D-aspartate Glutamate Receptor Research Articles

Short- and long-term enhancement of excitatory transmission in the spinal cord dorsal horn by nicotinic acetylcholine receptors.

Spinal administration of nicotinic agonists can produce both hyperalgesic and analgesic effects in vivo. The cellular mechanisms underlying these behavioral phenomena are not understood. As a possible explanation for nicotinic hyperalgesia, we tested whether nicotinic acetylcholine receptors (nAChRs) could enhance excitatory transmission onto spinal cord dorsal horn neurons. Whole-cell patch-clamp recordings were performed in neonatal rat spinal cord slices. Activation of nAChRs enhanced glutamatergic synaptic transmission in 59% of dorsal horn neurons tested, and this effect was blocked by methyllycaconitine (10 nM), suggesting a key role for alpha7 nAChRs. Inhibition of acetylcholinesterase with methamidophos also enhanced transmission, demonstrating a similar effect of endogenous acetylcholine. nAChR activation also enhanced transmission by dorsal root entry zone stimulation, suggesting that alpha7 nAChRs on the central terminals of DRG afferents mediate this effect. Paired pre- and postsynaptic stimulation induced long-term potentiation of excitatory inputs to some of the dorsal horn neurons. Long-term potentiation induction was much more prevalent when nicotine was applied during stimulation. This effect also depended on both alpha7 nAChRs and N-methyl-d-aspartate glutamate receptors. Our findings demonstrate that alpha7 nAChRs can contribute to both short- and long-term enhancement of glutamatergic synaptic transmission in the spinal cord dorsal horn and provide a possible mechanism for nicotinic hyperalgesia.

Topiramate protects against motor neuron degeneration in organotypic spinal cord cultures but not in G93A SOD1 transgenic mice

Topiramate is a novel anti-convulsant, structurally distinct from other known anti-convulsants. A number of independent studies suggest that topiramate has anti-excitotoxic properties. It has been found to diminish release of glutamate from neurons and block (-amino-3-hydoxy-5-methylisoxazole-4-proprionic acid glutamate receptor evoked currents. Since activation of non- N-methyl- d-aspartate glutamate receptors is thought to play a role in the selective loss of motor neurons in amyotrophic lateral sclerosis (ALS), we determined whether topiramate could protect against chronic glutamate-mediated motor neuron degeneration. An organotypic spinal cord culture system was used in which glutamate transport is inhibited by pharmacological blockade. After 3 weeks of treatment, topiramate was found to significantly prevent motor neuron degeneration in this culture model. However, the drug did not increase survival in G93A SOD1 transgenic mice, an animal model of ALS. These studies suggest that topiramate could be useful as a neuroprotectant, but were not effective in more complex motor injury paradigms such as the mouse model of ALS.

Acute and chronic alterations in calcium homeostasis in 3-nitropropionic acid-treated human NT2-N neurons

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, induced ATP depletion and both necrosis and apoptosis in human NT2-N neurons. Necrosis occurred predominantly within the first two days, and increased in a dose-dependent fashion with the concentration of 3-NP, whereas apoptosis was observed after 24 h or later at a similar rate in 0.1 mM and 5 mM 3-NP. We focused our efforts on intracellular calcium homeostasis during the first 48 h in 1 mM 3-NP, a period during which 10% of the neurons died by necrosis and 3% by apoptosis. All NT2-N neurons showed a stereotyped [Ca 2+] i rise, from 48±2 to 140±12 nM (mean ±S.E.M.), during the first 2 h in 3-NP. Despite severe ATP depletion, however, [Ca 2+] i remained above 100 nM in only 17% and 25% of the NT2-N neurons after 24 and 48 h in 3-NP, respectively, indicating that most neurons were able to recover from acute [Ca 2+] i rise, and suggesting that chronic [Ca 2+] i dysregulation is a better indicator of subsequent necrosis. Blockade of N-methyl- D-aspartate–glutamate receptor by MK-801 substantially ameliorated 3-NP-induced ATP depletion, subsequent chronic [Ca 2+] i elevation, and survival. Moreover, xestospongin C, an inhibitor of endoplasmic reticulum Ca 2+ release, enhanced the capacity of NT2-N neurons to maintain [Ca 2+] i homeostasis and resist necrosis while subjected to sustained energy deprivation. As far as we know, this report is the first to employ human neurons to study the pathophysiology of 3-NP neurotoxicity.

