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Glutamatergic systems have been increasingly recognized as mediators of methamphetamine’s (METH) pharmacological effects though little is known about the means by which METH interacts with glutamate receptors. The present studies examined effects of METH (0.1–100 μM) on [3H]MK-801 binding to membranes prepared from adult rat cortex, hippocampus and cerebellum, as well as the neurotoxicity produced by 24-h exposure to N-methyl-D-aspartate (5–10 μM; NMDA) employing organotypic hippocampal slice cultures of neonatal rat. Co-incubation of [3H]MK-801 with METH (0.1–100 μM) did not reduce dextromethorphan (1 mM)-displaceable ligand binding. Exposure of slice cultures to NMDA for 24-h produced increases in uptake of the non-vital fluorescent marker propidium iodide (PI) of 150–500% above control levels, most notably, in the CA1 region pyramidal cell layer. Co-exposure to METH (>1.0 μM) with NMDA (5 μM) reduced PI uptake by approximately 50% in each subregion, though the CA1 pyramidal cell layer was markedly more sensitive to the protective effects of METH exposure. In contrast, METH exposure did not reduce PI uptake stimulated by 24-h exposure to 10 μM NMDA. Co-exposure to the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (20 μM) prevented toxicity produced by exposure to 5 or 10 μM NMDA. These findings indicate that the pharmacological effects of short-term METH exposure involve inhibition of NMDA receptor-mediated neuronal signaling, not reflective of direct channel inhibition at an MK-801-sensitive site.

N-methyl-D-aspartate (NMDA) receptors are associated with many forms of synaptic plasticity. Their expression level and subunit composition undergo developmental changes in several brain regions. In the mouse cerebellum, beside a developmental switch between NR2B and NR2A/C subunits in granule cells, functional postsynaptic NMDA receptors are seen in Purkinje cells of neonate and adult but not juvenile rat and mice. A presynaptic effect of NMDA on GABA release by cerebellar interneurons was identified recently. Nevertheless whereas NMDA receptor subunits are detected on parallel fiber terminals, a presynaptic effect of NMDA on spontaneous release of glutamate has not been demonstrated. Using mouse cerebellar cultures and patch-clamp recordings we show that NMDA facilitates glutamate release onto Purkinje cells in young cultures via a presynaptic mechanism, whereas NMDA activates extrasynaptic receptors in Purkinje cells recorded in old cultures. The presynaptic effect of NMDA on glutamate release is also observed in Purkinje cells recorded in acute slices prepared from juvenile but not from adult mice and requires a specific protocol of NMDA application.

Agmatine is a polyamine and has been considered as a novel neurotransmitter or neuromodulator in the central nervous system. In the present study, the neuroprotective effect of agmatine against cell damage caused by N-methyl-d-aspartate (NMDA) and glutamate was investigated in cultured rat hippocampal neurons. Lactate dehydrogenase (LDH) activity assay, β-tubulin III immunocytochemical staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay were conducted to detect cell damage. Exposure of 12-day neuronal cultures of rat hippocampus to NMDA or glutamate for 1 h caused a concentration-dependent neurotoxicity, as indicated by the significant increase in released LDH activities. Addition of 100 µM agmatine into media ablated the neurotoxicity induced by NMDA or glutamate, an effect also produced by the specific NMDA receptor antagonist dizocilpine hydrogen maleate (MK801). Arcaine, an analog of agmatine with similar structure as agmatine, fully prevented the NMDA- or glutamate-induced neuronal damage. Spermine and putrescine, the endogenous polyamine and metabolic products of agmatine without the guanidine moiety of agmatine, failed to show this effect, indicating a structural relevance for this neuroprotection. Immunocytochemical staining and TUNEL assay confirmed the findings in the LDH measurement. That is, agmatine and MK801 markedly attenuated NMDA-induced neuronal death and significantly reduced TUNEL-positive cell numbers induced by exposure of cultured hippocampal neurons to NMDA. Taken together, these results demonstrate that agmatine can protect cultured hippocampal neurons from NMDA- or glutamate-induced excitotoxicity, through a possible blockade of the NMDA receptor channels or a potential anti-apoptotic property.

