Blockade Of NMDA Glutamate Receptors Research Articles

The Ability of NMDA-Type Glutamate Receptor Blockers to Prevent the Development of Pentylenetetrazole Kindling and Morphological Changes to Pyramidal Neurons in the Mouse Hippocampus

Experiments on mice addressed the link between convulsive syndrome and morphological changes in hippocampal neurons occurring on development of pentylenetetrazole (PTZ) kindling. Kindling was induced by i.p. PTZ (35 mg/kg) three times a week for one month. By the end of this period, 70% of the mice responded to administration of PTZ with severe clonic or clonic-tonic seizures. Hippocampal sections (stratum pyramidale, field CA1, Nissl staining) from convulsive mice showed large numbers of altered cells (24.7 ± 2.1%). Most of these were pyramidal neurons. These hyperchromic neurons had reduced body sizes, loss of turgor, wrinkling of the cell body, and deformation of dendritic processes. These dark-type changes were present in 2.3 ± 2.1% of neurons in the hippocampus of intact mice and mice resistant to the convulsogenic effect of PTZ (30% of the population). Immunohistochemical studies demonstrated normal expression of NeuN (Fox3) protein in all hippocampal cells, including dark hyperchromic neurons. This is evidence that neurons did not die en masse and were relatively viable. Prophylactic s.c. administration of NMDA receptor blockers (0.5 mg/kg memantine, 0.1 mg/kg IEM-1921, or 1 mg/kg IEM-1958) decreased the proportion of mice developing PTZ kindling from 70% to 40%. The proportion of altered neurons in the 60% of mice given NMDA blockers and not developing PTZ kindling or convulsions in the presence of blockers was 0.1 ± 0.06%, which was the same as in intact mice. Conversely, the hippocampus of mice demonstrating convulsions despite simultaneous administration of NMDA blockers showed 24.0 ± 5.6% hyperchromic neurons. These results provide evidence that pathologically altered neurons appeared after convulsive seizures in animals after PTZ kindling and that blockade of NMDA glutamate receptors could weaken both the development of convulsive syndrome and the concomitant morphological changes to hippocampal neurons.

Hyperoxia-induced developmental plasticity of the hypoxic ventilatory response in neonatal rats: Contributions of glutamate-dependent and PDGF-dependent mechanisms

Rats reared in hyperoxia exhibit a sustained (vs. biphasic) hypoxic ventilatory response (HVR) at an earlier age than untreated, Control rats. Given the similarity between the sustained HVR obtained after chronic exposure to developmental hyperoxia and the mature HVR, it was hypothesized that hyperoxia-induced plasticity and normal maturation share common mechanisms such as enhanced glutamate and nitric oxide signaling and diminished platelet-derived growth factor (PDGF) signaling. Rats reared in 21% O2 (Control) or 60% O2 (Hyperoxia) from birth until 4–5 days of age were studied after intraperitoneal injection of drugs targeting these pathways. Hyperoxia rats receiving saline showed a sustained HVR to 12% O2, but blockade of NMDA glutamate receptors (MK-801) restored the biphasic HVR typical of newborn rats. Blockade of PDGF-β receptors (imatinib) had no effect on the pattern of the HVR in Hyperoxia rats, although it attenuated ventilatory depression during the late phase of the HVR in Control rats. Neither nitric oxide synthase inhibitor used in this study (nNOS inhibitor I and l-NAME) altered the pattern of the HVR in Control or Hyperoxia rats. Drug-induced changes in the biphasic HVR were not correlated with changes in metabolic rate. Collectively, these results suggest that developmental hyperoxia hastens the transition from a biphasic to sustained HVR by upregulating glutamate-dependent mechanisms and downregulating PDGF-dependent mechanisms, similar to the changes underlying normal postnatal maturation of the biphasic HVR.

