N-methyl-D-aspartate Receptor Density Research Articles

Endocannabinoids Released in the Ventral Tegmental Area During Copulation to Satiety Modulate Changes in Glutamate Receptors Associated With Synaptic Plasticity Processes.

Endocannabinoids modulate mesolimbic (MSL) dopamine (DA) neurons firing at the ventral tegmental area (VTA). These neurons are activated by copulation, increasing DA release in nucleus accumbens (NAcc). Copulation to satiety in male rats implies repeated ejaculation within a short period (around 2.5 h), during which NAcc dopamine concentrations remain elevated, suggesting continuous neuronal activation. During the 72 h that follow copulation to satiety, males exhibit long-lasting changes suggestive of brain plasticity processes. Enhanced DA neuron activity triggers the synthesis and release of endocannabinoids (eCBs) in the VTA, which participate in several long-term synaptic plasticity processes. Blockade of cannabinoid type 1 receptors (CB1Rs) during copulation to satiety interferes with the appearance of the plastic changes. Glutamatergic inputs to the VTA express CB1Rs and contribute to DA neuron burst firing and synaptic plasticity. We hypothesized that eCBs, released during copulation to satiety, would activate VTA CB1Rs and modulate synaptic plasticity processes involving glutamatergic transmission. To test this hypothesis, we determined changes in VTA CB1R density, phosphorylation, and internalization in rats that copulated to satiety 24 h earlier as compared both to animals that ejaculated only once and to sexually experienced unmated males. Changes in glutamate AMPAR and NMDAR densities and subunit composition and in ERK1/2 activation were determined in the VTA of males that copulated to satiety in the presence or absence of AM251, a CB1R antagonist. The CB1R density decreased and the proportion of phosphorylated CB1Rs increased in the animals that copulated compared to control rats. The CB1R internalization was detected only in sexually satiated males. A decrease in α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) density, blocked by AM251 pretreatment, and an increase in the proportion of GluA2-AMPARs occurred in sexually satiated rats. GluN2A- N-methyl-D-aspartate receptor (NMDAR) expression decreased, and GluN2B-NMDARs increased in these animals, both of which were prevented by AM251 pre-treatment. An increase in phosphorylated ERK1/2 emerged in males copulating to satiety in the presence of AM251. Results demonstrate that during copulation to satiety, eCBs activate CB1Rs in the VTA, producing changes in glutamate receptors compatible with a reduced neuronal activation. These changes could play a role in the induction of the long-lasting physiological changes that characterize sexually satiated rats.

Exercise-induced gene expression changes in skeletal muscle of old mice

Exercise is believed to be beneficial for skeletal muscle functions across all ages. Regimented exercise is often prescribed as an effective treatment/prophylaxis for age-related loss of muscle mass and function, known as sarcopenia, and plays an important role in the maintenance of mobility and functional independence in the elderly. However, response to exercise declines with aging, resulting in limited gain of muscle strength and endurance. These changes likely reflect age-dependent alterations in transcriptional response underlying the muscular adaptation to exercise. The exact changes in gene expression accompanying exercise, however, are largely unknown, and elucidating them is of a great clinical interest for understanding and optimizing the exercise-based therapies for sarcopenia.In order to characterize the exercise-induced transcriptomic changes in aged muscle, a paired-end RNA sequencing was performed on rRNA-depleted total RNA extracted from the gastrocnemius muscles of 24 months-old mice after 8 weeks of regimented exercise (exercise group) or no formal exercise program (sedentary group).Differential gene expression analysis of aged skeletal muscle revealed upregulations in the group of genes involved in neurotransmission and neuroexcitation, as well as equally notable absence of anabolic gene upregulations in the exercise group. In particular, genes encoding the transporters and receptor components of glutaminergic transmission were significantly upregulated in exercised muscles, as exemplified by Gria 1, Gria 2 and Grin2c encoding glutamate receptor 1, 2 and 2C respectively, Grin1 and Grin2b encoding N-methyl-d-aspartate receptors (NMDARs), Nptx1 responsible for glutaminergic receptor clustering, and Slc1a2 and Slc17a7 regulating synaptic uptake of glutamate. These changes were accompanied by an increase in the post-synaptic density of NMDARs and acetylcholine receptors (AChRs), as well as their innervation at neuromuscular junctions (NMJs).These results suggest that neural responses predominate the adaptive response of aged skeletal muscle to exercise, and indicate a possibility that glutaminergic transmission at NMJs may be present and responsible for synaptic protection and neural remodeling accompanying the exercise-induced functional enhancement in aged skeletal muscle. In addition, the absence of upregulations in the anabolic pathways highlights them as the area of potential pharmacological targeting for optimizing exercise-led sarcopenia therapy.

