Direct inhibition of N-methyl-D-aspartate (NMDA)-receptor function by antiglaucomatous beta-antagonists

In the present study, we investigated the direct effects of antiglaucoma drugs (timolol, betaxolol, pilocarpine, and latanoprost) on N-methyl-D-aspartate (NMDA)-receptor function using a Xenopus oocytes expression system and electrophysiological techniques. In oocytes expressing wild-type NMDA (NR1a/NR2A) receptors, timolol and betaxolol significantly inhibited glutamate-evoked currents, whereas less inhibition was obtained with pilocarpine, and latanoprost had few effects. Moreover, the effect of timolol and betaxolol was noncompetitive with respect to glutamate. Mutations that changed Asn616 of the NR1a subunit, a critical residue for Mg(2+) blocking of NMDA receptors, to Arg (N616R) or Gln (N616Q) almost eliminated the inhibitory effects of timolol and betaxolol, as well as the blocking effect of Mg(2+). Experiments were also carried out to examine the protective effects of timolol and betaxolol against death of oocytes expressing NMDA receptors. During incubation of oocytes, especially in Mg(2+)-free medium, cell death was induced by addition of glutamate because of the continuous activation of the NMDA receptors expressed. Timolol and betaxolol significantly improved oocyte viability when they were added during the incubation period. These results suggest that timolol and betaxolol may have an additional role that they directly inhibit NMDA-receptor function, possibly via N616 of the NR1a subunit.     

Mutations within the selectivity filter of the NMDA receptor-channel influence voltage dependent block by 5-hydroxytryptamine

BACKGROUND AND PURPOSE: Voltage-dependent block by Mg2+ is a cardinal feature of NMDA receptors which acts as a coincidence detector to prevent the receptor from over-activation. Inhibition of NMDA receptor currents by 5-hydroxytryptamine (5-HT) indicated that 5-HT, similar to Mg2+, binds within the membrane electric field. In the present study, we assessed whether point mutations of critical asparagine residues located within the selectivity filter of NR1 and NR2A subunits of NMDA receptor-channel affect voltage-dependent block by 5-HT. EXPERIMENTAL APPROACH: The mode of action of 5-HT and Mg2+ on wild-type and mutated NMDA receptor-channels expressed in Xenopus oocytes was investigated using the two-electrode voltage clamp recording technique. KEY RESULTS: The mutation within the NR1 subunit NR1(N0S or N0Q) strongly reduced the voltage dependent block by 5-HT and increased the IC50. The corresponding mutations within the NR2 subunits NR2A(N0Q or N+1Q) reduced the block by 5-HT to a lesser extent. This is in contrast to the block produced by external Mg2+ where a substitution at the NR2A(N0) and NR2A(N+1) sites but not at the NR1(N0) site significantly reduced Mg2+ block.CONCLUSION AND IMPLICATIONS: The block of NMDA receptor-channels by 5-HT depends on the NR1-subunit asparagine residue and to a lesser extent on the NR2A-subunit asparagine residues. These data suggest that the interaction of 5-HT with functionally important residues in a narrow constriction of the pore of the NMDA receptor-channel provides a significant barrier to ionic fluxes through the open channel due to energetic factors governed by chemical properties of the binding site and the electric field.     

Input-specific targeting of NMDA receptor subtypes at mouse hippocampal CA3 pyramidal neuron synapses

Hippocampal CA3 pyramidal neurons receive synaptic inputs from commissural and associational fibers on both apical and basal dendrites. NMDA receptors at these synapses were examined in hippocampal slices of wild-type mice and GluRvarepsilon1 (NR2A) subunit knockout mice. Electrical stimulations at the CA3 stratum radiatum or stratum oriens activate both commissural and associational (C/A) synapses, whereas stimulations at ventral fimbria mainly activate commissural synapses. Ro 25-6981 and ifenprodil, the GluRepsilon2 (NR2B) subunit-selective NMDA receptor antagonists, suppressed NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) at the commissural-CA3 synapses on basal dendrites more strongly than those at the C/A-CA3 synapses on apical or basal dendrites. However, glutamate-evoked NMDA receptor currents were reduced by the GluRepsilon1 subunit knockout to a similar extent at both apical and basal dendrites. The GluRepsilon1 subunit knockout also reduced NMDA EPSCs at the C/A-CA3 synapses on basal dendrites, but did not affect NMDA EPSCs at the commissural-CA3 synapses on basal dendrites. These results confirmed our previous findings that NMDA receptors operating at different synapses in CA3 pyramidal cells have different GluRepsilon subunit compositions, and further show that the GluRepsilon subunit composition may be regulated depending on the types of synaptic inputs, even within a single CA3 pyramidal neuron.     