The mechanisms of neuronal death produced by mitochondrial toxin 3-nitropropionic acid: the roles of N-methyl- D-aspartate glutamate receptors and mitochondrial calcium overload

Previous studies showed that 3-nitropropionic acid, an irreversible inhibitor of succinate dehydrogenase, produced neuronal death secondary to perturbed intracellular calcium homeostasis. However, the response of intramitochondrial calcium ([Ca 2+] m) to 3-nitropropionic acid remains unknown. In this study, we investigated the roles of and relationships among [Ca 2+] m overload, mitochondrial reactive oxygen species, and mitochondrial membrane depolarization in 3-nitropropionic acid-induced neuronal death. Following 1 mM 3-nitropropionic acid treatment on primary rat neuronal cultures, there was a gradual increase of [Ca 2+] m beginning at 2–4 h post 3-nitropropionic acid application, and a twofold increase of mitochondrial reactive oxygen species at 4 h. These were followed by mitochondrial membrane depolarization at 6–8 h post-treatment. By inhibiting [Ca 2+] m uptake, Ruthenium Red attenuated the production of reactive oxygen species, and prevented the 3-nitropropionic acid-induced mitochondrial membrane depolarization and 70% of apoptotic neuronal death ( P<0.001). Inhibition of caspase activation attenuated the elevation of [Ca 2+] m ( P<0.001), indicating that caspase activation plays a role in the elevation of [Ca 2+] m. MK-801, an antagonist of N-methyl- D-aspartate (NMDA) glutamate receptors, prevented 3-nitropropionic acid-induced [Ca 2+] m elevation, caspase-3 activation, mitochondrial depolarization, and neuronal death. We conclude that the activation of NMDA glutamate receptor contributes to mitochondrial alterations induced by 3-nitropropionic acid. Inhibition of its activation and [Ca 2+] m overload with subsequent mitochondrial membrane depolarization can therefore attenuate the neuronal death induced by 3-nitropropionic acid.

Sexually dimorphic effects of NMDA receptor antagonism on brain–pituitary–gonad axis development in the platyfish

The N-methyl- d-aspartate glutamate receptor (NMDAR) is found in hypothalamic nuclei involved in the regulation of reproduction in several species of mammals and fishes. NMDAR is believed to affect reproductive development and function by regulating gonadotropin releasing hormone (GnRH)-producing cells. These pathways are likely to be sexually dimorphic, as are several other neurotransmitter systems involved in reproductive function. In this report, male and female platyfish received intraperitoneal injections of 0, 5, 10, 20, 40 or 60 μg/g body wt. of the non-competitive NMDAR antagonist MK-801. Injections began at 6 weeks of age and continued thrice weekly until control animals reached puberty, as evidenced by anal fin maturation. The percent of pubescent animals was significantly affected by sex and treatment, with fewer MK-801-injected females in puberty than control females at each dose ( P<0.001), and fewer pubescent females than males at 10, 20 and 40 μg/g ( P<0.05). There were no MK-801-related effects in males. Histological analyses revealed typical immature gonads and pituitary glands in treated females, and typical mature morphology in control females and all males. Immunocytochemical distribution of the R1 subunit of the NMDAR within the brain-pituitary-gonad (BPG) axis was limited to GnRH-containing brain cells in all animals; however, NMDAR1 distribution was in an immature pattern in treated females and a mature pattern in all others. Neural concentrations of GnRH were unaffected by MK-801 treatment in both sexes. These data suggest that in the platyfish, NMDAR influence on reproductive development is sexually dimorphic and occurs at, or above, the level of GnRH-containing cells of the BPG axis.

Selective antihyperalgesic effect of [Ser 1] histogranin on complete Freund's adjuvant-induced hyperalgesia in rats