Meclizine Enhancement of Sensorimotor Gating in Healthy Male Subjects with High Startle Responses and Low Prepulse Inhibition

Histamine H1 receptor systems have been shown in animal studies to have important roles in the reversal of sensorimotor gating deficits, as measured by prepulse inhibition (PPI). H1-antagonist treatment attenuates the PPI impairments caused by either blockade of NMDA glutamate receptors or facilitation of dopamine transmission. The current experiment brought the investigation of H1 effects on sensorimotor gating to human studies. The effects of the histamine H1 antagonist meclizine on the startle response and PPI were investigated in healthy male subjects with high baseline startle responses and low PPI levels. Meclizine was administered to participants (n=24) using a within-subjects design with each participant receiving 0, 12.5, and 25 mg of meclizine in a counterbalanced order. Startle response, PPI, heart rate response, galvanic skin response, and changes in self-report ratings of alertness levels and affective states (arousal and valence) were assessed. When compared with the control (placebo) condition, the two doses of meclizine analyzed (12.5 and 25 mg) produced significant increases in PPI without affecting the magnitude of the startle response or other physiological variables. Meclizine also caused a significant increase in overall self-reported arousal levels, which was not correlated with the observed increase in PPI. These results are in agreement with previous reports in the animal literature and suggest that H1 antagonists may have beneficial effects in the treatment of subjects with compromised sensorimotor gating and enhanced motor responses to sensory stimuli.

A cytokine network involving brain-borne IL-1β, IL-1ra, IL-18, IL-6, and TNFα operates during long-term potentiation and learning

We have previously shown that long-term potentiation (LTP) induces hippocampal IL-1β and IL-6 over-expression, and interfering their signalling either inhibits or supports, respectively, LTP maintenance. Consistently, blockade of endogenous IL-1 or IL-6 restricts or favours hippocampal-dependent memory, effects that were confirmed in genetically manipulated mice. Since cytokines are known for their high degree of mutual crosstalk, here we studied whether a network of cytokines with known neuromodulatory actions is activated during LTP and learning. We found that, besides IL-1β and IL-6, also IL-1 receptor antagonist (IL-1ra) and IL-18, but not TNFα are over-expressed during LTP maintenance in freely moving rats. The increased expression of these cytokines is causally related to an increase in synaptic strength since it was abrogated when LTP was interfered by blockade of NMDA-glutamate receptors. Likewise, IL-1 and IL-6 were found to be over-expressed in defined regions of the hippocampus during learning a hippocampus-dependent task. However, during learning, changes in IL-18 were restricted to the dorsal hippocampus, and no differences in TNFα and IL1-ra expression were noticed in the hippocampus. Noticeably, IL-1ra transcripts were significantly reduced in the prefrontal cortex. The relation between cytokine expression and learning was causal because such changes were not observed in animals from a pseudo-trained group that was subject to the same manipulation but could not learn the task. Taken together with previous studies, we conclude that activation of a cytokine network in the brain is a physiologic relevant phenomenon not only for LTP maintenance but also for certain types of learning.

Differential effects of the antidepressant mirtazapine on amphetamine- and dizocilpine-induced PPI deficits

Prepulse inhibition (PPI) refers to the decrease in motor startle response to salient sensory stimuli (pulses) when they are closely preceded in time by another more modest sensory stimulus (prepulse). PPI deficits can be induced by stimulation of dopamine receptors (e.g., amphetamine or apomorphine) or blockade of NMDA glutamate receptors (e.g., dizocilpine or PCP). Previously we found that antagonists of α2-noradrenergic and H1-histaminergic receptors significantly attenuate PPI impairments caused by amphetamine or dizocilpine. In the current study we assessed the effects of the antidepressant mirtazapine, which has combined antagonist effects at α2-noradrenergic, H1-histaminergic and 5-HT serotonergic receptors, on amphetamine- and dizocilpine-induced PPI deficits. In Experiment 1, rats were tested for PPI of the startle response to a tactile air-puff stimulus after auditory prepulses of three different intensities. Drug treatments consisted of combinations of amphetamine (0 and 1mg/kg) and mirtazapine (0, 0.5, 1, 2, and 5mg/kg), with all rats receiving all drug doses and combinations with different counterbalanced orders. In Experiment 2, a different group of rats was tested with drug treatments consisting of combinations of dizocilpine (0 and 0.05mg/kg) and mirtazapine (0, 0.5, 1, 2, and 5mg/kg). In Experiment 1 amphetamine (1mg/kg) significantly reduced PPI whereas mirtazapine caused the opposite effect, with the highest dose of mirtazapine (5mg/kg) effectively reversing the amphetamine-induced PPI deficit. In Experiment 2 dizocilpine (0.05mg/kg) significantly reduced PPI, but mirtazapine did not have a significant effect on the inhibition of the startle response. These results indicate that the potential beneficial effects of combined α-adrenergic, 5-HT, and H1 receptor blockade in counteracting PPI deficits may be associated to cases of sensorimotor gating disorders mediated by dopamine, but not necessarily to NMDA glutamate-induced PPI impairments.