Voltage-dependent block of NMDA responses by 5-HT agonists in ventral spinal cord neurones.

1. Modulation by 5-hydroxytryptamine receptor agonists of the NMDA responses of ventral spinal cord neurones was studied by use of the whole-cell patch-clamp technique. 2. In a Mg-free solution containing tetrodotoxin and glycine, 5-hydroxytryptamine (5-HT, 10-100 microM) reduced the NMDA response, the block increasing with hyperpolarization. Kainate responses were little affected. 3. Some classical agonists of 5-HT receptors induced similar blocking effects. At 10 microM, both a selective agonist of 5-HT2 receptors, (+/-)-2,5-dimethoxy-4 iodo amphetamine (DOI), and a selective agonist of some 5-HT1 receptors, (+/-)-8-hydroxy-2(n-dipropyl amino) tetralin (8-OH-DPAT), induced pronounced blocking effects, of 48% and 33% respectively at -100 mV, whereas another 5-HT1 agonist, 5-carboxamidotryptamine (5-CT) was ineffective. At 100 microM, 5-methoxytryptamine (5-MeOT) induced a complete block of the NMDA responses recorded at -100 mV. The order of potency was: 5-MeOT congruent to DOI > 8-OH-DPAT > 5-HT > 5-CT. 4. Neither spiperone nor ketanserin (1 microM) prevented the blocking effect of 5-HT or DOI. 5. Prolonged preincubations with 5-HT did not block the response if NMDA was applied without 5-HT. When 5-HT agonists were applied both by preincubation and with NMDA, the degree of block increased during the NMDA application. 6. Lowering the NMDA concentration (from 100 to 20 microM) slightly decreased the blocking effect of 5-MeOT. 7. External Mg2+ ions (1 mM) also reduced the blocking effects of 5-HT and 5-MeOT. 8. The blocking effects described appear to be independent of classical 5-HT receptors. Their voltage-dependence suggests a mechanism of open channel block consistent with all the results obtained.     

Effects of β-ODAP and its biosynthetic precursor on the electrophysiological activity of cloned glutamate receptors

3-N-Oxalyl- -2,3-diaminopropanoic acid (β-ODAP) induces neurolathyrism, a motor neuron disease. To elucidate the pathogenic mechanism of this process, the action of β-ODAP on the excitatory amino acid (EAA) receptor-mediated currents was examined using cloned EAA receptors expressed in Xenopus oocytes. On the voltage-clamp recordings of an AMPA receptor (₁/₂ heterooligomer), β-ODAP was a strong agonist on this receptor, the potency being almost the same as -glutamate. On the other hand, β-ODAP had little effect on the glutamate-evoked currents through the expressed NMDA receptor (NR1_A/NR2A), but showed a weak inhibitory effect on the glycine-modulatory site. β-ODAP may cause the neurodegenerative disease, neurolathyrism, mainly through the excitotoxic interaction with AMPA receptors.     