The search for alternative pharmacotherapies that target abnormal pain has focused on N-methyl- d-aspartate glutamate receptor (NMDAR) antagonists, since they are efficacious in various chronic pain models. However, adverse effects of currently available agents limit their therapeutic usefulness. The naturally derived NMDAR antagonist peptide, histogranin, is thought to interact at a novel site on the NMDAR subunit. Previous studies in our laboratory have suggested the potential for histogranin analogs to attenuate neuropathic pain. The ability of this peptide derivative to reduce inflammatory pain was evaluated in the present study. The effect of intrathecal (i.t.) injection of the stable analog [Ser 1] histogranin (SH) was evaluated in rats with a unilateral hind paw inflammation. Following injection of complete Freund's adjuvant into the hind paw, responsiveness to noxious thermal and mechanical stimuli were greatly enhanced (hyperalgesia). The i.t. injection of SH partially attenuated mechanical hyperalgesia for up to 2 h post-injection, with no effect on withdrawal thresholds of the non-inflamed paw. In contrast, SH had no effect on thermal hyperalgesia. No attendant motor abnormalities were noted. These results indicate that SH has selective and modest antinociceptive effects on inflammatory pain and suggests that novel histogranin analogs may be safe and useful adjuncts in the management of chronic pain.

Presynaptic N-methyl- d-aspartate receptor activation inhibits neurotransmitter release through nitric oxide formation in rat hippocampal nerve terminals

In brain synapses, nitric oxide synthase activation is coupled to N-methyl- d-aspartate-mediated calcium entry at postsynaptic densities through regulatory protein complexes, however a presynaptic equivalent to this signaling mechanism has not yet been identified. Novel evidence indicates that N-methyl- d-aspartate glutamate receptors may play a presynaptic role in synaptic plasticity. Thus, we investigated whether ionotropic glutamate receptor activation in isolated nerve terminals regulates neurotransmitter release, through nitric oxide formation. N-Methyl- d-aspartate dose-dependently inhibited the release of glutamate evoked by 4-aminopyridine (IC 50=155 μM), and this effect was reversed by the N-methyl- d-aspartate receptor antagonist d-(−)-2-amino-5-phosphopentanoic acid and by the nitric oxide synthase inhibitor, l-nitroarginine, in synaptosomes isolated from whole hippocampus, CA3 and CA1 areas, but not from the dentate gyrus. In contrast, the 4-aminopyridine-evoked release of glutamate was reduced by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate by a nitric oxide-independent mechanism, since it was not blocked by l-nitroarginine, and N-methyl- d-aspartate, but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate, significantly increased cGMP formation. Presynaptic N-methyl- d-aspartate receptors are probably involved since removing extracellular nitric oxide with the scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide did not block the depression of glutamate release by N-methyl- d-aspartate. The mechanism underlying this depression involves the inhibition of synaptic vesicle exocytosis since N-methyl- d-aspartate/nitric oxide inhibited the release of [ 3H]glutamate and [ 14C]GABA evoked by hypertonic sucrose. The results also suggest that presynaptic N-methyl- d-aspartate receptors may function as auto- and heteroreceptors.

Electrophysiology and pharmacology of projections from the suprachiasmatic nucleus to the ventromedial preoptic area in rat

Extracellular and whole-cell patch-clamp recordings were made from neurons in the ventromedial preoptic area in rat horizontal brain slices. Responses to single-pulse electrical stimulation of the ipsilateral suprachiasmatic nucleus were characterized using peristimulus time histograms or postsynaptic current recordings, and bath application of neurotransmitter receptor antagonists. Extracellular recordings showed that suprachiasmatic nucleus stimulation (50–150 μA) elicited a short-latency suppression in 35 of 64 neurons (55%), with the majority (29/35, 83%) showing a biphasic response consisting of a short-latency suppression followed by a long-duration activation. In addition, 14 cells (22%) showed activation only, while 15 (23%) were unresponsive. The GABA A receptor antagonist bicuculline (5–10 μM) reversibly blocked suppressions evoked by suprachiasmatic nucleus stimulation (20/20 cells). In the majority of these neurons (13/20), bicuculline also unmasked an activation in response to stimulation. Activations elicited by suprachiasmatic nucleus stimulation, either in the presence or absence of bicuculline, were blocked by the non- N-methyl- d-aspartate and N-methyl- d-aspartate glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (10/10 cells). 6,7-Dinitroquinoxaline-2,3-dione (10 μM) selectively and reversibly blocked the initial, short-duration (<50 ms) activation, but had no effect on the longer-duration activation. In contrast, (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (10 μM) appeared to inhibit the long-duration activation selectively without affecting the initial rapid activation. Combined applications of the two ionotropic glutamate receptor antagonists blocked stimulation-induced activations completely. All the pharmacological effects were concentration dependent. Whole-cell patch-clamp recordings showed that suprachiasmatic nucleus stimulation elicited inhibitory postsynaptic currents or a combination of inhibitory and excitatory postsynaptic currents in 25 of 33 neurons tested. The inhibitory postsynaptic currents had short onset latencies (4.9±0.3 ms) and a reversal potential of −56.0±3.8 mV ( n=5), and were reversibly blocked by bicuculline (5–10 μM, 4/4 cells). In the presence of bicuculline (5–10 μM), excitatory postsynaptic currents had short onset latencies (4.7±0.5 ms), and had a fast and a slow component. (±) 3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid blocked the slow, but not the fast, component, whereas 6,7-dinitroquinoxaline-2,3-dione blocked the fast, but not the slow, component ( n=7). These results suggest that the projection from the suprachiasmatic nucleus conveys both inhibitory (GABA) and excitatory (glutamate) inputs to the ventromedial preoptic area. GABA A receptor and both non- N-methyl- d-aspartate and N-methyl- d-aspartate glutamate receptors mediate these influences. These inputs may be responsible for conveying information related to circadian phase from the suprachiasmatic nucleus to regions of the preoptic area known to be involved in regulation of sleep/waking and other physiological functions.

