Abstract: Excitotoxic neuronal death, associated with neurodegenerative disorders and hypoxic insults, results from excessive exposure to excitatory neurotransmitters. Glutamate neurotoxicity is triggered primarily by massive Ca2+ influx arising from overstimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondria are primary targets in excitotoxicity by confocal imaging of intracellular Ca2+([Ca2+]i) and mitochondrial membrane potential (ΔΨ) on cultured rat hippocampal neurons. Sustained activation of NMDA receptors (20 min) elicits reversible elevation of [Ca2+]i. Longer activation (50 min) renders elevation of [Ca2+]i irreversible (Ca2+ overload). Susceptibility to NMDA-induced Ca2+ overload is increased when the 20 min stimuli are applied to neurons pretreated with electron transport chain inhibitors, thereby implicating mitochondria in [Ca2+]ihomeostasis during excitotoxic challenges. Remarkably, ΔΨ exhibits prominent and persistent depolarization in response to NMDA, which closely parallels the incidence of neuronal death. Blockade of the mitochondrial permeability transition pore by cyclosporin A allows complete recovery of ΔΨ and prevents cell death. These results suggest that early mitochondrial damage plays a key role in induction of glutamate neurotoxicity.

Abstract: In many regions of the cerebral cortex, Ca2+ influx through NMDA (N-methyl-D-aspartate) sensitive glutamate receptors (NMDA receptors) can trigger two forms of synaptic plasticity: long-term depression (LTD) and long-term potentiation (LTP). LTD is induced by low levels of postsynaptic NMDA-receptor activation, for instance in response to low-frequency stimulation, whereas LTP is induced by the stronger activation that occurs following high-frequency stimulation. Theoretical studies have shown that the properties of synaptic LTD and LTP can account for many aspects of experience-dependent plasticity in the developing visual cortex, provided that the LTD-LTP crossover point (the modification threshold, theta(m)) varies as a function of the history of cortical activity. Here we provide direct experimental evidence that the value of theta(m) depends on sensory experience. We find in visual cortex of light-deprived rats that LTP is enhanced and LTD diminished over a range of stimulation frequencies, and that these effects can be reversed by as little as two days of light exposure. Our findings support the idea that a variable synaptic modification threshold allows synaptic weights in neural networks to achieve a stable equilibrium.

Abstract: NMDA receptor antagonists can induce a schizophrenia-like psychosis, but the role of NMDA receptors in the pathophysiology of schizophrenia remains unclear. Expression patterns of mRNAs for five NMDA receptor subunits (NR1/NR2A-D) were determined by in situ hybridization in prefrontal, parieto-temporal, and cerebellar cortex of brains from schizophrenics and from neuroleptic-treated and nonmedicated controls. In the cerebral cortex of both schizophrenics and controls, mRNAs for NR1, NR2A, NR2B, and NR2D subunits were preferentially expressed in layers II/III, Va, and VIa, with much higher levels in the prefrontal than in the parieto-temporal cortex. Levels of mRNA for the NR2C subunit were very low overall. By contrast, the cerebellar cortex of both schizophrenics and controls contained very high levels of NR2C subunit mRNA, whereas levels for the other subunit mRNAs were very low, except NR1, for which levels were moderate. Significant alterations in the schizophrenic cohort were confined to the prefrontal cortex. Here there was a shift in the relative proportions of mRNAs for the NR2 subunit family, with a 53% relative increase in expression of the NR2D subunit mRNA. No comparable changes were found in neuroleptic-treated or untreated controls. These findings indicate regional heterogeneity of NMDA receptor subunit expression in human cerebral and cerebellar cortex. In schizophrenics, the alterations in expression of NR2 subunit mRNA in prefrontal cortex are potential indicators of deficits in NMDA receptor-mediated neurotransmission accompanying functional hypoactivity of the frontal lobes.

Abstract: Behavioral stress has detrimental effects on subsequent cognitive performance in many species, including humans. For example, humans exposed to stressful situations typically exhibit marked deficits in various learning and memory tasks. However, the underlying neural mechanisms by which stress exerts its effects on learning and memory are unknown. We now report that in adult male rats, stress (i.e., restraint plus tailshock) impairs long-term potentiation (LTP) but enhances long-term depression (LTD) in the CA1 area of the hippocampus, a structure implicated in learning and memory processes. These effects on LTP and LTD are prevented when the animals were given CGP39551 (the carboxyethylester of CGP 37849; DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, before experiencing stress. In contrast, the anxiolytic drug diazepam did not block the stress effects on hippocampal plasticity. Thus, the effects of stress on subsequent LTP and LTD appear to be mediated through the activation of the NMDA subtype of glutamate receptors. Such modifications in hippocampal plasticity may contribute to learning and memory impairments associated with stress.