Adenosine A2A receptor-induced inhibition of NMDA and GABAA receptor-mediated synaptic currents in a subpopulation of rat striatal neurons

The function of adenosine A(2A) receptors, localized at the enkephalin-containing GABAergic medium spiny neurons of the striatum, has been discussed controversially. Here we show that, in the absence of external Mg(2+), the adenosine A(2A) receptor agonist CGS 21680 postsynaptically depressed the NMDA, but not the non-NMDA (AMPA/kainate) receptor-mediated fraction of the electrically evoked EPSCs in a subpopulation of striatal neurons. Current responses to locally applied NMDA but not AMPA were also inhibited by CGS 21680. However, in the presence of external Mg(2+), the inhibition by CGS 21680 of the GABA(A) receptor-mediated IPSCs led to a depression of the EPSC/IPSC complexes. The current response to the locally applied GABA(A) receptor agonist muscimol was unaltered by CGS 21680. Whereas, the frequency of spontaneous (s)IPSCs was inhibited by CGS 21680, their amplitude was not changed. Hence, it is suggested that under these conditions the release rather than the postsynaptic effect of GABA was affected by CGS 21680. In conclusion, under Mg(2+)-free conditions, CGS 21680 appeared to postsynaptically inhibit the NMDA receptor-mediated component of the EPSC, while in the presence of external Mg(2+) this effect turned into a presynaptic inhibition of the GABA(A) receptor-mediated IPSC.     

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.     

Inhibition of the NMDA response by pregnenolone sulphate reveals subtype selective modulation of NMDA receptors by sulphated steroids

1. The neurosteroid pregnenolone sulphate (PS) potentiates N-methyl-D-aspartate (NMDA) receptor mediated responses in various neuronal preparations. The NR1 subunit can combine with NR2A, NR2B, NR2C, or NR2D subunits to form functional receptors. Differential NR2 subunit expression in brain and during development raises the question of how the NR2 subunit influences NMDA receptor modulation by neuroactive steroids. 2. We examined the effects of PS on the four diheteromeric NMDA receptor subtypes generated by co-expressing the NR1(100) subunit with each of the four NR2 subunits in Xenopus oocytes. Whereas PS potentiated NMDA-, glutamate-, and glycine-induced currents of NR1/NR2A and NR1/NR2B receptors, it was inhibitory at NR1/NR2C and NR1/NR2D receptors. 3. In contrast, pregnanolone sulphate (3alpha5betaS), a negative modulator of the NMDA receptor that acts at a distinct site from PS, inhibited all four subtypes, but was approximately 4 fold more potent at NR1/NR2C and NR1/NR2D than at NR1/NR2A and NR1/NR2B receptors. 4. These findings demonstrate that residues on the NR2 subunit are key determinants of modulation by PS and 3alpha5betaS. The modulatory effects of PS, but not 3alpha5betaS, on dose-response curves for NMDA, glutamate, and glycine are consistent with a two-state model in which PS either stabilizes or destabilizes the active state of the receptor, depending upon which NR2 subunit is present. 5. The selectivity of sulphated steroid modulators for NMDA receptors of specific subunit composition is consistent with a neuromodulatory role for endogenous sulphated steroids. The results indicate that it may be possible to develop therapeutic agents that target steroid modulatory sites of specific NMDA receptor subtypes.     

Generalized loss of inhibitory innervation reverses serotonergic inhibition into excitation in a rabbit model of TNBS-colitis

1. Inflammation may affect subpopulations of neurons of the myenteric plexus. 2. In the present study the effect of trinitrobenzene sulphonic acid (TNBS) induced colitis on nitrergic, purinergic and adrenergic inhibitory neurotransmission was studied as well as the consequences of the related changes on the response of 5-HT agonists using these neurotransmitters to mediate their effect. 3. Strips from normal and colitis rabbits (135 mg kg(-1) TNBS) were subjected to electrical field stimulation (EFS, 0.3 ms, 6V, 0.5 - 32 Hz, 10 s train). The response was measured isometrically in the absence or presence of L-NAME, suramin, guanethidine, the 5-HT agonists (5-HT(1/5A/7): 5-carboxamidotryptamine (5-CT), 5-HT(2): alpha-methyl-5-HT, 5-HT(3): 2-methyl-5-HT, 5-HT(4): 5-methoxytryptamine (5-MeOT)) or a combination. 4. In normal strips L-NAME (1 - 32 Hz), suramin (0.5 - 2, 8 Hz) and guanethidine (4, 16, 32 Hz) increased the response to EFS. This effect was abolished in inflamed strips and was accompanied by a decrease in nNOS expression. 5. In normal strips all 5-HT agonists induced pronounced (5-CT, alpha-methyl-5-HT) or small (2-methyl-5-HT, 5-MeOT) inhibitory neural responses. In inflamed strips this was reversed to cholinergic excitatory responses. 6. The effect of inflammation on the 5-HT(4) response was mimicked by preincubation of normal strips with L-NAME or suramin. Accordingly, in inflamed strips L-NAME or suramin did not affect the excitatory effects of 5-MeOT. 7. TNBS-colitis abolishes nitrergic, purinergic and adrenergic neurotransmission. This reverses serotonergic inhibition into excitation.     