Changes in expression of N-methyl- d-aspartate receptor subunits in the rat neostriatum after a single dose of antisense oligonucleotide specific for N-methyl- d-aspartate receptor 1 subunit

In order to investigate the process of gene expression of N-methyl- d-aspartate glutamate receptor (NMDAR) subunits in the rat neostriatum and how this relates to motor behaviors, a single dose of antisense phosphodiester oligodeoxynucleotide specific for NMDAR1 was unilaterally applied in the neostriatum in a stereotaxic apparatus. After one day of antisense treatment, ipsilateral rotation behaviors that were induced by apomorphine were found in the treated animals. Reductions in the levels of expression of NMDAR1 and NMDAR2A messenger RNAs (NMDAR1: 20.6%; NMDAR2A: 19.7%) were found in the antisense-treated striatal tissues by reverse transcriptase–polymerase chain reaction. There was no change in the levels of NMDAR2B, NMDAR2C and NMDAR2D messenger RNAs. After two days, western blotting experiments showed that there were decreases in the levels of expression of NMDAR1 (decreased 27.6%) and NMDAR2A (decreased 19.2%) proteins in the NMDAR1 antisense-treated striatal tissues. In addition, NMDAR1 immunoreactivity was found to decrease in intensity in the NMDAR1 antisense-treated neostriatum. At the cellular level, the intensity of NMDAR1 immunoreactivity in perikarya of presumed medium spiny neurons was found to decrease. These results indicate that a single dose of NMDAR1 antisense modifies the expression of NMDAR1 messenger RNA and protein in neurons in the neostriatum. The modification in the expression of NMDAR1 has differential effects in the expression of NMDAR2 subunits. Gene expression of the native NMDAR subunits is likely to be a dynamic process. The change in gene expression of the NMDAR subunits in the neostriatum may have a profound effect on the motor behaviors of rats.

Non- N-methyl- d-aspartate glutamate receptors mediate oxygen–glucose deprivation-induced oligodendroglial injury

Cells of oligodendroglial lineage are susceptible to oxygen and glucose deprivation. When oligodendrocyte-like cells differentiated from CG-4-immortalized rat O-2A progenitor cells were exposed to hypoxia alone or glucose deprivation alone for 48 h, release of lactate dehydrogenase (LDH) into the culture medium did not increase. However, when cells were deprived of both oxygen and glucose for 6 or 12 h preceding reoxygenation for 2 h, LDH release increased. Adding glucose to the medium protected against cell death and increased lactate production in a concentration-dependent manner. Cell damage induced by deprivation of oxygen and glucose was prevented by calcium-free medium or by non- N-methyl- d-aspartate glutamate receptor (GluR) antagonists, such as 6-cyano-7-nitroquinoxaline-2,3-dione or LY293558, but not by the voltage-dependent calcium channel blocker, nimodipine, or by the N-methyl- d-aspartate GluR antagonist, MK-801. The glutamate concentration in the medium from cells exposed to oxygen–glucose deprivation for 12 h was 49.70±3.04 μM/l, which is sufficient to activate GluRs during deprivation of oxygen and glucose. Apoptotic cells detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) or Hoechst 33258 staining did not increase in cells exposed to oxygen–glucose deprivation for 12 h and subsequent reoxygenation for 2 h. No DNA laddering was detected by agarose gel electrophoresis from cells exposed to deprivation of oxygen and glucose. Neither acetyl-YVAD-CHO, an inhibitor of caspase-1-like proteases, nor acetyl-DEVD-CHO, an inhibitor of caspase-3-like proteases, prevented oxygen–glucose deprivation-induced injury. Thus, oxygen and glucose deprivation causes calcium-influx-induced necrotic cell damage in cells of oligodendroglial lineage via non- N-methyl- d-aspartate GluR channels.