One of the most consistent findings in schizophrenia is the decreased expression of the GABA synthesizing enzymes GAD67 and GAD65 in specific interneuron populations. This dysfunction is observed in distributed brain regions including the prefrontal cortex, hippocampus, and cerebellum. In an effort to understand the mechanisms for this GABA deficit, we investigated the effect of the N-methyl-D-aspartate receptor (NMDAR) antagonist phencyclidine (PCP), which elicits schizophrenia-like symptoms in both humans and animal models, in a chronic, low-dose exposure paradigm. Adult rats were given PCP at a dose of 2.58 mg/kg/day i.p. for a month, after which levels of various GABAergic cell mRNAs and other neuromodulators were examined in the cerebellum by RT-qPCR. Administration of PCP decreased the expression of GAD67, GAD65, and the presynaptic GABA transporter GAT-1, and increased GABAA receptor subunits similar to those seen in patients with schizophrenia. Additionally, we found that the mRNA levels of two Golgi cell selective NMDAR subunits, NR2B and NR2D, were decreased in PCP treated rats. Furthermore, we localized the deficits in GAD67 expression solely to these interneurons. Slice electrophysiological studies showed that spontaneous firing of Golgi cells was reduced by acute exposure to low dose PCP, suggesting that these neurons are particularly vulnerable to NMDA receptor antagonism. In conclusion, our results demonstrate that chronic exposure to low levels of PCP in rats mimics the GABAergic alterations reported in the cerebellum of patients with schizophrenia (Bullock et al., Am J Psychiatry 165: 1594-1603, 2008), further supporting the validity of this animal model.

Background

Acute exposure of ethanol (alcohol) inhibits NMDA receptor function. Our previous study showed that acute ethanol inhibited the pressor responses induced by NMDA applied intrathecally; however, prolonged ethanol exposure may increase the levels of phosphorylated NMDA receptor subunits leading to changes in ethanol inhibitory potency on NMDA-induced responses. The present study was carried out to examine whether acute ethanol exposure influences the effects of ketamine, a noncompetitive NMDA receptor antagonist, on spinal NMDA-induced pressor responses.

Methods

The blood pressure responses induced by intrathecal injection of NMDA were recorded in urethane-anesthetized rats weighing 250-275 g. The levels of several phosphorylated residues on NMDA receptor GluN1 subunits were determined by western blot analysis.

Results

Intravenous injection of ethanol or ketamine inhibited spinal NMDA-induced pressor responses in a dose-dependent and reversible manner. Ketamine inhibition of NMDA-induced responses was synergistically potentiated by ethanol when ethanol was applied just before ketamine. However, ketamine inhibition was significantly reduced when applied at 10 min after ethanol administration. Western blot analysis showed that intravenous ethanol increased the levels of phosphoserine 897 on GluN1 subunits (pGluN1-serine 897), selectively phosphorylated by protein kinase A (PKA), in the lateral horn regions of spinal cord at 10 min after administration. Intrathecal administration of cAMPS-Sp, a PKA activator, at doses elevating the levels of pGluN1-serine 897, significantly blocked ketamine inhibition of spinal NMDA-induced responses.

Conclusions

The results suggest that ethanol may differentially regulate ketamine inhibition of spinal NMDA receptor function depending on ethanol exposure time and the resulting changes in the levels of pGluN1-serine 897.

Lead is a male reproductive toxicant. Data suggest that rats dosed with relatively high levels of lead acetate for short periods of time induced changes in the hypothalamic gonadotropin-releasing hormone (GnRH) at the molecular level, but these changes were attenuated with increased concentration of exposure. The current study evaluated whether exposure to low levels of lead acetate over longer periods of time would produce a similar pattern of adaptation to toxicity at the molecular and biologic levels. Adult 100-day-old Sprague-Dawley male rats were dosed with 0, 0.025, 0.05, 0.1, and 0.3% lead acetate in water. Animals were killed after 1, 4, 8, and 16 weeks of treatment. Luteinzing hormone (LH) and GnRH levels were measured in serum, and lead levels were quantified in whole blood. Hypothalamic GnRH mRNA levels were also quantified. We found no significant differences in serum LH and GnRH among the groups of animals treated within each time period. A significant dose-related increase of GnRH mRNA concentrations with lead dosing occurred in animals treated for 1 week. Animals treated for more than 1 week also exhibited a significant increase in GnRH mRNA, but with an attenuation of the increase at the higher concentrations of lead with increased duration of exposure. We conclude that the signals within and between the hypothalamus and pituitary gland appear to be disrupted by long-term, low-dose lead exposure.