The α2-adrenergic antagonist idazoxan counteracts prepulse inhibition deficits caused by amphetamine or dizocilpine in rats

Prepulse inhibition (PPI) is the reduction in startle response magnitude when intense stimuli are closely preceded by other weak stimuli. Animal models used to investigate sensorimotor gating deficits include both the stimulation of dopamine receptors (e.g., amphetamine or apomorphine) and the blockade of NMDA-glutamate receptors (e.g., dizocilpine or phencyclidine). We assessed the effects of idazoxan (an α(2)-adrenergic antagonist) on amphetamine- and dizocilpine-induced PPI disruptions in adult female Sprague-Dawley rats. In experiment 1, rats were tested for PPI in a bimodal paradigm with an acoustic prepulse and a tactile startle stimulus. Interactions of amphetamine (1 mg/kg) and idazoxan (0.5, 1, and 2 mg/kg) were assessed, with all rats receiving all drug doses in a counterbalanced order. In experiment 2, dizocilpine (0.05 mg/kg) and idazoxan (0.5, 1, and 2 mg/kg) interactions were analyzed. Amphetamine (1 mg/kg) caused a significant reduction in PPI. Both the 1- and 2-mg/kg doses of idazoxan significantly counteracted this effect. Dizocilpine (.05 mg/kg) effectively inhibited PPI, and the 2-mg/kg idazoxan dose significantly counteracted this impairment. These results suggest that the effectiveness of atypical antipsychotics such as clozapine in counteracting sensorimotor gating deficits reported in previous studies (e.g., Swerdlow and Geyer, Pharmacol Biochem Behav 44:741-744, 1993; Bakshi et al., J Pharmacol Exp Ther 271:787-794, 1994) may be related to their α(2)-antagonist effects, which may be a critical mechanism of the therapeutic effects of atypical antipsychotics in schizophrenia.

Chronic nicotine and dizocilpine effects on nicotinic and NMDA glutamatergic receptor regulation: Interactions with clozapine actions and attentional performance in rats

Blockade of NMDA glutamate receptors with dizocilpine (MK-801) has been shown to cause substantial cognitive deficits and has been used to model symptoms of schizophrenia. Nicotine or nicotinic agonists, in contrast, may enhance cognitive or attentional functions and be of therapeutic potential in schizophrenia. Nicotinic–glutamatergic interactions, therefore, may have important implications in cognitive functions and antipsychotic treatments. Clozapine, a widely used antipsychotic drug, has been shown in some studies to be effective in ameliorating the cognitive deficits associated with schizophrenia. However, there is some evidence to suggest that clozapine similar to haloperidol may impair sustained attention in rats. In this study, we sought to determine whether chronic nicotine or dizocilpine may modify the effects of acute clozapine on attentional parameters and whether the behavioral effects would correlate with nicotinic or NMDA receptor densities in discrete brain regions. Adult female rats trained on an operant visual signal detection task were given 4 weeks of nicotine (5 mg/kg/day), dizocilpine (0.15 mg/kg/day), the same doses of both nicotine and dizocilpine as a mixture, or saline by osmotic minipump. While on chronic treatment, rats received acute injections of various doses of clozapine (0, 0.625, 1.25, 2.5 mg/kg, sc) 10 min prior to tests on attentional tasks. The pumps were removed on day 28 and 24 h later the animals were sacrificed for measurements of receptor densities in specific brain regions. The percent correct hit as a measure of sustained attention was significantly impaired by clozapine in a dose-related manner. Neither chronic nicotine nor dizocilpine affected this measure on their own or modified the effects of clozapine. Both nicotine and dizocilpine affected the receptor bindings in a region specific manner and their combination further modified the effects of each other in selective regions. Attentional performance was inversely correlated with alpha-bungarotoxin binding in the frontal cortex only. In conclusion, the data suggest attentional impairments with clozapine alone and no modification of this effect with nicotine or dizocilpine. Moreover, cortical low affinity nicotinic receptors may have a role in attentional functions.