Glutamate-induced increase of extracellular glutamate through N-methyl- d-aspartate receptors in ethanol withdrawal

Ethanol withdrawal is a physiopathological state associated with increased number and function of N-methyl- d-aspartate glutamate receptors. We assessed the effect of N-methyl- d-aspartate receptor stimulation on the extracellular levels of glutamate in vivo by the focal application of N-methyl- d-aspartate in the striatum of dependent rats following withdrawal from chronic treatment with ethanol. In control, chronic sucrose-treated rats, 800 μM N-methyl- d-aspartate increased glutamate levels to 268% of baseline values. In ethanol-withdrawn animals, 12 h after interruption of the chronic treatment, the application of N-methyl- d-aspartate increased glutamate levels to 598% of baseline values. In ethanol-intoxicated rats N-methyl- d-aspartate was ineffective. Concentration–response curves showed that in ethanol withdrawn animals N-methyl- d-aspartate was five-fold more potent than in controls. In withdrawn animals, the non-competitive N-methyl- d-aspartate receptor antagonist dizocilpine (1.0 mg/kg i.p.) or ethanol (5 g/kg i.g.) markedly reduced the N-methyl- d-aspartate-induced increase in glutamate levels. These results are consistent with the up-regulation of N-methyl- d-aspartate receptors by chronic ethanol and add biochemical evidence for the presence of N-methyl- d-aspartate receptors facilitating glutamate release through a positive feedback mechanism. The glutamate-induced, N-methyl- d-aspartate receptor-mediated elevations of extracellular glutamate may constitute a neurochemical substrate for the neuropathological alterations associated with alcoholism.

A role for nucleus accumbens glutamate transmission in the relapse to cocaine-seeking behavior

This study investigated the effect of ionotropic glutamate receptor agonist or antagonist administration into the nucleus accumbens on the maintenance of cocaine self-administration and the reinstatement of cocaine-seeking behavior. The stimulation of α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid or N-methyl- d-aspartate glutamate receptors in the nucleus accumbens with either α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid or 1-aminocyclobutane- cis-1,3-dicarboxylic acid, respectively, decreased the number of cocaine-reinforced responses, suggesting an enhancement in the rewarding properties of cocaine. In contrast, blockade of α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptors with N-methyl- d-aspartate, or N-methyl- d-aspartate receptors with dizocilpine maleate or 2-amino-5-phosphonovaleric acid had no selective effect on the maintenance of cocaine self-administration. Following one week of extinction from the reinforcing cue of the drug-paired lever, both α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid and 1-aminocyclobutane- cis-1,3-dicarboxylic acid treatment in the nucleus accumbens reinstated cocaine-seeking behavior. However, α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid treatment increased responding only on the drug-paired lever, while 1-aminocyclobutane- cis-1,3-dicarboxylic acid increased responding on both the drug-paired and non-drug-paired levers. These results suggest that stimulation of glutamate receptors in the nucleus accumbens augments the reinforcing effect of cocaine, yet glutamate transmission is not required to maintain cocaine self-administration. In addition, increased glutamate transmission in the nucleus accumbens may be involved in facilitating the relapse to cocaine-seeking behavior.

Time dependence and role of N-methyl- d-aspartate glutamate receptors in the priming of D2-mediated rotational behavior and striatal Fos expression in 6-hydroxydopamine lesioned rats