Abstract: The effects of gonadal steroid hormones on dendritic spines were studied in hippocampal neurons that were dissociated and grown in culture for 2–3 weeks. Exposure to estradiol caused up to a twofold increase in dendritic spine density in these neurons. The effect of estradiol was stereospecific and blocked by the steroid antagonist tamoxifen. The estradiol-induced rise in spine density was blocked by the NMDA antagonist APV, but not by the AMPA/KA antagonist DNQX. The estradiol-induced rise in spine density was blocked by the serine/threonine kinase inhibitor H7, but not by the tyrosine kinase inhibitor genestein, and was partially mimicked by PMA, an activator of protein kinase C. Estradiol also caused an increase in the fluorescence intensity of synaptophysin-immunoreactive terminals, corresponding to presynaptic boutons. Finally, estradiol caused a rise in [Ca]i reactivity of the cultured neurons to topical application of glutamate. These studies are the first to examine receptor and second messenger regulation of dendritic spines, and they illustrate the viability of cultured neurons as a powerful test system to address issues related to the regulation of dendritic spine maturation.

Abstract: Most reported actions of kainate are mediated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptors. Here we report that, unlike AMPA which stimulates, kainate elicits a dose-dependent decrease in L-glutamate release from rat hippocampal synaptosomes and also depresses glutamatergic synaptic transmission. Brief exposure to kainate inhibited Ca(2+)-dependent [3H]L-glutamate release by up to 80%. Inhibition was reversed by kainate antagonists but not by the AMPA-selective non-competitive antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466). A corresponding reversible kainate-evoked depression of NMDA (N-methyl-D-aspartate) receptor-mediated excitatory postsynaptic currents (e.p.s.cs) was observed when AMPA receptors were blocked by GYKI 52466. The synaptic depression was preceded by a brief period of enhanced release and a small inward current was also observed. The effects of kainate were unaffected by metabotropic glutamate (mGlu), GABAA, GABAB, glycine and adenosine receptor antagonists. These results indicate that glutamate release can be modulated directly by kainate autoreceptors.

Abstract: Estradiol treatment increases the number of NMDA receptor binding sites, and changes evoked synaptic currents in a manner consistent with a steroid-induced functional enhancement of NMDA receptors in rat hippocampus. In this study, we investigate the cellular mechanisms of estradiol-induced NMDA receptor regulation at the protein and mRNA levels in ovariectomized rats treated with ovarian steroids using immunocytochemical and in situ hybridization techniques. Confocal laser scanning microscopy was used to quantify alterations in immunofluorescence intensity levels of NMDAR1 subunit proteins within neuronal somata and dendrites of discrete hippocampal fields, whereas in parallel, in situ hybridization was used to examine NMDAR1 mRNA levels in corresponding hippocampal regions. The data indicate that estradiol treatment in ovariectomized rats significantly increases immunofluorescence intensity levels in comparison with nonsteroid treated ovariectomized rats within the somata and dendrites of CA1 pyramidal cells and, to a lesser extent, within the granule cell somata of the dentate gyrus. In contrast, such alterations in immunofluorescence intensity occur without concomitant changes in mRNA hybridization levels. Thus, these data suggest that estradiol modulates NMDA receptor function via post-transcriptional regulation of the NMDAR1 subunit protein. The increase in immunofluorescence intensity may reflect an increase in the concentration of the subunit protein, which could account for estrogen-induced changes in pharmacological and physiological properties of the NMDA receptor.

Abstract: The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits. Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis. Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity. These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins. Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly. These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.