Using a new method which combines a brain microdialysis technique and measurement of nitrite/nitrate levels by the Griess reaction, it has been proven that activation of N-methyl-D-aspartate (NMDA) receptors in the cerebelli of rats which had been under non-anesthetic and freely moving conditions induces the release of nitric oxide (NO). Since L-NG-monomethylarginine (L-NMMA), which competitively blocks NO synthesis from L-arginine, significantly inhibited the release of nitrite/nitrate from the rat cerebellum, these results indicate that the new method is capable of measuring NO formation from L-arginine following the stimulation of NMDA receptors. This method should prove useful for investigating the relation between brain functions such as behavior, learning and memory and NO in the central nervous system.

Glycogen synthase kinase-3 (GSK3), which is inhibited by serine-phosphorylation, is involved in the neuropathology of Alzheimer's disease (AD). We tested if the two therapeutic strategies used for AD, inhibition of acetylcholinesterase and of N-methyl-d-aspartate (NMDA) receptors, modulate the phosphorylation state of the two isoforms of GSK3 in mouse brain. Large, rapid increases in the levels of phospho-Ser21-GSK3α and phospho-Ser9-GSK3β occurred in mouse hippocampus, cerebral cortex, and striatum after treatment of mice with the muscarinic agonist pilocarpine or the acetylcholinesterase inhibitor physostigmine. Treatment with memantine, an NMDA receptor antagonist, also increased the serine-phosphorylation of both GSK3 isoforms in mouse brain. Co-administration of physostigmine and memantine increased serine-phosphorylated GSK3 levels equally to that achieved by either agent alone, indicating that the actions of these two drugs converge on overlapping pools of GSK3. Thus, drugs in each class of therapeutic agents used for AD have the common property of increasing the regulatory serine-phosphorylation of GSK3 within common pools of the enzyme.

The elevated nitric oxide/peroxynitrite and the neural sensitization theories of multiple chemical sensitivity (MCS) are extended here to propose a central mechanism for the exquisite sensitivity to organic solvents apparently induced by previous chemical exposure in MCS. This mechanism is centered on the activation of N-methyl-D-aspartate (NMDA) receptors by organic solvents producing elevated nitric oxide and peroxynitrite, leading in turn to increased stimulating of and hypersensitivity of NMDA receptors. In this way, organic solvent exposure may produce progressive sensitivity to organic solvents. Pesticides such as organophosphates and carbamates may act via muscarinic stimulation to produce a similar biochemical and sensitivity response. Accessory mechanisms of sensitivity may involve both increased blood-brain barrier permeability, induced by peroxynitrite, and cytochrome P450 inhibition by nitric oxide. The NMDA hyperactivity/hypersensitivity and excessive nitric oxide/peroxynitrite view of MCS provides answers to many of the most puzzling aspects of MCS while building on previous studies and views of this condition.

Domoic acid (DomA) is a naturally occurring shellfish toxin that can induce brain damage in mammalians. Neonates have shown increased sensitivity to DomA-induced toxicity, and prenatal exposure has been associated with e.g. decreased brain GABA levels, and increased glutamate levels. Here, we evaluated DomA-induced toxicity in immature and mature primary cultures of neurons and glial cells from rat cerebellum by measuring the mRNA levels of selected genes. Moreover, we assessed if the induced toxicity was mediated by the activation of the AMPA/KA and/or the NMDA receptor. The expression of all studied neuronal markers was affected after DomA exposure in both immature and mature cultures. However, the mature cultures seemed to be more sensitive to the treatment, as the effects were observed at lower concentrations and at earlier time points than for the immature cultures. The DomA effects were completely prevented by the antagonist of the AMPA/KA receptor (NBQX), while the antagonist of the NMDA receptor (APV) partly blocked the DomA-induced effects. Interestingly, the DomA-induced effect was also partly prevented by the neurotransmitter GABA. DomA exposure also affected the mRNA levels of the astrocytic markers in mature cultures. These DomA-induced effects were reduced by the addition of NBQX, APV, and GABA.