Administration of dopamine agonists to 6-hydroxydopamine (6-OHDA) lesioned rats enhances the rotational response to subsequent administration of dopamine agonist, an effect called `priming'. Previously, we have shown that 6-OHDA rats primed with three injections of the D1/D2 dopamine agonist apomorphine (0.5 mg/kg) permitted a challenge with an otherwise inactive dose of the D2 agonist quinpirole (0.25 mg/kg) to elicit robust rotational behavior and to induce Fos expression in striatoentopeduncular neurons. In this study, the time-course and role of N-methyl- d-aspartate (NMDA) glutamate receptors on apomorphine-priming of these D2 responses were investigated. The enhanced rotational behavior and striatal Fos expression observed following challenge with quinpirole (0.25 mg/kg) peaked 1 day following the third apomorphine priming injection and persisted, in reduced form, for at least 4 months. Pretreatment with the NMDA antagonists MK-801 or 3-[(+)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate (CPP) dose-dependently attenuated apomorphine-priming of quinpirole-mediated rotational behavior and striatal Fos induction compared to 6-OHDA rats primed with apomorphine alone. Taken together, these data suggest that priming of these D2-mediated responses in 6-OHDA rats develops rapidly, persists for several months, and is dependent on concomitant NMDA receptor stimulation. Since this priming effect resembles response fluctuations observed in patients with Parkinson's disease receiving long-term l-dihydroxyphenylalanine therapy, the results of the present study suggest that interventions that prevent the development of this enhanced response, such as NMDA antagonists, could prove useful in reducing the incidence these response fluctuations.

Dynamic properties of corticothalamic excitatory postsynaptic potentials and thalamic reticular inhibitory postsynaptic potentials in thalamocortical neurons of the guinea-pig dorsal lateral geniculate nucleus

The properties of postsynaptic potentials evoked by stimulation of cortical, retinal and GABAergic thalamic afferents were examined in vitro in thalamocortical neurons of the guinea-pig dorsal lateral geniculate nucleus. Brief trains of stimulation (2–10 stimuli) delivered to corticothalamic fibers led to a frequency-dependent increase in excitatory postsynaptic potential amplitude associated with an increase in activation of both N-methyl- d-aspartate and non- N-methyl- d-aspartate glutamate receptors. In addition, repetitive stimulation of corticothalamic fibers also gave rise to a slow excitatory postsynaptic potential that was blocked by local application of the glutamate metabotropic receptor antagonist α-methyl-4-carboxyphenylglycine. In contrast, repetitive stimulation of optic tract fibers resulted in monosynaptic excitatory postsynaptic potentials that did not potentiate and were not followed by the generation of a slow excitatory postsynaptic potential. Repetitive activation of the optic radiation also evoked both GABA A and GABA B receptor-mediated inhibitory postsynaptic potentials. These inhibitory postsynaptic potentials exhibited frequency-dependent depression during repetitive activation. The presence of frequency-dependent facilitation of corticothalamic excitatory postsynaptic potentials and frequency-dependent decrement of inhibitory postsynaptic potentials, as well as the ability of corticothalamic fibers to activate glutamate metabotropic receptors, suggests that sustained activation of corticothalamic afferents in vivo may result in postsynaptic responses in thalamocortical cells that are initially dominated by GABAergic inhibitory postsynaptic potentials followed by prominent monosynaptic excitatory postsynaptic potentials as well as a slow depolarization of the membrane potential. Therefore, the corticothalamic system may inhibit or enhance the excitability and responsiveness of thalamocortical neurons, based both on the spatial and temporal features of thalamocortical interactions.

Trigeminovascular nociceptive transmission involves N-methyl- d-aspartate and non- N-methyl- d-aspartate glutamate receptors

Interest in the fundamental mechanisms underlying headache, particularly the pathophysiology of migraine and cluster headache, has lead to the study of the physiology and pharmacology of the trigeminovascular system and its central ramifications. Cats were anaesthetized (60 mg/kg α-chloralose, i.p., along with halothane for all surgical procedures) and prepared for physiological monitoring. The animals were placed in a stereotaxic frame and ventilated. A midline craniotomy and C 2 laminectomy were performed for access to the superior sagittal sinus and C 2 dorsal horn, respectively. The sinus was isolated from the underlying cortex and stimulated electrically after the animals had been paralysed with gallamine (6 mg/kg, i.v.). Units linked to stimulation were recorded with a tungsten-in-glass microelectrode placed in the most caudal part of the trigeminal nucleus, the trigeminocervical complex. Signals from the neurons were amplified, filtered and passed to a microcomputer, where post-stimulus histograms were constructed on-line to analyse the responses to stimulation. Units responded to sagittal sinus stimulation with a typical latency of 8–10 ms. All units studied had a probability of firing of 0.6 or greater. Intravenous injection of the non-competitive N-methyl- d-aspartate receptor antagonist, dizocilpine maleate (4 mg/kg, i.v.), resulted in a substantial and prolonged blockade of firing of units in the trigeminocervical complex. Similarly, administration of the non- N-methyl- d-aspartate excitatory amino acid receptor blocker, GYKI 52466, lead to a dose-dependent inhibition of trigeminovascular-evoked responses in the trigeminocervical complex. These data demonstrate the participation of both N-methyl- d-aspartate- and non- N-methyl- d-aspartate-mediated mechanisms in transmission within the trigeminocervical complex, and suggest a clear pre-clinical role of glutamatergic mechanisms in primary headache syndromes, such as migraine and cluster headache.