Effects of Blockade of NMDA Receptors and NO Synthase on the Expression of Associative and Non-Associative Sensitization to Effects of Cocaine

After repeated administration of psychostimulant drugs, a sensitization rather than a tolerance to the behavioral effects can be observed. In our own previous studies, it was shown that both blockade of NMDA glutamate receptors and inhibition of NO synthase selectively inhibited the expression of associative but not non-associative sensitization to D-amphetamine. The present experiments were performed in order to study whether a similar selective inhibition of expression of associative sensitization to cocaine can be observed after blockade of NMDA receptors by MK-801 or inhibition of NO synthase by L-NAME. MK-801 as well as L-NAME inhibited the locomotor activity in acutely cocaine-treated rats. Both drugs did not prevent the sensitization either in the associative or the non-associative group. The results suggest that the acute locomotor effects of cocaine were inhibited by both drugs whereas both the non-associative and the associative sensitization to locomotor effects were not inhibited by blockade of NMDA receptors or inhibition of NO synthase. Accordingly, the expression of neither type of sensitization to cocaine was inhibited by any of these drugs.

NMDA receptors regulate developmental gap junction uncoupling via CREB signaling

Signaling through gap junctions (electrical synapses) is important in the development of the mammalian central nervous system. Abundant between neurons during postnatal development, gap junction coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. Here we report that developmental uncoupling of gap junctions in the rat hypothalamus in vivo and in vitro is associated with a decrease in connexin 36 (Cx36) protein expression. Both developmental gap junction uncoupling and Cx36 downregulation are prevented by the blockade of NMDA glutamate receptors, action potentials and the calcium-cyclic AMP response element binding protein (CREB), and are accelerated by CREB overexpression. Developmental gap junction uncoupling and Cx36 downregulation are not affected by blockade of non-NMDA glutamate receptors, and do not occur in hypothalamic neurons from NMDA receptor subunit 1 (NMDAR1) knockout mice. These results demonstrate that NMDA receptor activity contributes to the developmental uncoupling of gap junctions via CREB-dependent downregulation of Cx36.

Chronic nicotine and dizocilpine effects on regionally specific nicotinic and NMDA glutamate receptor binding

Chronic nicotine administration has long been known to increase the number of high-affinity α4β2 nicotinic receptors with lesser effects on low-affinity α7 nicotinic receptors. Nicotine has been shown to promote the release of a variety of neurotransmitters including glutamate. Nicotine may also interact directly with the glutamatergic receptors. Nicotinic–glutamate interactions may be critical to the long-term effects of nicotine. Conversely, glutamatergic drugs may interact with the nicotinic system. Such interactions have important implications in interpretation of the mechanism of drug actions, especially when the drugs are given together. The current study examined the effects of chronic administration of nicotine (5 mg of the nicotine base/kg/day for 28 days), dizocilpine (MK-801) (0.3 mg/kg/day for 28 days), an NMDA receptor antagonist, as well as the combination of the two drugs on nicotinic and NMDA receptor densities in discrete brain regions. The chronic dose of dizocilpine used was behaviorally active causing a dramatic reduction in prepulse inhibition (PPI) of acoustic startle response. The nicotine dose used did not significantly affect PPI but previously we have found it to be behaviorally active in improving working memory function. High-affinity nicotinic receptor binding, as has been seen previously, was significantly increased by chronic nicotine in most areas. Chronic dizocilpine alone did not affect high-affinity nicotinic receptor binding, but it did modify the effects of chronic nicotine, attenuating nicotine-induced increases in the frontal cortex and striatum. Low-affinity nicotinic binding was significantly increased by chronic nicotine in only one area, the cerebellum. Chronic dizocilpine significantly increased low-affinity nicotinic binding in several brain areas, the colliculi, hippocampus, and the hypothalamus. The combination of nicotine and dizocilpine attenuated the effects of each with diminished nicotine-induced increased nicotinic low-affinity binding in the cerebellum and diminished dizocilpine-induced increased nicotinic low-affinity binding in the hippocampus and hypothalamus. In contrast, chronic nicotine and dizocilpine had a mutually potentiating effect of increasing nicotinic low-affinity binding in the frontal cortex. NMDA receptor binding was affected only in the hippocampus, where both dizocilpine and nicotine significantly increased binding. Chronic nicotine effects on receptor regulation are significantly affected by concurrent blockade of NMDA glutamate receptors.