Lack of interaction between nitric oxide and the redox modulatory site of the NMDA receptor

1. The inhibitory effects of nitric oxide (NO) on N-methyl-D-aspartate (NMDA) receptor function have been proposed to be mediated via the interaction of this gas with a redox-sensitive thiol moiety on the receptor. Here, we evaluated this suggested mechanism by examining the actions of various NO donors on native neuronal receptors as well as in wild-type and cysteine-mutated recombinant NMDA receptors expressed in Chinese hamster ovary (CHO) cells. 2. The NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydraxino)ethanamine (NOC-12; 100 microM) produced a rapid and readily reversible inhibition of whole-cell currents induced by NMDA (30 microM) in cultured cortical neurons. The inhibition was apparent at all holding potentials, though a more pronounced block was observed at negative voltages. The effects of NOC-12 disappeared when the donor was allowed to expire. A similar receptor block was observed with another NO-releasing agent, S-nitroso-N-acetylpenicillamine (SNAP; 1 mM). 3. The blocking effects of NO released by SNAP, 3-morpholinosydnonimine (SIN-1; 1 mM), and 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamin e (NOC-5; 100 microM) on currents mediated by recombinant NRI/NR2B receptors were virtually indistinguishable from those observed on native receptors. Furthermore, mutating cysteines 744 and 798 of NR1, which constitute the principal redox modulatory site of the NR1/NR2B receptor configuration, did not affect the inhibition produced by NO. 4. The NR2A subunit may contribute its own redox-sensitive site. However, the effects of NO on NR1/NR2A receptors were very similar to those seen for all other receptor configurations evaluated. Hence, we conclude that NO does not exert its inhibition of NMDA-induced responses via a modification of any of the previously described redox-sensitive sites on the receptor.     

Chronic benzodiazepine administration alters hippocampal CA1 neuron excitability: NMDA receptor function and expression(1)

Rats are tolerant to benzodiazepine (BZ) anticonvulsant actions two days after ending one-week administration of the BZ, flurazepam (FZP). Concurrently, GABA(A) receptor-mediated inhibition is reduced and AMPA receptor-mediated excitation is selectively enhanced in CA1 pyramidal neurons in hippocampal slices. In the present study, the effects of chronic FZP exposure on NMDA receptor (NMDAR) currents were examined in CA1 pyramidal neurons in hippocampal slices and following acute dissociation. In CA1 neurons from chronic FZP-treated rats, evoked NMDAR EPSC amplitude was significantly decreased (52%) in slices, and the maximal current amplitude of NMDA-induced currents in dissociated neurons was also significantly reduced (58%). Evoked NMDAR EPSCs were not altered following acute desalkyl-FZP treatment. Using in situ hybridization and immunohistochemical techniques, a selective reduction in NR2B subunit mRNA and protein expression was detected in the CA1 and CA2 regions following FZP treatment. However, total hippocampal NMDAR number, as assessed by autoradiography with the NMDAR antagonist, [(3)H]MK-801, was unchanged by FZP treatment. These findings suggest that reduced NMDAR-mediated currents associated with chronic BZ treatment may be related to reduced NR2B subunit-containing NMDARs in the CA1 and CA2 regions. Altered NMDAR function and expression after chronic BZ exposure may contribute to BZ anticonvulsant tolerance or dependence.     