Developmental Changes of Glutamate and GABA Receptor Densities in Wistar Rats.

Neurotransmitters and their receptors are key molecules of signal transduction and subject to various changes during pre- and postnatal development. Previous studies addressed ontogeny at the level of neurotransmitters and expression of neurotransmitter receptor subunits. However, developmental changes in receptor densities to this day are not well understood. Here, we analyzed developmental changes in excitatory glutamate and inhibitory γ-aminobutyric acid (GABA) receptors in adjacent sections of the rat brain by means of quantitative in vitro receptor autoradiography. Receptor densities of the ionotropic glutamatergic receptors α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) as well as of the ionotropic GABAA and metabotropic GABAB receptors were investigated using specific high-affinity ligands. For each receptor binding site, significant density differences were demonstrated in the investigated regions of interest [olfactory bulb, striatum, hippocampus, and cerebellum] and developmental stages [postnatal day (P) 0, 10, 20, 30 and 90]. In particular, we showed that the glutamatergic and GABAergic receptor densities were already present between P0 and P10 in all regions of interest, which may indicate the early relevance of these receptors for brain development. A transient increase of glutamatergic receptor densities in the hippocampus was found, indicating their possible involvement in synaptic plasticity. We demonstrated a decline of NMDA receptor densities in the striatum and hippocampus from P30 to P90, which could be due to synapse elimination, a process that redefines neuronal networks in postnatal brains. Furthermore, the highest increase in GABAA receptor densities from P10 to P20 coincides with the developmental shift from excitatory to inhibitory GABA transmission. Moreover, the increase from P10 to P20 in GABAA receptor densities in the cerebellum corresponds to a point in time when functional GABAergic synapses are formed. Taken together, the present data reveal differential changes in glutamate and GABA receptor densities during postnatal rat brain development, which may contribute to their specific functions during ontogenesis, thus providing a deeper understanding of brain ontogenesis and receptor function.

Fluorinated GluN2B Receptor Antagonists with a 3-Benzazepine Scaffold Designed for PET Studies.

To analyze the N-methyl-d-aspartate (NMDA) receptor distribution in the central nervous system, fluorinated ligands that selectively address the ifenprodil binding site of GluN2B-subunit-containing NMDA receptors were developed. Various strategies to introduce a fluorine atom into the potent GluN2B ligand 2 (3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepin-1,7-diol) were pursued, including replacement of the benzylic OH moiety with a fluorine atom (13) and introduction of fluoroethoxy moieties at various positions (14 (7-position), 17 (9-position), 18a-c (1-position)). With respect to GluN2B affinity and selectivity over related receptors, the fluoroethoxy derivatives 14 and 18a are the most promising ligands. Radiosynthesis of fluoroethoxy derivative [18 F]14 was performed by nucleophilic substitution of the phenol 2 with 2-[18 F]fluoroethyl tosylate. On rat brain slices the fluorinated PET tracer [18 F]14 accumulated in regions with high density of NMDA receptors containing GluN2B subunits. The bound radioactivity could not be replaced by (S)-glutamate. However, the GluN2B ligands eliprodil, Ro 25-6981, and the non-labeled 3-benzazepine 14 were able to abolish the specific binding of [18 F]14.

Depletion of Progranulin Reduces GluN2B-Containing NMDA Receptor Density, Tau Phosphorylation, and Dendritic Arborization in Mouse Primary Cortical Neurons.

Loss-of-function mutations in the progranulin (PGRN) gene are a common cause of familial frontotemporal lobar degeneration (FTLD). This age-related neurodegenerative disorder, characterized by brain atrophy in the frontal and temporal lobes and such typical symptoms as cognitive and memory impairment, profound behavioral abnormalities, and personality changes is thought to be related to connectome dysfunctions. Recently, PGRN reduction has been found to induce a behavioral phenotype reminiscent of FTLD symptoms in mice by affecting neuron spine density and morphology, suggesting that the protein can influence neuronal structural plasticity. Here, we evaluated whether a partial haploinsufficiency-like PGRN depletion, achieved by using RNA interference in primary mouse cortical neurons, could modulate GluN2B-containing N-methyl-d-aspartate (NMDA) receptors and tau phosphorylation, which are crucially involved in the regulation of the structural plasticity of these cells. In addition, we studied the effect of PGRN decrease on neuronal cell arborization both in the presence and absence of GluN2B-containing NMDA receptor stimulation. We found that PGRN decline diminished GluN2B-containing NMDA receptor levels and density as well as NMDA-dependent tau phosphorylation. These alterations were accompanied by a marked drop in neuronal arborization that was prevented by an acute GluN2B-containing NMDA receptor stimulation. Our findings support that PGRN decrease, resulting from pathogenic mutations, might compromise the trophism of cortical neurons by affecting GluN2B-contaning NMDA receptors. These mechanisms might be implicated in the pathogenesis of FTLD.