Abstract: 1. Ifenprodil is a selective, atypical non-competitive antagonist of NMDA receptors that contain the NR2B subunit with an undefined mechanism of action. Ifenprodil is neuroprotective in in vivo models of cerebral ischaemia but lacks many of the undesirable side-effects associated with NMDA antagonist. 2. Using whole-cell voltage-clamp recordings, we have studied the mechanism of inhibition of NMDA-evoked currents by ifenprodil in rat cultured cortical neurones in the presence of saturating concentrations of glycine. 3. Ifenprodil antagonized NMDA receptors in an activity-dependent manner, whilst also increasing the receptor affinity for glutamate recognition-site agonists. Ifenprodil inhibition curves against 10 and 100 microM NMDA-evoked currents yielded IC50 values of 0.88 and 0.17 microM, respectively. Thus, the apparent affinity of ifenprodil for the NMDA receptor is increased in an NMDA concentration-dependent manner. 4. Currents evoked by 0.3 and 1 microM NMDA were potentiated to approximately 200% of control levels in the presence of 3 microM ifenprodil. Thus, with increasing concentration of NMDA the effect of ifenprodil on NMDA-evoked currents changed from one of potentiation to one of increasing inhibition. 5. These results are predicted by a reaction scheme in which ifenprodil exhibits a 39- and 50-fold higher affinity for the agonist-bound activated and desensitized states of the NMDA receptor, respectively, relative to the resting, agonist-unbound state. Furthermore, ifenprodil binding to the NMDA receptor results in a 6-fold higher affinity for glutamate site agonists. 6. This represents a novel mechanism of NMDA receptor antagonism that, together with the subunit selectivity, probably contributes to the attractive neuropharmacological profile of this and related compounds.

Abstract: This report describes the activity of the antiepileptic agent gabapentin (Neurontin) in animal models predictive of anxiolysis and analgesia. Gabapentin displayed anxiolytic-like action in the rat conflict test, the mouse light/dark box and the rat elevated X-maze with respective minimum effective doses (MEDs) of 3, 10 and 30 mg/kg. Furthermore, gabapentin also induced behavioural changes suggestive of anxiolysis in the marmoset human threat test with a MED of 30 mg/kg. In the rat formalin test of tonic nociception, gabapentin dose-dependently (30–300 mg/kg) and selectively blocked the late phase with a MED of 100 mg/kg. However, it failed to block carrageenan-induced paw oedema. The intracerebroventricular (ICV) administration of the glycine/NMDA receptor agonistd-Serine, dose-dependently (10–100 μg/animal) reversed the antinociceptive action of gabapentin (200 mg/kg, SC).d-Serine (30 μg/animal, ICV) also reversed the anxiolytic-like effects (in the light/dark box and the rat elevated X-maze) of gabapentin (30 mg/kg). In contrast,l-Serine (100 μg, ICV) failed to block the antinociceptive action of gabapentin. The antinociceptive action of (+)-HA-966 (25 mg/kg, SC), a partial agonist at the glycine/NMDA receptor, was reversed byd-Serine (100 μg/animal, ICV). However,d-Serine (100 μg/animal, ICV) failed to affect the antinociceptive action of a competitive NMDA receptor antagonist CGS 19755 (3 mg/kg, SC). Gabapentin has negligible affinity for the strychnine insensitive [3H]glycine binding site. This indicates that the interaction between gabapentin andd-Serine may not involve the NMDA receptor complex. Gabapentin may represent a novel type of anxiolytic and analgesic agent.

Abstract: The present study investigated the role of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor subtypes in peripheral pain transmission. Activation of NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate acid (KA) receptors in glabrous skin of the rat hindpaw resulted in mechanical allodynia and mechanical hyperalgesia. These agonist-induced pain behaviors were attenuated following peripheral injection of appropriate antagonists (MK-801 and CNQX). Thus, activation of NMDA, AMPA or KA receptors at the level of the peripheral nerve terminal can produce nociceptive behavior. These data suggest that topical application of glutamate receptor antagonists may be useful in treating pain disorders. Since all three receptor subtypes are involved in peripheral pain transmission, however, it will be necessary to antagonize multiple glutamate receptor subtypes to achieve effective pain relief.

Abstract: The present experiments were designed to further examine the hypothesis that receptor subtype determines the direction of dopamine's (DA) ability to modulate neostriatal neuronal responses. We have reported that DA potentiates responses mediated by activation of N-methyl-d-aspartate (NMDA) receptors, but attenuates responses mediated by activation of non-NMDA receptors in neocortex [Cepeda et al. (1992b) Synapse, 11:330-341] and neostriatum [Cepeda et al. (1993) Proc. Natl. Acad. Sci. U.S.A., 90:9576-9580]. In these studies, responses to excitatory amino acids (EAAs) were evoked by microphoretic application of agonists. The present studies examined whether this differential modulation also applies to components of synaptic responses evoked by electrical stimulation of neostriatal afferents and mediated by activation of specific subtypes of EAA receptors. Using brain slices, the actions of DA and its receptor specific agonists on components of neostriatal synaptic responses that were mediated either by NMDA or non-NMDA receptors were assessed. Responses mediated by NMDA receptors were potentiated by DA while those mediated by non-NMDA receptors were attenuated. These findings provide further support for the hypothesis that the direction of modulatory action of DA is determined by the specific subtype of EAA receptor activated. In addition, the enhancement of NMDA receptor-mediated responses was mimicked by application of SKF 38393, a D1 receptor agonist. Quinpirole, a D2 receptor agonist, consistently attenuated responses mediated by activation of non-NMDA receptors. Thus, the complex modulatory actions of DA are dependent upon combinations of co-activation of specific subtypes of EAA and DA receptors. These findings are of clinical relevance since the actions of DA and EAAs have been implicated in neurological and affective disorders.