Direct pharmacological monitoring of the developmental switch in NMDA receptor subunit composition using TCN 213, a GluN2A-selective, glycine-dependent antagonist

BACKGROUND AND PURPOSE

Developmental switches in NMDA receptor subunit expression have been inferred from studies of GluN2 expression levels, changes in kinetics of glutamatergic synaptic currents and sensitivity of NMDA receptor-mediated currents to selective GluN2B antagonists. Here we use TCN 213, a novel GluN2A-selective antagonist to identify the presence of this subunit in functional NMDA receptors in developing cortical neurones.

EXPERIMENTAL APPROACH

Two-electrode voltage-clamp (TEVC) recordings were made from Xenopus laevis oocytes to determine the pharmacological activity of TCN 213 at recombinant NMDA receptors. TCN 213 antagonism was studied in cultures of primary cortical neurones, assessing the NMDA receptor dependency of NMDA-induced excitotoxicity and monitoring developmental switches in NMDA receptor subunit composition.

KEY RESULTS

TCN 213 antagonism of GluN1/GluN2A NMDA receptors was dependent on glycine but independent of glutamate concentrations in external recording solutions. Antagonism by TCN 213 was surmountable and gave a Schild plot with unity slope. TCN 213 block of GluN1/GluN2B NMDA receptor-mediated currents was negligible. In cortical neurones, at a early developmental stage predominantly expressing GluN2B-containing NMDA receptors, TCN 213 failed to antagonize NMDA receptor-mediated currents or to prevent GluN2B-dependent, NMDA-induced excitoxicity. In older cultures (DIV 14) or in neurones transfected with GluN2A subunits, TCN 213 antagonized NMDA-evoked currents. Block by TCN 213 of NMDA currents inversely correlated with block by ifenprodil, a selective GluN2B antagonist.

CONCLUSIONS AND IMPLICATIONS

TCN 213 selectively blocked GluN1/GluN2A over GluN1/GluN2B NMDA receptors allowing direct dissection of functional NMDA receptors and pharmacological profiling of developmental changes in native NMDA receptor subunit composition.     

Introduction of the 5-HT3B subunit alters the functional properties of 5-HT3 receptors native to neuroblastoma cells

The identification of a second 5-HT(3) (5-HT(3B)) subunit provides an explanation for 5-HT(3) receptor heterogeneity. We investigated whether introduction of recombinant 5-HT(3B) subunits would alter the functional properties of mouse neuroblastoma 5-HT(3) receptors. RT-PCR analysis revealed that NB41A3 cells contain mRNAs encoding 5-HT(3A) and 5-HT(3B) subunits. 5-HT increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and caused the concentration-dependent activation of inward currents recorded at -60 mV. Both actions of 5-HT were antagonized by ondansetron. The 5-HT concentration-response relationship of NB41A3 cells was indistinguishable from that of the related NG108-15 cell line. The selective 5-HT(3)-receptor agonist mCPBG also elevated [Ca(2+)](i) and activated inward currents. 2-M-5HT was less efficacious than 5-HT as an activator of 5-HT(3) receptors in NB41A3 cells and did not significantly increase [Ca(2+)](i). The 5-HT induced increase in [Ca(2+)](i) did not involve caffeine- or thapsigargin-sensitive intracellular Ca(2+) stores. The introduction of the 5-HT(3B) subunit by transient transfection of NB41A3 cells caused 5-HT to become less potent as an activator of 5-HT(3) receptors and altered the kinetics of 5-HT activated currents so that they resembled currents mediated by 5-HT(3AB) receptors. The 5-HT(3B) subunit also abolished the 5-HT induced [Ca(2+)](i) increase seen in untransfected NB41A3 cells. These data are consistent with the hypothesis that NB41A3 cells predominantly express homomeric 5-HT(3A) receptors that become heteromeric 5-HT(3AB) receptors upon introduction of the recombinant 5-HT(3B) subunit.     