Trigeminal brainstem modulation of persistent orbicularis oculi muscle activity in a rat model of dry eye

Altered corneal reflex activity is a common feature of dry eye disease (DE). Trigeminal sensory nerves supply the ocular surface and terminate at the trigeminal interpolaris/caudalis (ViVc) transition and spinomedullary (VcC1) regions. Although both regions contribute to corneal reflexes, their role under dry eye conditions is not well defined. This study assessed the influence of local inhibitory and excitatory amino acid neurotransmission at the ViVc transition and VcC1 regions on hypertonic saline (HS) evoked orbicularis oculi muscle activity (OOemg) in urethane-anesthetized male rats after exorbital gland removal (DE). HS increased the magnitude of long-duration OOemg activity (OOemgL, >200ms) in DE compared to sham rats, while short-duration OOemg activity (OOemgS, <200ms) was similar for both groups. Inhibition of the ViVc transition by muscimol, a GABAA receptor agonist, greatly reduced HS-evoked OOemgL activity in DE rats, whereas injections at the VcC1 region had only minor effects in both groups. Blockade of GABAA receptors by bicuculline methiodide at the ViVc transition or VcC1 region increased HS-evoked OOemgL activity in DE rats. Blockade of N-methyl-D-aspartate (NMDA) receptors at either region reduced HS-evoked OOemgL activity in DE and sham rats. GABAαβ3 receptor density was reduced at the ViVc transition, while NMDA receptor density was increased at both regions in DE rats. Loss of GABAergic inhibition at the ViVc transition coupled with enhanced NMDA excitatory amino acid neurotransmission at the ViVc transition and the VcC1 region likely contribute to altered corneal reflexes under dry eye conditions.

The Density of N-Methyl-D-Aspartate Receptor in Rat Striatum Varies with Differing Psychotropic Drug Treatments

Background: A number of studies in rodents suggest that antipsychotic drug treatment can either alter the levels or functionality of the N-methyl-D-aspartate receptor (NMDAR). This raises the possibility that some of the therapeutic actions or side-effects of antipsychotic drugs could be mediated through the NMDAR. Objective: To determine if treatments with combinations of psychotropic drugs change the levels of the NMDAR in rat Central Nervous System (CNS). Methods: In situ radioligand binding and autoradiography were used to quantify [3H]MK-801 binding to NMDAR in frozen CNS sections from rats treated with vehicle, haloperidol (0.1 mg/kg/day), olanzapine (1.0 mg/kg/ day), lithium (25.5 mg/kg/day) or, at the same doses, haloperidol and lithium or olanzapine and lithium for 28 days. Results: There was a significant variation of [3H]MK-801 binding with treatment in the rat striatum due to higher levels of radioligand in rats treated with haloperidol compared to those treated with olanzapine and lithium (p < 0.01; Cohen’s d = 1.47). Levels of radioligand binding did not vary with drug treatment in the cortex. Conclusion: These data argue for some effect of polypharmacy on NMDAR levels, possibly localized to the striatum. Further studies of mood stabilisers with known effects on NMDAR functionality, such as valproate and lamotrigine, with and without antipsychotic drugs could be worthwhile as they may show a greater effect on NMDAR density in the CNS. Keywords: Glutamate, haloperidol, lithium, olanzapine, NMDA, psychotropic drug.

Circuit-specific changes in D-serine-dependent activation of the N-methyl-D-aspartate receptor in the aging hippocampus.

