 nM) or diphenhydramine (1 microM). 6. We conclude that histamine facilitates the NMDA depolarization at cortical neurones via two distinct mechanisms. One mechanism involves activation of the histamine H1 receptor and is sensitive to Mg2+. The second mechanism is independent of histamine cell surface receptor activation and may reflect a direct action of histamine at the NMDA receptor.

Action of 5-hydroxytryptamine in facilitating N-methyl-D-aspartate depolarization of cortical neurones mimicked by calcimycin, cyclopiazonic acid and thapsigargin.

1. The ability of calcimycin, cyclopiazonic acid and thapsigargin to facilitate the N-methyl-D-aspartate (NMDA)-mediated depolarization of cortical projection neurones was investigated by use of grease-gap recording and the results compared with the facilitation that results from activation of 5-hydroxytryptamine2A receptors. 2. Calcimycin (0.25 to 3 microM), cyclopiazonic acid (5 to 30 microM), and thapsigargin (10 to 300 nM) reversibly facilitated the NMDA (50 microM)-induced depolarization in the presence of tetrodotoxin. The concentration-response relationships were bell-shaped with a mean enhancement of 550% for calcimycin (1 microM) and approximately 400% for cyclopiazonic acid (20 microM) and thapsigargin (100 nM). At the highest concentration of each agent tested, no facilitation was observed. 3. Chlorpromazine (1 microM) partially restored a facilitation at 3 microM calcimycin and 300 nM thapsigargin. Myo-inositol (10 mM) and 100 nM staurosporine were both ineffective in this regard. 4. The depolarization elicited by 10 microM quisqualate or 5 microM kainate was not facilitated by 10 microM cyclopiazonic acid. 5. Calcimycin (0.5 microM), cyclopiazonic acid (20 microM), and thapsigargin (100 nM) elicited a significant facilitation in the presence of an antagonist cocktail consisting of D,L-2-amino-3-phosphonopropionic acid, prazosin, ritanserin, and scopolamine, although the magnitude of the facilitation was reduced. 6. Facilitation of the NMDA depolarization elicited by both 30 microM 5-hydroxytryptamine and 10 microM phenylephrine was eliminated in nominally Mg(2+)-free medium. In contrast, the facilitation induced by 0.5 microM calcimycin remained intact. 7. Bis-(o-aminophenoxy)-ethane-N,N,N,N, tetraacetic acid aminoethoxy (50 microM) or perfusion with nominally Ca(2+)-free medium eliminated facilitation of the NMDA depolarization induced by 30 microM 5-hydroxytryptamine and 100 nM thapsigargin. 8. The facilitation induced by both 30 microM 5-hydroxytryptamine and 1 microM calcimycin was reduced in a concentration-dependent manner by nifedipine (1 to 10 microM). 9. Calcimycin, cyclopiazonic acid and thapsigargin facilitate the NMDA depolarization in a manner which closely mimics the facilitation induced by 5-hydroxytryptamine. It is concluded that enhancement of the NMDA depolarization at cortical projection neurones results from an elevation of Ca2+ in the cytosol and that several sources of Ca2+ contribute to the facilitation.

Characterization of metabotropic glutamate receptor-mediated facilitation of N-methyl-D-aspartate depolarization of neocortical neurones.

1. Facilitation of the N-methyl-D-aspartate (NMDA) receptor-mediated depolarization of cortical neurones induced by metabotropic glutamate receptor (mGluR) agonists in the presence of tetrodotoxin has been examined by use of grease-gap recording. 2. Quisqualate (1-2 microM) and 10 to 100 microM 1S,3R-I-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) facilitated the NMDA-, but not the kainate-induced depolarization with an EC50 of 16 microM for 1S,3R-ACPD. The facilitation induced by quisqualate was reduced, but not blocked, by 4 microM 6-cyano-7-nitroquinoxaline-2,3-dione. 3. D,L-2-Amino-3-phosphonopropionic acid and D,L-2-amino-4-phosphonobutyric acid antagonized the 1S,3R-ACPD facilitation in a non-competitive manner with IC50 values of 0.24 microM and 4.4 microM respectively. 4. Homologous desensitization of the 1S,3R-ACPD induced facilitation was not observed. The facilitation was not altered by 10 nM staurosporine or 3 microM phorbol diacetate. 5. Substitution of 20 microM 8-bromo-cyclic adenosine monophosphate, 20 microM 8-bromo-cyclic guanosine monophosphate, or 10 microM arachidonic acid for 1S,3R-ACPD did not induce facilitation of the NMDA response. However, the 1S,3R-ACPD facilitation was potentiated by 10 mM myo-inositol and exhibited heterologous desensitization following exposure to 100 microM 5-hydroxytryptamine. 6. The 1S,3R-ACPD-induced facilitation persisted in both 10 microM nifedipine and nominally Ca(2+)-free medium and was only gradually eliminated following addition of 100 microM bis-(-o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid in Ca(2+)-free medium. Facilitation of the NMDA response induced by carbachol, but not phenylephrine, was also observed in nominally Ca(2+)-free medium. Perfusing 50 microM bis-(-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid aminoethoxy eliminated the 1S,3R-ACPD facilitation. 7. These experiments have shown that mGluR agonists selectively facilitate the NMDA depolarization of cortical wedges, most likely by activating one or more mGluR subtypes that couple to phospholipase C. We conclude the facilitation results from a Ca(2+)-sensitive mechanism dependent on activation of phospholipase C and release of internal Ca2+. The facilitation is not contingent on activation of protein kinase C or entry of Ca2+ through nifedipine-sensitive Ca2+ channels.