Investigation into the 5-hydroxytryptamine-induced atropine-resistant neurogenic contraction of guinea-pig proximal colon.

1. The aim of this study was to characterize the receptors mediating the atropine-resistant neurogenic contraction to 5-hydroxytryptamine (5-HT) in the longitudinal muscle of the guinea-pig proximal colon and to determine the type of tachykinin receptors involved in the contractile response to 5-HT by the use of selective antagonists. 2. In the presence of atropine (0.3 microM), guanethidine (5 microM), hexamethonium (100 microM), ketanserin (0.1 microM) and indomethacin (3 microM), 5-HT (0.01-3 microM) produced concentration-dependent neurogenic contractions of colonic strips and at 0.3 microM produced a maximal effect (pEC50 = 7.39 +/- 0.09, n = 18). The 5-HT4 receptor stimulant, 5-methoxytryptamine (5-MeOT, 0.03-10 microM) also produced neurogenic contractions with similar maximum effect to those of 5-HT (pEC50 = 6.89 +/- 0.16). 3. The 5-HT4 receptor antagonist, DAU 6285 (3 microM) shifted the concentration-response curves to both 5-HT and 5-MeOT to the right without significant depression of the maximum, but the 5-HT1/5-HT2 receptor antagonist, metitepine (0.1 microM) and the 5-HT3 receptor antagonist, ondansetron (0.3 microM) had no effect on the control curves to 5-HT and 5-MeOT. 4. The selective NK1 receptor antagonist, FK 888 (1 microM) markedly attenuated the contractions to 5-HT and 5-MeOT. In contrast, the selective NK2 receptor antagonist, SR 48968 (10 nM) and the selective NK3 receptor antagonist, SR 142801 (10 nM) had no effect on the contractions to 5-HT and 5-MeOT. 5. These results indicate that the 5-HT-induced atropine-resistant neurogenic contraction of guinea-pig proximal colon is due to activation of 5-HT4 receptors, presumably located on excitatory motor neurones, innervating the longitudinal muscle. The contraction evoked by activation of the 5-HT4 receptors is mediated primarily via NK1 receptors but not NK2 or NK3, suggesting that the 5-HT4 receptor-mediated contraction is evoked indirectly via tachykinin release from tachykinin-releasing excitatory neurones.     

Chronic intermittent ethanol treatment selectively alters N-methyl-D-aspartate receptor subunit surface expression in cultured cortical neurons

A chronic intermittent ethanol (CIE) exposure regimen consists of repeated episodes of ethanol intoxication and withdrawal. CIE treatment has been reported to result in a significant enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in vivo, and trafficking of NMDA receptors is emerging a key regulatory mechanism that underlies the channel function. Therefore, in the present study, we examined the effects of CIE on NMDA receptor subunit surface expression. Cultured cortical neurons were exposed to 75 mM ethanol for 14 h followed by 10 h of withdrawal, repeated this cycle five times, and followed by 2 or 5 days of withdrawal. Surface-expressed NMDA receptor subunits and their endocytosis were measured by biotinylation and Western blots. CIE significantly increased NMDA receptor (NR) 1 and NR2B but not NR2A subunit surface expression after 5 days of treatment. However, CIE treatment did not reduce the NMDA receptor endocytosis. Quantification of immunocytochemistry confirmed CIE-induced increase in both the total number of NR1 and NR2B subunit clusters and their targeting to synaptic sites. It is noteworthy that this effect persisted even after ethanol withdrawal with a peak expression occurring between 0 and 2 days after withdrawal, and the expression on the plasma membrane was still at high levels after 5 days of withdrawal. In addition, this was accompanied by significant increases in postsynaptic density protein 95 clusters. Protein kinase A inhibitor completely reversed CIE-induced increase in NR1 and partially in NR2B surface level and a long-lasting effect. These changes may contribute to the development of ethanol-induced neurotoxicity and ethanol dependence.     

Point mutations identify the glutamate binding pocket of the N-methyl-D-aspartate receptor as major site of conantokin-G inhibition.