N-methyl-D-aspartate (NMDA) receptors are present in high density within the cerebral cortex and hippocampus and play an important role in learning and memory. NMDA receptors are negatively affected by aging, but these effects are not uniform in many different ways. This review discusses the selective age-related vulnerabilities of different binding sites of the NMDA receptor complex, different subunits that comprise the complex, and the expression and functions of the receptor within different brain regions. Spatial reference, passive avoidance, and working memory, as well as place field stability and expansion all involve NMDA receptors. Aged animals show deficiencies in these functions, as compared to young, and some studies have identified an association between age-associated changes in the expression of NMDA receptors and poor memory performance. A number of diet and drug interventions have shown potential for reversing or slowing the effects of aging on the NMDA receptor. On the other hand, there is mounting evidence that the NMDA receptors that remain within aged individuals are not always associated with good cognitive functioning. This may be due to a compensatory response of neurons to the decline in NMDA receptor expression or a change in the subunit composition of the remaining receptors. These studies suggest that developing treatments that are aimed at preventing or reversing the effects of aging on the NMDA receptor may aid in ameliorating the memory declines that are associated with aging. However, we need to be mindful of the possibility that there may also be negative consequences in aged individuals.

An association between age-related memory impairments and changes in functional plasticity in the aging brain has been under intense study within the last decade. In this article, we show that an impaired activation of the strychnine-insensitive glycine site of N-methyl-d-aspartate receptors (NMDA-R) by its agonist d-serine contributes to deficits of synaptic plasticity in the hippocampus of memory-impaired aged rats. Supplementation with exogenous d-serine prevents the age-related deficits of isolated NMDA-R-dependent synaptic potentials as well as those of theta-burst-induced long-term potentiation and synaptic depotentiation. Endogenous levels of d-serine are reduced in the hippocampus with aging, that correlates with a weaker expression of serine racemase synthesizing the amino acid. On the contrary, the affinity of d-serine binding to NMDA-R is not affected by aging. These results point to a critical role for the d-serine-dependent pathway in the functional alterations of the brain underlying memory impairment and provide key information in the search for new therapeutic strategies for the treatment of memory deficits in the elderly.

Chronic N-Methyl-D-aspartate (NMDA) administration, a model of excitotoxicity, and chronic intracerebroventricular lipopolysaccharide infusion, a model of neuroinflammation, are reported to upregulate arachidonic acid incorporation and turnover in rat brain phospholipids as well as enzymes involved in arachidonic acid metabolism. This suggests cross-talk between signaling pathways of excitotoxicity and of neuroinflammation, involving arachidonic acid. To test whether chronic NMDA administrations to rats can upregulate brain markers of neuroinflammation, NMDA (25 mg/kg i.p.) or vehicle (1 ml saline/kg i.p.) was administered daily to adult male rats for 21 days. Protein and mRNA levels of cytokines and other inflammatory markers were measured in the frontal cortex using immunoblot and real-time PCR. Compared with chronic vehicle, chronic NMDA significantly increased protein and mRNA levels of interleukin-1beta, tumor necrosis factor alpha, glial fibrillary acidic protein and inducible nitric oxide synthase. Chronic NMDA receptor overactivation results in increased levels of neuroinflammatory markers in the rat frontal cortex, consistent with cross-talk between excitotoxicity and neuroinflammation. As both processes have been reported in a number of human brain diseases, NMDA receptor inhibitors might be of use in treating neuroinflammation in these diseases.