Age-related memory deficits have recently been associated with the impaired expression of D-serine-dependent synaptic plasticity in neuronal networks of the hippocampal CA1 area. However, whether such functional alterations are common to the entire hippocampus during aging remains unknown. Here, we found that D-serine was also required for the induction of N-methyl-D-aspartate receptor (NMDA-R)-dependent long-term potentiation (LTP) at perforant path-granule cell synapses of the dentate gyrus. LTP as well as isolated NMDA-R synaptic potentials were impaired in slices from aged rats, but in contrast to the CA1, this defect was not reversed by exogenous D-serine. The lower activation of the glycine-binding site by the endogenous co-agonist does not therefore appear to be a critical mechanism underlying age-related deficits in NMDA-R activation in the dentate gyrus. Instead, our data highlight the role of changes in presynaptic inputs as illustrated by the weaker responsiveness of afferent glutamatergic fibers, as well as changes in postsynaptic NMDA-R density. Thus, our study indicates that although NMDA-R-dependent mechanisms driving synaptic plasticity are quite similar between hippocampal circuits, they show regional differences in their susceptibility to aging, which could hamper the development of effective therapeutic strategies aimed at reducing cognitive aging.

Activity‐dependent regulation of NMDA receptors in substantia nigra dopaminergic neurones

N-Methyl-d-aspartate receptors (NMDARs) are Ca2+-permeable glutamate receptors that play a critical role in synaptic plasticity and promoting cell survival. However, overactive NMDARs can trigger cell death signalling pathways and have been implicated in substantia nigra pars compacta (SNc) pathology in Parkinson's disease. Calcium ion influx through NMDARs recruits Ca2+-dependent proteins that can regulate NMDAR activity. The surface density of NMDARs can also be regulated dynamically in response to receptor activity via Ca2+-independent mechanisms. We have investigated the activity-dependent regulation of NMDARs in SNc dopaminergic neurones. Repeated whole-cell agonist applications resulted in a decline in the amplitude of NMDAR currents (current run-down) that was use dependent and not readily reversible. Run-down was reduced by increasing intracellular Ca2+ buffering or by reducing Ca2+ influx but did not appear to be mediated by the same regulatory proteins that cause Ca2+-dependent run-down in hippocampal neurones. The NMDAR current run-down may be mediated in part by a Ca2+-independent mechanism, because intracellular dialysis with a dynamin-inhibitory peptide reduced run-down, suggesting a role for clathrin-mediated endocytosis in the regulation of the surface density of receptors. Synaptic NMDARs were also subject to current run-down during repeated low-frequency synaptic stimulation in a Ca2+-dependent but dynamin-independent manner. Thus, we report, for the first time, regulation of NMDARs in SNc dopaminergic neurones by changes in intracellular Ca2+ at both synaptic and extrasynaptic sites and provide evidence for activity-dependent changes in receptor trafficking. These mechanisms may contribute to intracellular Ca2+ homeostasis in dopaminergic neurones by limiting Ca2+ influx through the NMDAR.

Effect of prenatal stress on density of NMDA receptors in rat brain

N-methyl-d-aspartate (NMDA) receptors are important excitatory receptors which contribute to many brain functions. Altered NMDA receptor levels cause maldevelopment of corticostriatal and corticolimbic pathways, which is a neurobiological predisposing factor for development of epilepsy, schizophrenia and other idiopathic psychotic disorders. It was hypothesized that prenatal stress could play a role in pathophysiology of these disorders by affecting expression of the receptors through releasing corticosterone. Sixty-eight virgin female Wistar rats were selected and mated with male rats with the same genotype. Then, the pregnant rats were subjected to restraint or predator stress on 15th, 16th and 17th gestation days. Prenatal stress consisted of restraint or predator stresses of the dams under normal room conditions. After parturition, the pups were studied in terms of density of NMDA receptors in brain at different time points. Meanwhile, blood sample was obtained and corticosterone blood level (CBL) was measured. The pups were then compared with the pups born to unstressed dams. Stress induced significant rise in CBL and NMDA receptors in brain of the offspring. CBL was significantly higher among the stressed rats compared to the control ones; there was significant difference between the two stresses and between the two sexes. The male pups were affected more severely. Stressful events during gestation had important effects on NMDA receptors of the offspring. It can be concluded that stress-induced elevation of NMDA receptors and corticosterone might mediate altered susceptibility to epilepsy and decrease ability of learning and memory and other stress-induced neurologic disorders.