Ethanol-induced apoptotic neurodegeneration in the developing C57BL/6 mouse brain

Recent studies have shown that administration of ethanol to infant rats during the synaptogenesis period (first 2 weeks after birth), triggers extensive apoptotic neurodegeneration throughout many regions of the developing brain. While synaptogenesis is largely a postnatal phenomenon in rats, it occurs prenatally (last trimester of pregnancy) in humans. Recent evidence strongly supports the interpretation that ethanol exerts its apoptogenic action by a dual mechanism—blockade of NMDA glutamate receptors and hyperactivation of GABA A receptors. These findings in immature rats represent a significant advance in the fetal alcohol research field, in that previous in vivo animal studies had not demonstrated an apoptogenic action of ethanol, had not documented ethanol-induced cell loss from more than a very few brain regions and had not provided penetrating insight into the mechanisms underlying ethanol’s neurotoxic action. To add to the mechanistic insights recently gained, it would be desirable to examine gene-regulated aspects of ethanol-induced apoptotic neurodegeneration, using genetically altered strains of mice. The feasibility of such research must first be established by demonstrating that appropriate mouse strains are sensitive to this neurotoxic mechanism. In the present study, we demonstrate that mice of the C57BL/6 strain, a strain frequently used in transgenic and gene deletion research, are exquisitely sensitive to the mechanism by which ethanol induces apoptotic neurodegeneration during the synaptogenesis period of development.

Glutamate and GABA receptor dysfunction in the fetal alcohol syndrome.

The brain damaging effects of ethanol, as the most disabling component of the fetal alcohol syndrome FAS), have been recognized for 3 decades, but the mechanism underlying these effects has remained elusive. Recently, we discovered that ethanol triggers widespread apoptotic neurodegeneration throughout the developing brain when administered to infant rodents during the period of synaptogenesis, also known as the brain growth spurt period. These findings provide a more likely explanation than has heretofore been available for the reduced brain mass and lifelong neurobehavioral disturbances associated with the human FAS. We propose that a dual mechanism - blockade of NMDA glutamate receptors and hyperactivation of GABA(A) receptors - mediates ethanol's apoptogenic action, based on established evidence that ethanol has both NMDA antagonist and GABAmimetic properties, and our recent finding that other drugs with either NMDA antagonist or GABAmimetic properties robustly trigger apoptotic neurodegeneration in the developing brain. The brain growth spurt occurs in different species at different times relative to birth. In rats and mice it is a postnatal event, but in humans it extends from the sixth month of gestation to several years after birth. Thus, there is a period in pre and postnatal human development, lasting for several years, during which immature CNS neurons are prone to commit suicide if exposed to intoxicating concentrations of drugs with NMDA antagonist or GABAmimetic properties. These findings are important, not only because of their relevance to the FAS, but because there are many agents in the human environment, other than ethanol, that have NMDA antagonist or GABAmimetic properties. Such agents include drugs that may be abused by pregnant mothers [ethanol, phencyclidine (angel dust), ketamine (Special K), nitrous oxide (laughing gas), barbiturates, benzodiazepines], and many medicinals used in obstetric and pediatric neurology (anticonvulsants), and anesthesiology (all general anesthetics are either NMDA antagonists or GABAmimetics).