The present study was undertaken to examine whether genetically predetermined differences in components of the endocannabinoid system were present in the brain of Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats, a pair of rat lines selectively bred for opposite alcohol preference. The effects of acquisition and maintenance of alcohol drinking, alcohol withdrawal, and alcohol re-exposure on the endocannabinoid system was also assessed in the striatum of sP rats. The findings revealed significantly higher density of the CB1 receptors and levels of CB1 receptor mRNA, CB1 receptor-mediated G-protein coupling, and endocannabinoids in the cerebral cortex, hippocampus and striatum of alcohol-naive sP rats than sNP rats. A significantly lower expression of mFAAH enzyme was evident in the hippocampus of alcohol-naive sP rats. Alcohol drinking (during both acquisition and maintenance phases) in sP rats resulted in a significant reduction in striatal CB1 receptor-mediated G-protein coupling whereas alcohol withdrawal attenuated this effect. Alcohol consumption was also associated with markedly increased levels of endocannabinoids in the striatum. Co-administration of the CB1 receptor antagonist, rimonabant (SR141716A) reduced alcohol intake, and reversed alcohol-induced changes in CB1 receptor-mediated G-protein activation. These findings provided a new insight into a potential genetic basis of excessive alcohol consumption, suggesting innate differences in the endocannabinoid system might be associated with higher alcohol preference in sP rats. The data also indicate a modulation of CB1 receptor-mediated signaling following alcohol consumption, and further strengthen the potential of the endocannabinoid system as a target for the treatment of alcohol related behaviors.

Excessive burst firing of action potentials in subthalamic nucleus (STN) neurons has been correlated with the bradykinesia and rigidity seen in Parkinson's disease. Consequently, there is much interest in characterizing mechanisms that promote burst firing, such as the regulation of N-methyl-D-aspartate (NMDA) receptor function. Using whole-cell recording techniques in rat brain slices, we report that inward currents evoked by NMDA are greatly potentiated by ATP-sensitive K+ (K-ATP) channel blocking agents in STN neurons but not in dopamine neurons in the substantia nigra. Moreover, we found that the ability of NMDA to evoke K-ATP current was blocked by inhibitors of nitric oxide synthase, guanylyl cyclase, and calcium/calmodulin. By altering firing patterns of STN neurons, this NMDA/K-ATP interaction may exert an important influence on basal ganglia output and thereby affect the clinical expression of Parkinson's disease.

N-Methyl-d-aspartate (NMDA) receptors regulate structural plasticity by modulating actin organization within dendritic spines. Herein, we report identification and characterization of p250GAP, a novel GTPase-activating protein for Rho family proteins that interacts with the GluRε2 (NR2B) subunit of NMDA receptors in vivo. The p250GAP mRNA was enriched in brain, with high expression in cortex, corpus striatum, hippocampus, and thalamus. Within neurons, p250GAP was highly concentrated in the postsynaptic density and colocalized with the GluRε2 (NR2B) subunit of NMDA receptors and with postsynaptic density-95. p250GAP promoted GTP hydrolysis of Cdc42 and RhoA in vitro and in vivo. When overexpressed in neuroblastoma cells, p250GAP suppressed the activities of Rho family proteins, which resulted in alteration of neurite outgrowth. Finally, NMDA receptor stimulation led to dephosphorylation and redistribution of p250GAP in hippocampal slices. Together, p250GAP is likely to be involved in NMDA receptor activity-dependent actin reorganization in dendritic spines.

Long-term memory formation requires selective changes in gene expression. Here, we determined the contribution of chromatin remodeling to learning-induced changes in brain-derived neurotrophic factor (bdnf) gene expression in the adult hippocampus. Contextual fear learning induced differential regulation of exon-specific bdnf mRNAs (I, IV, VI, IX) that was associated with changes in bdnf DNA methylation and altered local chromatin structure. Infusions of Zebularine (a DNA methyltransferase inhibitor) significantly altered bdnf DNA methylation, and triggered changes in exon-specific bdnf mRNA levels, indicating that altered DNA methylation is sufficient to drive differential bdnf transcript regulation in the hippocampus. In addition, N-methyl-D-aspartic acid (NMDA) receptor blockade prevented memory-associated alterations in bdnf DNA methylation, resulting in a block of altered bdnf gene expression in hippocampus and a deficit in memory formation. These results suggest epigenetic modification of the bdnf gene as a mechanism for isoform-specific gene readout during memory consolidation.