Changes in synaptic transmission and protein expression in the brains of adult offspring after prenatal inhibition of the kynurenine pathway

During early brain development, N-methyl-d-aspartate (NMDA) receptors are involved in cell migration, neuritogenesis, axon guidance and synapse formation, but the mechanisms which regulate NMDA receptor density and function remain unclear. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at NMDA receptors and we have previously shown that inhibition of the pathway using the kynurenine-3-monoxygenase inhibitor Ro61-8048 in late gestation produces rapid changes in protein expression in the embryos and effects on synaptic transmission lasting until postnatal day 21 (P21). The present study sought to determine whether any of these effects are maintained into adulthood. After prenatal injections of Ro61-8048 the litter was allowed to develop to P60 when some offspring were euthanized and the brains removed for examination. Analysis of protein expression by Western blotting revealed significantly reduced expression of the GluN2A subunit (32%) and the morphogenetic protein sonic hedgehog (31%), with a 29% increase in the expression of doublecortin, a protein associated with neurogenesis. No changes were seen in mRNA abundance using quantitative real-time polymerase chain reaction. Neuronal excitability was normal in the CA1 region of hippocampal slices but paired-pulse stimulation revealed less inhibition at short interpulse intervals. The amount of long-term potentiation was decreased by 49% in treated pups and recovery after low-frequency stimulation was delayed. The results not only strengthen the view that basal, constitutive kynurenine metabolism is involved in normal brain development, but also show that changes induced prenatally can affect the brains of adult offspring and those changes are quite different from those seen previously at weaning (P21). Those changes may be mediated by altered expression of NMDAR subunits and sonic hedgehog.

NMDA Receptors in GABAergic Synapses during Postnatal Development

GABA (gamma-aminobutyric-acid), the main inhibitory neurotransmitter in the adult brain, exerts depolarizing (excitatory) actions during development and this GABAergic depolarization cooperates with NMDARs (N-methyl-D-aspartate receptors) to drive spontaneous synchronous activity (SSA) that is fundamentally important for developing neuronal networks. Although GABAergic depolarization is known to assist in the activation of NMDARs during development, the subcellular localization of NMDARs relative to GABAergic synapses is still unknown. Here, we investigated the subcellular distribution of NMDARs in association with GABAergic synapses at the developmental stage when SSA is most prominent in mice. Using multiple immunofluorescent labeling and confocal laser-scanning microscopy in the developing mouse hippocampus, we found that NMDARs were associated with both glutamatergic and GABAergic synapses at postnatal day 6–7 and we observed a direct colocalization of GABAA- and NMDA-receptor labeling in GABAergic synapses. Electron microscopy of pre-embedding immunogold-immunoperoxidase reactions confirmed that GluN1, GluN2A and GluN2B NMDAR subunits were all expressed in glutamatergic and GABAergic synapses postsynaptically. Finally, quantitative post-embedding immunogold labeling revealed that the density of NMDARs was 3 times higher in glutamatergic than in GABAergic synapses. Since GABAergic synapses were larger, there was little difference in the total number of NMDA receptors in the two types of synapses. In addition, receptor density in synapses was substantially higher than extrasynaptically. These data can provide the neuroanatomical basis of a new interpretation of previous physiological data regarding the GABAAR-NMDAR cooperation during early development. We suggest that during SSA, synaptic GABAAR-mediated depolarization assists NMDAR activation right inside GABAergic synapses and this effective spatial cooperation of receptors and local change of membrane potential will reach developing glutamatergic synapses with a higher probability and efficiency even further away on the dendrites. This additional level of cooperation that operates within the depolarizing GABAergic synapse, may also allow its own modification triggered by Ca2+-influx through the NMDA receptors.

Antidepressant-like effects of low ketamine dose is associated with increased hippocampal AMPA/NMDA receptor density ratio in female Wistar–Kyoto rats

Preclinical as well as limited clinical studies indicate that ketamine, a non-competitive glutamate N-methyl-d-aspartate (NMDA) receptor antagonist, may exert a quick and prolonged antidepressant effect. It has been postulated that ketamine action is due to inhibition of NMDA and stimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. Here, we sought to determine whether ketamine would exert antidepressant effects in Wistar–Kyoto (WKY) rats, a putative animal model of depression and whether this effect would be associated with changes in AMPA/NMDA receptor densities in the hippocampus. Adult female WKY rats and their control Wistar rats were subjected to acute and chronic ketamine doses and their locomotor activity (LMA) and immobility in the forced swim test (FST) were evaluated. Hippocampal AMPA and NMDA receptor densities were also measured following a chronic ketamine dose. Ketamine, both acutely (0.5–5.0mg/kg i.p.) and chronically (0.5–2.5mg/kg daily for 10days) resulted in a dose-dependent and prolonged decrease in immobility in FST in WKY rats only, suggesting an antidepressant-like effect in this model. Chronic treatment with an effective dose of ketamine also resulted in an increase in AMPA/NMDA receptor density ratio in the hippocampus of WKY rats. LMA was not affected by any ketamine treatment in either strain. These results indicate a rapid and lasting antidepressant-like effect of a low ketamine dose in WKY rat model of depression. Moreover, the increase in AMPA/NMDA receptor density in the hippocampus could be a contributory factor to behavioral effects of ketamine. These findings suggest potential therapeutic benefit in simultaneous reduction of central NMDA and elevation of AMPA receptor function in treatment of depression.