Glutamate signaling and the fetal alcohol syndrome.

It has been known for three decades that ethanol, the most widely abused drug in the world, has deleterious effects on the developing human brain, but progress has been slow in developing animal models that are optimal for studying this problem, and the underlying mechanisms have remained elusive. Recently, we have shown that during the synaptogenesis period, also known as the brain growth spurt period, ethanol has the potential to trigger widespread neuronal suicide (apoptosis), deleting many millions of neurons from the in vivo mammalian brain. It appears that ethanol triggers apoptotic neurodegeneration by a dual mechanism (blockade of NMDA glutamate receptors and excessive activation of GABA(A) receptors), in that ethanol has both NMDA antagonist and GABAmimetic properties; we have shown that other drugs which have either of these properties trigger apoptotic neurodegeneration in the developing brain. The brain growth spurt period in humans spans the last trimester of pregnancy and the first several years after birth. Thus, our findings provide a likely explanation for the reduced brain mass and neurobehavioral disturbances associated with the human fetal alcohol syndrome. Furthermore, since NMDA antagonist and GABAmimetic drugs are sometimes abused by pregnant women and also are used as anticonvulsants, sedatives, or anesthetics in pediatric medicine, our findings suggest the possibility that exposure of the developing brain to these various drugs either pre or postnatally could contribute to mental disability syndromes that have heretofore been attributed to unknown causes. In addition, the observation that ethanol and related drugs trigger massive neuronal apoptosis in the developing brain provides an unprecedented opportunity to study both neuropathological aspects and molecular mechanisms of apoptotic neurodegeneration in the in vivo mammalian brain.

Enhanced [3H]DOPA and [3H]dopamine turnover in striatum and frontal cortex in vivo linked to glutamate receptor antagonism.

We tested the hypothesis that blockade of NMDA glutamate receptors in brain enhances dopamine turnover. We blocked this class of glutamate receptors in the rat brain in vivo with dizocilpine (MK-801) and measured the accumulation of radiolabeled DOPA and its metabolites as functions of time after intravenous bolus injection. Using the time courses of the accumulated metabolites, we calculated the turnover constants of enzymes mediating dopamine synthesis and catabolism. Dizocilpine treatment for 8 days enhanced the rates of DOPA decarboxylation and dopamine oxidation (monoamine oxidation) 4- and 16-fold, respectively, in neostriatum and 10- and 3-fold, respectively, in frontal cortex. The findings are not inconsistent with the hypothesis that the psychotomimetic properties of dizocilpine may be the manifestation of denervation hypersensitivity linked to activation of key enzymes of dopamine turnover in striatum.

NMDA-glutamate receptors regulate phosphorylation of dendritic cytoskeletal proteins in the hippocampus

Most forms of synaptic potentiation need the activation of the N-methyl- d-aspartate (NMDA) subtype of glutamate receptors which generate changes in dendritic morphology of postsynaptic neurons. Since microtubule proteins have an essential role in dendritic morphology, they may be involved and regulated during the modifications of dendritic morphology associated with synaptic potentiation. The phosphorylation of the microtubule-associated proteins (MAPs) has been analyzed in situ after activation or blockade of NMDA-glutamate receptors in hippocampal slices. The phosphorylation of MAP1B and MAP2 has been studied by using several antibodies raised against phosphorylation-sensitive epitopes. Whereas antibodies 125 and 305 recognize phosphorylated epitopes on MAP1B and MAP2, respectively, Ab 842 recognizes a phosphorylatable sequence on MAP1B only when it is dephosphorylated. NMDA treatment decreased the phosphorylation state of the epitope recognized by the antibody 305 on MAP2 and caused a slight dephosphorylation of MAP1B sequences recognized by Ab 125 and 842. Moreover, exposure to APV (an antagonist of NMDA-glutamate receptors) counteracted the effect of NMDA and induced an increase in the phosphorylation state of these sequences in MAP2. Since phosphorylation regulates the interaction of MAPs with cytoskeleton, the results suggest that the modulation of the phosphorylated state of MAP2 by NMDA-glutamate receptors may be implicated in dendritic plasticity.