Recent findings suggest that methamphetamine (METH) functions acutely to inhibit N-methyl-d-aspartate (NMDA) receptor function. Protracted withdrawal from METH exposure may increase the sensitivity of NMDA receptors to agonist exposure, promoting neuronal excitability. However, the relevance of METH effects on NMDA receptor activity with regard to neuronal viability has not been studied. The present studies examined the effects of protracted METH exposure (6 or 7 days; 1.0-100 μM) and withdrawal (1 or 7 days) on NMDA receptor-dependent neurotoxicity, determined with use of the non-vital fluorescent marker propidium iodide, in organotypic slice cultures of male and female rats. Prolonged exposure to METH (100 μM) produced only modest toxicity in the granule cell layer of the dentate gyrus. Withdrawal from METH exposure (1 or 7 days) did not produce overt neuronal injury in any region of slice cultures. Exposure to NMDA (5 μM) produced marked neurotoxicity in the CA1 pyramidal cell layer. Neither co-exposure to METH nor 1 day of METH withdrawal in combination with NMDA exposure altered NMDA-induced neurotoxicity. In contrast, protracted withdrawal from METH exposure (7 days) was associated with a marked (~400%) increase in NMDA-induced neurotoxicity in CA1 region pyramidal cells. This potentiation of neurotoxicity was prevented by co-exposure to the selective NMDA receptor antagonist 5-2-amino-5-phoshonovaleric acid (20 μM) and was markedly attenuated by co-exposure of slices to xestospongin C (1 μM), an antagonist of IP3 receptors. The results of the present studies suggest that long-term METH withdrawal functionally sensitizes the NMDA receptor to agonist exposure and requires the co-activation of NMDA and IP3 receptors.

Hyperbilirubinemia may lead to neurotoxicity and neuronal death. Although the mechanisms of nerve cell damage by unconjugated bilirubin (UCB) appear to involve a disruption of the redox status and excitotoxicity, the contribution of nitric oxide (NO·) and of N-methyl-d-aspartate (NMDA) glutamate receptors is unclear. We investigated the role of NO· and NMDA glutamate receptors in the pathways of nerve cell demise by UCB. Neurons were incubated with 100 μmol/L UCB, in the presence of 100 μmol/L human serum albumin for 4 h at 37ºC, alone or in combination with N-ω-nitro-l-arginine methyl ester (l-NAME) (an inhibitor of neuronal nitric oxide synthase [nNOS]), hemoglobin (an NO· scavenger) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (an NMDA-receptor antagonist). Exposure to UCB led to increased expression of nNOS and production of both NO· and cyclic guanosine 3′,5′-monophosphate (cGMP), along with protein oxidation and depletion of glutathione. These events concurred for cell dysfunction and death and were counteracted by l-NAME. Moreover, the UCB-induced loss of neuronal viability was abolished by hemoglobin, whereas the activation of nNOS and production of both NO· and cGMP were counteracted by MK-801, resulting in significant protection from cell dysfunction and death. These results reinforce the involvement of oxidative stress by showing that nerve cell damage by UCB is mediated by NO· and therefore is counteracted by NO· inhibitors or scavengers. Our findings strongly suggest that the activation of nNOS and neurotoxicity occur through the engagement of NMDA receptors. These data reveal a role for overstimulation of glutamate receptors in mediating oxidative damage by UCB.

Estrogens and nitric oxide (NO) exert wide-ranging effects on brain function. Recent evidence suggested that one important mechanism for the regulation of NO production may reside in the differential coupling of the calcium-activated neuronal NO synthase (nNOS) to glutamate N-Methyl-D-Aspartate (NMDA) receptor channels harboring NR2B subunits by the scaffolding protein postsynaptic density-95 (PSD95), and that estrogens promote the formation of this ternary complex. Here, we demonstrate that 30-min estradiol-treatment triggers the production of NO by physically and functionally coupling NMDA receptors to nNOS in primary neurons of the rat preoptic region in vitro. The ability of estradiol to activate neuronal NO signaling in preoptic neurons and to promote changes in protein-protein interactions is blocked by ICI 182,780, an estrogen receptor antagonist. In addition, blockade of NMDA receptor NR2B subunit activity with ifenprodil or disruption of PSD95 synthesis in preoptic neurons by treatment with an antisense oligodeoxynucleotide inhibited the estradiol-promoted stimulation of NO release in cultured preoptic neurons. Thus, estrogen receptor-mediated stimulation of the nNOS/PSD95/NMDA receptor complex assembly is likely to be a critical component of the signaling process by which estradiol facilitates coupling of glutamatergic fluxes for NO production in neurons.