Abstract: In in vitro receptor binding and synaptosomal uptake experiments the (+)-enantiomer of tramadol (CAS 148229-78-1) is specific for the mu-opioid receptor site and for the serotonin (5-HT) carrier, whereas the (-)-enantiomer (CAS 148229-79-2) has a higher affinity to the noradrenaline (NA) transporter. The antinociceptive active tramadol metabolite O-demethyltramadol (M1) shows a pronounced mu-selectivity. With respect to in vitro receptor binding experiments, the affinity of (+)-M1 to this opioid receptor subtype is more than two orders of magnitude higher than that of (+)-tramadol and approximately 1/10 that of morphine. Tramadol and M1 (and the enantiomers thereof) have no affinity to other receptor or uptake sites tested, e.g. 5-HT1A, 5-HT2, 5-HT3, NMDA (ligand: MK801), dopamine (DA)-D1, DA-D2, benzodiazepine, muscarine M1 and DA uptake (Ki > or = 2 x 10(-5) mol/l). Ex vivo neurotransmitter determinations show that tramadol (46.4 mg/kg i.p.) elevates the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid and enhances DA release in definite brain areas. The active enantiomer of the racemic tramadol is the (+)-enantiomer. (+)-Tramadol significantly enhances the turnover rate of DA. The enantioselective elevation of DOPAC by (+)-tramadol is antagonized by naloxone (2 x 5 mg/kg i.p.). Morphine (21.5 mg/kg i.p.) enhances the turnover of NA in definite brain areas. Neither the NA-specific uptake inhibition nisoxetine (31.6 mg/kg i.p.) nor tramadol (or its (+)- and (-)-enantiomers) have any influence on the NA turnover. Tramadol reduces the levels of 5-HT and its metabolite 5-hydroxyindoleacetic acid. Morphine enhances, whereas tramadol reduces, 5-HT utilisation in the brain areas under assay. The 5-HT specific uptake inhibitor fluoxetine (20 mg/kg i.p.) shows the same influence on 5-HT turnover as tramadol. The results indicate that tramadol enhances DA turnover via an opioid mechanism. The interaction with the noradrenergic and serotonergic neurotransmission is clearly different from that of an opioid receptor agonist and closely resembles that of NA and 5-HT uptake inhibitors.

Abstract: 1. Intracellular sharp electrode and whole-cell patch-clamp recording from characterized paraventricular nucleus (PVN) neurones in rat hypothalamic slices were used to study the synaptic mechanism and associated neurotransmitters that mediate their response to suprachiasmatic nucleus (SCN) stimulation. 2. Electrical stimulation restricted to SCN evoked short-latency inhibitory postsynaptic potentials (IPSPs) or combinations of IPSPs and excitatory postsynaptic potentials (EPSPs) in all (n = 59) PVN neurones tested. Type I neurones (n = 18) were magnocellular and a majority (13/18) demonstrated monosynaptic IPSPs that reversed polarity at the chloride equilibrium potential and were sensitive to bicuculline. 3. Type II (n = 10) and III parvocellular (n = 13), and unclassifiable neurones (n = 18) displayed combinations of IPSPs and EPSPs following similar stimuli applied to SCN. IPSP blockade with bicuculline uncovered SCN-evoked monosynaptic dual-component EPSPs that were sensitive to N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists. In addition, chemical microstimulation within SCN was associated with transient increases in spontaneous EPSPs recorded from these PVN neurones. 4. These data imply that the amino acids GABA and glutamate are important mediators of fast monosynaptic transmission from SCN to defined neurones in PVN, and are candidates for conveying circadian rhythmicity to PVN regulation of neuroendocrine and autonomic processes.