Ca 2+ mobilizing agents mimic serotonin 5-HT 2 A facilitation of N-methyl-D-aspartate depolarization

Serotonin activation of 5-HT 2 A receptors facilitates depolarization of neocortical neurons by N-methyl-D-aspartate (NMDA). Using grease-gap recordings from an in vitro cortical wedge preparation we have examined whether agents which raise the concentration of intracellular Ca 2+ mimic the facilitation. Perfusing A23187, cyclopiazonic acid or thapsigargin selectively facilitate the NMDA depolarization of cortical neurons in a concentration-dependent manner. Buffering intracellular Ca 2+ by perfusing BAPTA-AM eliminates the serotonin, cyclopiazonic acid and thapsigargin induced facilitation. We conclude that a rise in intracellular Ca 2+ is both necessary and sufficient to account for facilitating the NMDA depolarization following activation of 5-HT 2 A receptors

Activation of 5-HT 1C/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord

Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and α-methyl-5-HT (α-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 μM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or α-Me-5-HT (both 100 μM). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of l-glutamate and l-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists N-methyl- d-aspartate (NMDA), quisqualate, α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate. α-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HT-induced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HT 1C/2 antagonist ketanserin and the 5-HT 1A/2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA NMDA-depolarizations. The relatively selective 5-HT 1A antagonist spiroxatrine and the selective 5-HT 3 blocker MDL 72222 were without effect against the depressive effects of 5-HT. These observations support the notion that 5-HT putatively released from descending bulbospinal fibers can alter frog motoneuronal output. The data indicate that 5-HT 1C/2 receptors affect the afferent input to motoneurons and do so by attenuation of NMDA receptor-mediated synaptic processes.

NMDA depolarizations and long-term potentiation are reduced in the aged rat neocortex

N-Methyl- d-aspartate (NMDA) responses were recorded intracellularly in layer V neocortical neurons in in vitro slices taken from young (4–6 months) and aged (27–29 months) Fischer 344 rats. Increasing amounts of NMDA produced membrane depolarizations in both groups of cells. The regression analysis showed significantly reduced sensitivity to NMDA in old neurons compared to young. A significant long-term potentiation of the field potential evoked by subcortical white matter stimulation was present in young but not in old slices. These results suggest that aging results in a decreased sensitivity to NMDA and impaired synaptic plasticity in the neocortex.

A potent antagonist of the strychnine insensitive glycine receptor has anticonvulsant properties

5,7-Dinitro-quinoxaline-2,3-dione (MNQX) displaced [ 3H]glycine binding to cortical membranes but had no effect on [ 3 H]3-((±)-2- carboxypiperazin-4- ly)- propyl-1- phosphonic acid ([ 3 H] CPPP ) binding. MNQX potently antagonized N-methyl-D-aspartate (NMDA)-evoked release of [ 3H]GABA from cultured cortical neurones, NMDA evoked spreading depression and NMDA depolarizations in the rat neo-cortex. All of these responses were reversed by addition of glycine to the perfusion media. These results suggested that MNQX is an antagonist at the strychnine-in-sensitive glycine receptor associated with the NMDA receptor/ionophore complex. Furthermore the compound was found to antagonise audiogenic seizures in DBA-2 mice indicating the potential of glycine antagonists of this type in anticonvulsant therapy.

An intracellular analysis of amino acid induced excitations of deep dorsal horn neurones in the rat spinal cord slice

The rat spinal cord slice preparation has been used to investigate the sensitivity of deep dorsal horn neurones to the excitatory amino acids N-methyl-D-aspartate (NMDA), quisqualate and L-glutamate. Intracellular recordings were made from 44 neurones in laminae III-VI of 14- to 16-day rats. Superfusion of quisqualate (30 microM) excited all neurones, NMDA (50 microM) excited 72% and L-glutamate (0.5-1 mM) 63% of the neurones. Depolarizations were retained after tetrodotoxin but with a reduced amplitude. The NMDA antagonist D-aminophosphonovalerate (D-APV, 10 microM) reduced NMDA and L-glutamate depolarizations by 66% and by 40%, respectively, while the quisqualate responses were enhanced by 27%. Dorsal root stimulation elicited two main patterns of activity; short-latency single/double spikes followed by subthreshold excitatory postsynaptic potentials (EPSPs) or a burst of spikes rising from a long duration composite EPSP. D-APV reduced the long-latency components of the first type and reduced the amplitude and duration of the composite EPSP of the second. These results support a specialized role for NMDA receptors in synaptic transmission in the dorsal horn.

L-Glutamate and [formula omitted] actions on membrane potential and conductance of cat abducens motoneurones

The actions of l-glutamate and N- methyl- d-aspartate (NMDA) were studied with intracellular recordings from cat abducens motoneurones. Amino acids were electrophoresed extracellularly from the same 7-barreled electrode types as those used in the spinal cord. Depolarization development, conductance changes and firing pattern induced by amino acids were very similar to those described for spinal motoneurones. The shape of NMDA depolarizations suggests a uniform distribution of the involved receptors on the membrane of the motoneurone.