Developmental vitamin D deficiency alters MK-801-induced behaviours in adult offspring

Developmental vitamin D (DVD) deficiency is a candidate risk factor for developing schizophrenia in humans. In rodents DVD deficiency induces subtle changes in the way the brain develops. This early developmental insult leads to select behavioural changes in the adult, such as an enhanced response to amphetamine-induced locomotion in female DVD-deficient rats but not in male DVD-deficient rats and an enhanced locomotor response to the N-methyl-D: -aspartate (NMDA) receptor antagonist, MK-801, in male DVD-deficient rats. However, the response to MK-801-induced locomotion in female DVD-deficient rats is unknown. Therefore, the aim of the current study was to further examine this behavioural finding in male and female rats and assess NMDA receptor density. DVD-deficient Sprague Dawley rats were assessed for locomotion, ataxia, acoustic startle response (ASR) and prepulse inhibition (PPI) of the ASR to multiple doses of MK-801. The NMDA receptor density in relevant brain regions was assessed in a drug-naive cohort. DVD deficiency increased locomotion in response to MK-801 in both sexes. DVD-deficient rats also showed an enhanced ASR compared with control rats, but PPI was normal. Moreover, DVD deficiency decreased NMDA receptor density in the caudate putamen of both sexes. These results suggest that a transient prenatal vitamin D deficiency has a long-lasting effect on NMDA-mediated signalling in the rodent brain and may be a plausible candidate risk factor for schizophrenia and other neuropsychiatric disorders.

Temporal and Regional Alterations in NMDA Receptor Expression in Mecp2‐Null Mice

Our previous postmortem study of girls with Rett Syndrome (RTT), a development disorder caused by MECP2 mutations, found increases in the density of N-Methyl-D-aspartate (NMDA) receptors in the prefrontal cortex of 2-8-year-old girls, whereas girls older than 10 years had reductions in NMDA receptors compared with age-matched controls (Blue et al., Ann Neurol 1999b;45:541-545). Using [(3)H]-CGP to label NMDA-type glutamate receptors in 2- and 7-week old wild-type (WT), Mecp2-null, and Mecp2-heterozygous (HET) mice (Bird model), we found that frontal areas of the brain also exhibited a bimodal pattern in NMDA expression, with increased densities of NMDA receptors in Mecp2-null mice at 2 weeks of age but decreased densities at 7 weeks of age. Visual cortex showed a similar pattern, while other cortical regions only exhibited changes in NMDA receptor densities at 2 weeks (retrosplenial granular) or 7 weeks (somatosensory). In thalamus of null mice, NMDA receptors were increased at 2 and 7 weeks. No significant differences in density were found between HET and WT mice at both ages. Western blots for NMDAR1 expression in frontal brain showed higher levels of expression in Mecp2-null mice at 2 weeks of age but not at 1 or 7 weeks of age. Our mouse data support the notion that deficient MeCP2 function is the primary cause of the NMDA receptor changes we observed in RTT. Furthermore, the findings of regional and temporal differences in NMDA expression illustrate the importance of age and brain region in evaluating different genotypes of mice.

Chronic administration of MK-801 and the NMDA receptor: Further evidence for reduced sensitivity of the primary acceptor site from studies with the cortical wedge preparation

Cortical slices removed from rats pre-treated with MK-901 0.5 mg kg-1 twice a day for 7 days had reduced responses to N-methyl-D-aspartate (NMDA) relative to quisqualate and glutamate compared with control animals. Potencies of competitive (CPMP) and non-competitive (ketamine) NMDA antagonists appeared unchanged. These changes are consistent with a reduced density of NMDA receptors.