We previously showed that the serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the AMPA-type glutamate receptor GluA1 protein in the plasma membrane. The activation of AMPA receptors by NMDA-type glutamate receptors eventually leads to postsynaptic neuronal plasticity. Here, we show that SGK3 mRNA is upregulated in the hippocampus of new-born wild type Wistar rats after NMDA receptor activation. We further demonstrate in the Xenopus oocyte expression system that delivery of GluA1 protein to the plasma membrane depends on the small GTPase RAB11. This RAB-dependent GluA1 trafficking requires phosphorylation and activation of phosphoinositol-3-phosphate-5-kinase (PIKfyve) and the generation of PI(3,5)P2. In line with this mechanism we could show PIKfyve mRNA expression in the hippocampus of wild type C57/BL6 mice and phosphorylation of PIKfyve by SGK3. Incubation of hippocampal slices with the PIKfyve inhibitor YM201636 revealed reduced CA1 basal synaptic activity. Furthermore, treatment of primary hippocampal neurons with YM201636 altered the GluA1 expression pattern towards reduced synaptic expression of GluA1. Our findings demonstrate for the first time an involvement of PIKfyve and PI(3,5)P2 in NMDA receptor-triggered synaptic GluA1 trafficking. This new regulatory pathway of GluA1 may contribute to synaptic plasticity and memory.

Delineating the mechanisms of survival pathways that exist in neurons will provide important insight into how neurons utilize intracellular proteins as neuroprotectants against the causes of acute neurodegeneration. We have employed cultured rat cerebellar granule cells as a model for determining the mechanisms of these intraneuronal survival pathways. Glutamate has long been known to kill neurons by an N-methyl-d-aspartate (NMDA) receptor-mediated mechanism. Paradoxically, subtoxic concentrations of NMDA protect neurons against glutamate-mediated excitotoxicity. Because NMDA protects neurons in physiologic concentrations of glucose and oxygen, we refer to this phenomenon as physiologic preconditioning. One of the major mechanisms of NMDA neuroprotection involves the activation of NMDA receptors leading to the rapid release of brain-derived neurotrophic factor (BDNF). BDNF then binds to and activates its cognate receptor, receptor tyrosine kinase B (TrkB). The efficient utilization of these two receptors confers remarkable resistance against millimolar concentrations of glutamate that kill more than eighty percent of the neurons in the absence of preconditioning the neurons with a subtoxic concentration of NMDA. Exactly how the neurons mediate neuroprotection by activation of both receptors is just beginning to be understood. Both NMDA and TrkB receptors activate nuclear factor kappaB (NF-kB), a transcription factor known to be involved in protecting neurons against many different kinds of toxic insults. By converging on survival transcription factors, such as NF-kB, NMDA and TrkB receptors protect neurons. Thus, crosstalk between these very different receptors provides a rapid means of neuronal communication to upregulate survival proteins through release and transcriptional activation of messenger RNA.