Conantokin-G (Con-G), a gamma-carboxylglutamate (Gla) containing peptide derived from the venom of the marine cone snail Conus geographus, acts as a selective and potent inhibitor of N-methyl-D-aspartate (NMDA) receptors. Here, the effect of Con-G on recombinant NMDA receptors carrying point mutations within the glycine and glutamate binding pockets of the NR1 and NR2B subunits was studied using whole-cell voltage-clamp recording from cRNA injected Xenopus oocytes. At wild-type receptors, glutamate-induced currents were inhibited by Con-G in a dose-dependent manner at concentrations of 0.1-100 microM. Substitution of selected residues within the NR2B subunit reduced the inhibitory potency of Con-G, whereas similar mutations in the NR1 subunit had little effect. These results indicate a selective interaction of Con-G with the glutamate binding pocket of the NMDA receptor. Homology-based molecular modeling of the glutamate binding region based on the known structure of the glutamate binding site of the AMPA receptor protein GluR2 suggests how selected amino acid side chains of NR2B might interact with specific residues of Con-G.     

Adenosine A2A receptors inhibit the N-methyl-D-aspartate component of excitatory synaptic currents in rat striatal neurons

The effects of the adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS 21680) on currents mediated by excitatory amino acid receptors were examined in rat striatal brain slices. In a Mg(2+)-free superfusion medium, CGS 21680 decreased the amplitude of excitatory postsynaptic currents (EPSCs) in about 70% of striatal neurons. The inhibitory effect of CGS 21680 disappeared both in the presence of the adenosine A(2A) receptor antagonist 8-(3-chlorostyryl) caffeine and the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5). NMDA-induced currents were also depressed by CGS 21680 in a subset of striatal cells, whereas alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced currents were not affected. The results suggest that adenosine A(2A) receptor agonists inhibit the NMDA component of the EPSC.     

NMDA receptor subunit and CaMKII changes in rat hippocampus induced by acute MDMA treatment: a mechanism for learning impairment

Rationale Cognitive deficits have been reported in recreational 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) users. In rats and other animal species, acute MDMA administration produces an impairment in passive avoidance and other learning tasks. Different studies have shown that this learning deficit is not strictly related to the pronounced serotonin (5-HT) depletion induced by the drug.Objectives This study was aimed at determining if acute MDMA administration induces in the rat hippocampus early molecular changes related to memory impairment in a passive avoidance task. The membrane expression of key molecules in memory consolidation, such as the NR1 and NR2B subunits of the N-methyl-D-aspartate (NMDA) receptor, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase 1 (PP1) was measured. Some of these studies were also performed after 5-HT depletion induced by the 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA).Methods Neurochemical studies were performed in rats treated with MDMA and killed 90 min later and also in rats subjected to passive avoidance 30 min after MDMA treatment. Western blotting was used for measuring the levels of NMDA receptor subunits, CAMKII and PP1. Enzyme activity assays were also performed.Results In hippocampal membrane extracts, passive avoidance training increased NMDA receptor NR1 subunit expression as well as CaMKII levels and phosphorylated CaMKII. In untrained rats, MDMA reduced NR1 and NR2B protein levels, membrane CaMKII levels and enzyme activity, and enhanced PP1 levels and activity. In trained rats, MDMA prevented the learning-specific increase in NR1 subunit expression and membrane CaMKII/pCaMKII levels. After pronounced 5-HT depletion by PCPA, MDMA impaired passive avoidance retention to a similar extent and also prevented the training-associated changes in NR1 levels and CaMKII activity.Conclusions Diminished function of hippocampal CaMKII and reduced levels of synaptic NMDA receptor subunits appear to be involved in the impairment of passive avoidance learning induced in rats by acute MDMA treatment.     