The effects of sub-chronic clozapine and haloperidol administration on isolation rearing induced changes in frontal cortical N-methyl- d-aspartate and D 1 receptor binding in rats

Glutamate and dopamine disturbances are implicated in frontal cortical dysfunction in schizophrenia. Little, however, is known about the nature of dopamine D 1 and N-methyl- d-aspartate (NMDA) receptor interactions in the illness, nor of the extent of their co-involvement in antipsychotic drug response. It is well known that early life adversity may pre-date the development of schizophrenia. Using a neurodevelopmental model of schizophrenia, namely post weaning social isolation rearing (SIR), we studied the effect of SIR (post natal day 21–61) on frontal cortical NMDA and D 1 receptor binding characteristics with/without chronic haloperidol (0.1 mg/kg/day i.p.) or clozapine (5 mg/kg/day i.p.) treatment, undertaken from post-natal day 50–60. SIR increased frontal cortical NMDA-density, with decreased affinity (decreased pK D), but reduced D 1 receptor density (without effects on pK D). In socially reared animals, clozapine but not haloperidol increased NMDA receptor density without effects on pK D. Neither drug markedly affected D 1 receptor density, although clozapine increased D 1 affinity. Increased NMDA density in SIR animals was unaffected by haloperidol, but further increased by clozapine. However, SIR-associated decrease in NMDA affinity remained unaltered despite drug treatment. Reduced D 1 receptor density in SIR animals was exacerbated by haloperidol, but unaltered by clozapine, without changes in pK D. SIR thus induces opposing effects on frontal cortical NMDA and D 1 radio-receptor binding characteristics, which has direct bearing on the mutual interplay of these receptors in schizophrenia. The ability of SIR to affect NMDA receptor affinity warrants deeper study. Furthermore, at the doses examined, in contrast to haloperidol, clozapine bolsters frontal cortical glutamatergic but stabilizes D 1 dopaminergic pathways in a neurodevelopmental animal model of schizophrenia, possibly explaining the atypical clinical characteristics of this drug.

Selective vulnerability of hippocampal cornu ammonis 1 pyramidal cells to excitotoxic insult is associated with the expression of polyamine-sensitive N-methyl- d-asparate-type glutamate receptors

Excess glutamate release and stimulation of post-synaptic glutamatergic receptors have been implicated in the pathophysiology of many neurological diseases. The hippocampus, and the pyramidal cell layer of the cornu ammonus 1 (CA1) region in particular, has been noted for its selective sensitivity to excitotoxic insults. The current studies examined the role of N-methyl- d-aspartate (NMDA) receptor subunit composition and sensitivity to stimulatory effects of the polyamine spermidine, an allosteric modulator of NMDA NR2 subunit activity, in hippocampal CA1 region sensitivity to excitotoxic insult. Organotypic hippocampal slice cultures of 8 day-old neonatal rat were obtained and maintained in vitro for 5 days. At this time, immunohistochemical analysis of mature neuron density (NeuN); microtubule associated protein-2(a,b) density (MAP-2); and NMDA receptor NR1 and NR2B subunit density in the primary cell layers of the dentate gyrus (DG), CA3, and CA1 regions, was conducted. Further, autoradiographic analysis of NMDA receptor distribution and density (i.e. [ 125I]MK-801 binding) and spermidine (100 μM)-potentiated [ 125I]MK-801 binding in the primary cell layers of these regions was examined. A final series of studies examined effects of prolonged exposure to NMDA (0.1–10 μM) on neurodegeneration in the primary cell layers of the DG, CA3, and CA1 regions, in the absence and presence of spermidine (100 μM) or ifenprodil (100 μM), an allosteric inhibitor of NR2B polypeptide subunit activity. The pyramidal cell layer of the CA1 region demonstrated significantly greater density of mature neurons, MAP-2, NR1 and NR2B subunits, and [ 125I]MK-801 binding than the CA3 region or DG. Twenty-four hour NMDA (10 μM) exposure produced marked neurodegeneration (∼350% of control cultures) in the CA1 pyramidal cell region that was significantly reduced by co-exposure to ifenprodil or dl-2-Amino-5-phosphonopentanoic acid (APV). The addition of spermidine significantly potentiated [ 125I]MK-801 binding and neurodegeneration induced by exposure to a non-toxic concentration of NMDA, exclusively in the CA1 region. This neurodegeneration was markedly reduced with co-exposure to ifenprodil. These data suggest that selective sensitivity of the CA1 region to excitotoxic stimuli may be attributable to the density of mature neurons expressing polyamine-sensitive NR2B polypeptide subunits.