Most vagal afferent neurons in rat nodose ganglia express mRNA coding for the NR1 subunit of the heteromeric NMDA receptor ion channel. NMDA receptor subunit immunoreactivity has been detected on axon terminals of vagal afferents in the dorsal hindbrain, suggesting a role for presynaptic NMDA receptors in viscerosensory function. Although NMDA receptor subunits (NR1, NR2B, NR2C, and NR2D) have been linked to distinct neuronal populations in the brain, the NMDA receptor subunit phenotype of vagal afferent neurons has not been determined. Therefore, we examined NMDA receptor subunit (NR1, NR2B, NR2C, and NR2D) immunoreactivity in vagal afferent neurons. We found that, although the left nodose contained significantly more neurons (7603), than the right (5978), the proportions of NMDA subunits expressed in the left and right nodose ganglia were not significantly different. Immunoreactivity for NMDA NR1 subunit was present in 92.3 % of all nodose neurons. NR2B immunoreactivity was present in 56.7% of neurons, NR2C expressing nodose neurons comprised 49.4% of the total population, and NR2D subunit immunoreactivity was observed in just 13.5% of all nodose neurons. Double labeling revealed that 30.2% of nodose neurons expressed immunoreactivity to both NR2B and NR2C, while NR2B and NR2D immunoreactivity were co-localized in 11.5% of nodose neurons. NR2C immunoreactivity co-localized with NR2D in 13.1% of nodose neurons. Our results indicate that the majority of vagal afferent neurons express NMDA receptor ion channels composed of NR1, NR2B and NR2C subunits, and that a minority phenotype that expresses NR2D also expresses NR1, NR2B, and NR2C.

Background:

Extended exposure to low levels of lead causes high blood pressure in human and laboratory animals. The mechanism is not completely recognized, but it is relatively implicated with generation of free radicals, oxidant agents such as ROS, and decrease of available nitric oxide (NO). In this study, we have demonstrated the effect of ascorbic acid as an antioxidant on nitric oxide metabolites and systolic blood pressure in rats exposed to low levels of lead.

Materials and Methods:

The adult male Wistar rats weighing 200-250 g were divided into four groups: control, lead acetate (receiving 100 ppm lead acetate in drinking water), lead acetate plus ascorbic acid (receiving 100 ppm lead acetate and 1 g/l ascorbic acid in drinking water), and ascorbic acid (receiving 1 g/l ascorbic acid in drinking water) groups. The animals were anesthetized with ketamin/xylazine (50 and 7 mg/kg, respectively, ip) and systolic blood pressure was then measured from the tail of the animals by a sphygmomanometer. Nitric oxide levels in serum were measured indirectly by evaluation of its stable metabolites (total nitrite and nitrate (NOχ)).

Results:

After 8 and 12 weeks, systolic blood pressure in the lead acetate group was significantly elevated compared to the control group. Ascorbic acid supplementation could prevent the systolic blood pressure rise in the lead acetate plus ascorbic acid group and there was no significant difference relative to the control group. The serum NOχ levels in lead acetate group significantly decreased in relation to the control group, but this reduction was not significantly different between the lead acetate plus ascorbic acid group and the control group.

Conclusion:

Results of this study suggest that ascorbic acid as an antioxidant prevents the lead induced hypertension. This effect may be mediated by inhibition of NOχ oxidation and thereby increasing availability of NO.

Ketamine, an N-methyl-D-aspartate (NMDA) receptor ion channel blocker, is a widely used anesthetic recently reported to enhance neuronal death in developing rodents and nonhuman primates. This study evaluated dose-response and time-course effects of ketamine, levels of ketamine in plasma and brain, and the relationship between altered NMDA receptor expression and ketamine-induced neuronal cell death during development. Postnatal day 7 rats were administered 5, 10, or 20 mg/kg ketamine using single or multiple injections (subcutaneously) at 2-h intervals, and the potential neurotoxic effects were examined 6 h after the last injection. No significant neurotoxic effects were detected in layers II or III of the frontal cortex of rats administered one, three, or six injections of 5 or 10 mg/kg ketamine. However, in rats administered six injections of 20 mg/kg ketamine, a significant increase in the number of caspase-3- and Fluoro-Jade C–positive neuronal cells was observed in the frontal cortex. Electron microscopic observations showed typical nuclear condensation and fragmentation indicating enhanced apoptotic characteristics. Increased cell death was also apparent in other brain regions. In addition, apoptosis occurred after plasma and brain levels of ketamine had returned to baseline levels. In situ hybridization also showed a remarkable increase in mRNA signals for the NMDA NR1 subunit in the frontal cortex. These data demonstrate that ketamine administration results in a dose-related and exposure-time dependent increase in neuronal cell death during development. Ketamine-induced cell death appears to be apoptotic in nature and closely associated with enhanced NMDA receptor subunit mRNA expression.