Co-expression of TrkB and the N-methyl- d-aspartate receptor subunits NR1-C1, NR2A and NR2B in the rat visual cortex

In the visual cortex, brain-derived neurotrophic factor expression is modulated through glutamate receptors, including the N-methyl- d-aspartate glutamate receptor. It has been proposed that the N-methyl- d-aspartate glutamate receptor subunit composition itself might be regulated by brain-derived neurotrophic factor. Here, we investigated the co-expression of the neurotrophin-4/brain-derived neurotrophic factor receptor TrkB with the N-methyl- d-aspartate glutamate receptor subunits NR1-C1, NR2A and NR2B, on postnatal days 10 and 22 and in the adult rat primary visual cortex. At both postnatal days 10 and 22, TrkB is co-expressed in all cortical layers with the studied N-methyl- d-aspartate glutamate receptor subunits. In the adult, in layers IV–V, co-expression is restricted to a subpopulation of neurons, while in layers II–III, VI nearly all neurons co-express TrkB with NR1-C1, NR2A and NR2B. We conclude that in layers IV–V, the co-expression of TrkB with subunits NR2B and NR2A is developmentally regulated.

Effects of N-methyl- d-aspartate (dizocilpine) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (LY215490) receptor antagonists on the voiding reflex induced by perineal stimulation in the neonatal rat

The present study was undertaken to examine the role of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl- d-aspartate glutamate receptors in the regulation of voiding reflexes induced by perineal stimulation in the neonatal rat. Four-, six- and 10-day-old awake rats were used in the experiments and perineal stimulation was applied using the tip of a 1-ml syringe to evoke voiding. Voided volume and residual volume were measured. In 10-day-old rats, LY215490 (3–10 mg/kg, i.p.), a competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist, significantly inhibited reflex voiding, increasing the residual volume 34–53-fold. A 3 mg/kg dose decreased the urine release by 55%, whereas 10 mg/kg totally suppressed the voiding reflex induced by the perineal stimulation. LY215490 (10 mg/kg, i.p.) produced similar effects in four- and six-day-old rats. Dizocilpine (1–3 mg/kg, i.p.), a non-competitive N-methyl- d-aspartate receptor antagonist, also significantly decreased the urine release (62–82%) and increased residual volume (180–230-fold). Combined administration of LY215490 (1 mg/kg, i.p.) and dizocilpine (0.3 mg/kg, i.p.) to 10-day-old rats, in doses that individually had no effect on perineal stimulation-evoked voiding, depressed voided volume by 65%. These results indicate that, in neonatal rats, glutamatergic transmission in the spinal cord has an essential role in reflex micturition induced by perineal stimulation, and that facilitatory interactions between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl- d-aspartate glutamatergic mechanisms are important for voiding, as noted previously in adult rats.

Accurate quantification of the mRNA of NMDAR1 splice variants measured by competitive RT-PCR