Intra-cornu ammonis 1 administration of the human immunodeficiency virus-1 protein trans-activator of transcription exacerbates the ethanol withdrawal syndrome in rodents and activates N-methyl- d-aspartate glutamate receptors to produce persisting spatial learning deficits

Human immunodeficiency virus-1 (HIV-1) infection may produce neurological deficits, such as cognitive decline, that may be worsened by concurrent ethanol (EtOH) abuse. Among the many biochemical cascades likely mediating HIV-1-associated neuronal injury is enhancement of N-methyl- d-aspartate (NMDA) receptor function and progression to excitotoxicity, an effect that may be directly or indirectly related to accumulation in brain of the HIV-1 trans-activator of transcription (Tat) factor. The present studies were designed to examine the hypothesis that binge-like EtOH pre-exposure would enhance effects of Tat on NMDA receptor function. These studies employed a modified in vivo binge EtOH exposure regimen designed to produce peak blood EtOH levels (BEL) of <200 mg/dl in adult male rats and were designed to examine effects of intra-hippocampal injection of Tat (0.5 μl/500 pM/2 min) on EtOH withdrawal-related behavior, spatial learning, and histological measures. Unilateral cannulae were implanted into the cornu ammonis 1 (CA1) pyramidal cell layer of animals prior to beginning a 4-day binge EtOH regimen. EtOH was administered via intragastric intubation (∼3.0–5.0 g/kg) with dose determined by behavioral ratings of intoxication daily for 4 days (at 08:00, 16:00, and 24:00 h). EtOH withdrawal behaviors were monitored 12 h after the last administration of EtOH. Morris water maze learning was assessed during the following 4 days, at which times brains were harvested for autoradiographic measurement of NMDA receptor density and neuroinflammation. Maximal BELs of 187.69 mg/dl were observed 60 min after EtOH administration on day 2 of the regimen. In contrast, peak BELs of approximately 100 mg/dl were observed 60 min after EtOH administration on day 4 of the regimen, suggesting development of metabolic tolerance. Significant behavioral abnormalities were observed in EtOH withdrawn animals, including tremor and seizures. Intra-CA1 region injection of Tat significantly potentiated EtOH withdrawal behavioral abnormalities, an effect that was reduced by MK-801 pre-exposure. While EtOH withdrawn animals showed learning similar to control animals, EtOH withdrawn animals that received intra-CA1 Tat injection demonstrated persisting deficits in spatial learning on days 3 and 4 of training, effects that were markedly reduced by administration of the competitive NMDA receptor antagonist MK-801 30 min prior to Tat injection. No changes in [ 3H]MK-801 binding were observed. Binding density of [ 3H]PK11195, a ligand for peripheral benzodiazepine receptors expressed on activated microglia, was elevated proximal to cannula tracks in all animals, but was not altered by EtOH or Tat exposure. These findings suggest that EtOH abuse and/or dependence in HIV-positive individuals may promote HIV-1-associated cognitive deficits by altering NMDA receptor function in the absence of microglial activation or neuroinflammation.

Chronic administration of valproic acid reduces brain NMDA signaling via arachidonic acid in unanesthetized rats.

Evidence that brain glutamatergic activity is pathologically elevated in bipolar disorder suggests that mood stabilizers are therapeutic in the disease in part by downregulating glutamatergic activity. Such activity can involve the second messenger, arachidonic acid (AA, 20:4n - 6). We tested this hypothesis with regard to valproic acid (VPA), when stimulating glutamatergic N-methyl-D: -aspartate (NMDA) receptors in rat brain and measuring AA and related responses. An acute subconvulsant dose of NMDA (25 mg/kg i.p.) or saline was administered to unanesthetized rats that had been treated i.p. daily with VPA (200 mg/kg) or vehicle for 30 days. Quantitative autoradiography following intravenous [1-(14)C]AA infusion was used to image regional brain AA incorporation coefficients k*, markers of AA signaling. In chronic vehicle-pretreated rats, NMDA compared with saline significantly increased k* in 41 of 82 examined brain regions, many of which have high NMDA receptor densities, and also increased brain concentrations of the AA metabolites, prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TXB(2)). VPA pretreatment reduced baseline concentrations of PGE(2) and TXB(2), and blocked the NMDA induced increases in k* and in eicosanoid concentrations. These results, taken with evidence that carbamazepine and lithium also block k* responses to NMDA in rat brain, suggest that mood stabilizers act in bipolar disorder in part by downregulating glutamatergic signaling involving AA.