Mechanisms intrinsic to 5-HT2B receptor-induced potentiation of NMDA receptor responses in frog motoneurones

In the presence of NMDA receptor open-channel blockers [Mg(2+); (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801); 1-amino-3,5-dimethyladamantane (memantine)] and TTX, high concentrations (30-100 microm) of either 5-hydroxytryptamine (5-HT) or alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT) significantly potentiated NMDA-induced depolarizations of frog spinal cord motoneurones. Potentiation was blocked by LY-53,857 (10-30 microm), SB 206553 (10 microm), and SB 204741 (30 microm), but not by spiroxatrine (10 microm), WAY 100,635 (1-30 microm), ketanserin (10 microm), RS 102221 (10 microm), or RS 39604 (10-20 microm). Therefore, alpha-Me-5-HT's facilitatory effects appear to involve 5-HT(2B) receptors. These effects were G-protein dependent as they were prevented by prior treatment with guanylyl-5'-imidodiphosphate (GMP-PNP, 100 microm) and H-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH(2) (GP antagonist 2A, 3-6 microm), but not by pertussis toxin (PTX, 3-6 ng ml(-1), 48 h preincubation). This potentiation was not reduced by protein kinase C inhibition with staurosporine (2.0 microm), U73122 (10 microm) or N-(2-aminoethyl)-5-isoquinolinesulfonamide HCl (H9) (77 microm) or by intracellular Ca(2+) depletion with thapsigargin (0.1 microm) (which inhibits Ca(2+)/ATPase). Exposure of the spinal cord to the L-type Ca(2+) channel blockers nifedipine (10 microm), KN-62 (5 microm) or gallopamil (100 microm) eliminated alpha-Me-5-HT's effects. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) (100 microm) diminished the potentiation. However, the calcium/calmodulin-dependent protein kinase II (CaM Kinase II) blocker KN-93 (10 microm) did not block the 5-HT enhancement of the NMDA responses. In summary, activation of 5-HT(2B) receptors by alpha-Me-5-HT facilitates NMDA-depolarizations of frog motoneurones via a G-protein, a rise in [Ca(2+)](i) from the entry of extracellular Ca(2+) through L-type Ca(2+) channels, the binding of Ca(2+) to calmodulin and a lessening of the Mg(2+) -produced open-channel block of the NMDA receptor.     

The NR2B subtype of NMDA receptor: a potential target for the treatment of alcohol dependence

Ethanol is a small molecule acting on several neurotransmitter systems in the brain. Accumulating evidences suggest that the primary excitatory--i.e. the glutamatergic--neurotransmitter system is a particularly important site of ethanol's action. Several studies showed that ethanol is a potent and selective inhibitor of the N-methyl-D-aspartate (NMDA) receptors and prolonged ethanol exposition leads to a compensatory "up-regulation" of these receptors resulting in enhanced NMDA receptor-mediated functions after removal of ethanol. These alterations are supposed to contribute to the development of ethanol tolerance, dependence as well as the acute and delayed signs of ethanol withdrawal. In recent papers, alterations in subunit composition of NMDA receptors were reported after long term ethanol exposure. mRNA and/or protein levels of NR2A and NR2B types of subunits were found elevated both by in vivo and in vitro experiments. Our results showed that especially the NR2B subunit expression is increased in cultured hippocampal and cortical neurones after 3 days of intermittent ethanol treatment. According to the high calcium permeability, the increased agonist sensitivity and the relatively slow closing kinetics of NMDA ion channels composed of NR2B subunits, the above mentioned changes may underlie the enhanced NMDA receptor activation observed after long term ethanol exposure. Accordingly, we have tested NR2B subunit selective NMDA receptor antagonists in primary cultures of rat cortical neurones pre-treated with ethanol intermittently for 3 days and found that these compounds potently inhibited the neurotoxic effect of ethanol withdrawal. Hypothesising the involvement of enhanced NR2B subunit expression in development of alcohol dependence and withdrawal symptoms and considering the tolerable side effect profile of the NR2B subunit selective NMDA receptor antagonists, the NR2B type of NMDA receptor subunit may serve as a possible drug target in pharmacological interventions for alcoholism. The aim of this review is to give an update on the role of altered structure and function of NMDA receptors after ethanol exposure and to summarise the recent data about the activity of NR2B subunit selective NMDA receptor antagonists in model systems related to alcoholism.