N-methyl- d-aspartate receptors (NMDAR) belong to the subclass of ionotropic glutamate receptors and are widely distributed in the vertebrate brain. Molecular cloning has revealed the existence of six NMDAR subunits: one NMDAR1 (NR1), four different NMDAR2 (NR2A–D) and one NMDAR3A (NR3A). Alternative splicing of the single NR1 gene generates eight isoforms with distinct functional properties [M. Hollmann, J. Boulter, C. Maron, L. Beasley, J. Sullivan, G. Pecht, S. Heinemann, Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor, Neuron 10 (1993) 943–954 [8]; R.S. Zukin, M.V.L. Bennett, Alternatively spliced isoforms of the NMDAR1 receptor subunit, TiNS 18 (1995) 306–313 [20]]. Despite the progress made in the functional analysis of NMDARs the molecular architecture of this receptor remains to be elucidated. In situ hybridization studies have already indicated that splicing of the NR1 gene is regionally regulated in the rodent brain, which may contribute to functional diversity of NMDARs in distinct brain areas [D.J. Laurie, P.H. Seeburg, Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA, J. Neurosci. 14 (1994) 3180–3194 [10]; D.G. Standaert, C.M. Testa, A.B. Young, J.B. Penney Jr., Organization of N-methyl- d-aspartate glutamate receptor gene expression in the basal ganglia of the rat, J. Comp. Neurology 343 (1994) 1–16 [18]; M. Hollmann, S. Heinemann, Cloned glutamate receptors, Ann. Rev. Neurosci. 17 (1994) 31–108 [9]]. Since in situ hybridization techniques do not allow accurate quantification of distinct NR1 splice variants and are also very time-consuming, an accurate and sensitive competitive RT-PCR assay was developed. This method was then used to study the distribution of three NR1 splice variants in the rat brain, and the results are compared with former in situ hybridization studies. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Excitatory amino acid receptors: structure, function and expression

Dizocilpine maleate, MK-801, but not 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline, NBQX, prevents transneuronal degeneration of nigral neurons after neurotoxic striatal–pallidal lesion

Unilateral neurotoxin lesion of rat caudate–putamen and globus pallidus resulted in delayed, transneuronal degeneration of GABAergic substantia nigra pars reticulata neurons. To explore whether the disinhibition of endogenous glutamate excitatory input played a role in the degeneration of substantia nigra pars reticulata neurons, animals with unilateral striatal–pallidal lesions received three daily intraperitoneal injections of either dizocilpine maleate (MK-801, 1 or 10 mg/kg), an N-methyl- d-aspartate glutamate receptor blocker, or 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX, 30 mg/kg), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor blocker, that began 24 h after the striatal–pallidal neurotoxin lesion. Drug treatment affected neither the volume of the initial lesion nor the volume of striatal–pallidal glial fibrillary acidic protein immunoreactivity. Neuron number in the substantia nigra pars reticulata ipsilateral to the lesioned striatopallidum was reduced on average by 37% in untreated control rats, in low dose MK-801, and NBQX-treated rats ( P<0.0001). However, in animals treated with high doses of MK-801 there was no difference in the number of neurons in the substantia nigra pars reticulata ipsilateral or contralateral to the neurotoxin lesion. These data demonstrate that dose-related treatment with N-methyl- d-aspartate glutamate receptor blockers protects substantia nigra pars reticulata neurons, and suggests that glutamatergic mechanisms play a role in delayed transneuronal degeneration.

Cellular mechanisms underlying spontaneous interictal spikes in an acute model of focal cortical epileptogenesis

The cellular mechanisms involved in the generation of spontaneous epileptiform potentials were investigated in the pirifom cortex of the in vitro isolated guinea-pig brain. A single, unilateral injection of bicuculline (150–200 nmol) in the anterior piriform cortex induced locally spontaneous interictal spikes that recurred with a period of 8.81±4.47 s and propagated caudally to the ipsi- and contralateral hemispheres. Simultaneous extra- and intracellular recordings from layer II and III principal cells showed that the spontaneous interictal spike correlates to a burst of action potentials followed by a large afterdepolarization. Intracellular application of the sodium conductance blocker, QX-314 (80 mM), abolished bursting activity and unmasked a high-threshold slow spike enhanced by the calcium chelator EGTA (50 mM). The slow spike was abolished by membrane hyperpolarization and by local perfusion with 2 mM cadmium. The depolarizing potential that followed the primary burst was reduced by arterial perfusion with the N-methyl- d-aspartate receptor antagonist, dl-2-amino-5-phosphonopentanoic acid (100–200 μM). The non- N-methyl- d-aspartate glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (20 μM), completely and reversibly blocked the spontaneous spikes. The interictal spikes were terminated by a large afterpotential blocked either by intracellular QX-314 (80 mM) or by extracellular application of phaclofen and 2-hydroxysaclofen (10 and 4 mM, respectively). The present study demonstrates that, in an acute model of epileptogenesis, spontaneous interictal spikes are fostered by a primary burst of fast action potentials that ride on a regenerative high-threshold, possibly calcium-mediated spike, which activates a recurrent, glutamate-mediated potential responsible for the entrainment of adjacent and remote cortical regions. The bursting activity is controlled by a GABA B receptor-mediated inhibitory synaptic potential.