D-aspartate Glutamate Receptor Research Articles

Inhibition of STEP61 ameliorates deficits in mouse and hiPSC-based schizophrenia models

The brain-specific tyrosine phosphatase, STEP (STriatal-Enriched protein tyrosine Phosphatase) is an important regulator of synaptic function. STEP normally opposes synaptic strengthening by increasing N-methyl D-aspartate glutamate receptor (NMDAR) internalization through dephosphorylation of GluN2B and inactivation of the kinases extracellular signal–regulated kinase 1/2 and Fyn. Here we show that STEP61 is elevated in the cortex in the Nrg1+/− knockout mouse model of schizophrenia (SZ). Genetic reduction or pharmacological inhibition of STEP prevents the loss of NMDARs from synaptic membranes and reverses behavioral deficits in Nrg1+/− mice. STEP61 protein is also increased in cortical lysates from the central nervous system-specific ErbB2/4 mouse model of SZ, as well as in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons and Ngn2-induced excitatory neurons, from two independent SZ patient cohorts. In these selected SZ models, increased STEP61 protein levels likely reflect reduced ubiquitination and degradation. These convergent findings from mouse and hiPSC SZ models provide evidence for STEP61 dysfunction in SZ.

The N-Methyl d-Aspartate Glutamate Receptor Antagonist Ketamine Disrupts the Functional State of the Corticothalamic Pathway.

The non-competitive N-methyl d-aspartate glutamate receptor (NMDAR) antagonist ketamine elicits a brain state resembling high-risk states for developing psychosis and early stages of schizophrenia characterized by sensory and cognitive deficits and aberrant ongoing gamma (30-80 Hz) oscillations in cortical and subcortical structures, including the thalamus. The underlying mechanisms are unknown. The goal of the present study was to determine whether a ketamine-induced psychotic-relevant state disturbs the functional state of the corticothalamic (CT) pathway. Multisite field recordings were performed in the somatosensory CT system of the sedated rat. Baseline activity was challenged by activation of vibrissa-related prethalamic inputs. The sensory-evoked thalamic response was characterized by a short-latency (∼4 ms) prethalamic-mediated negative sharp potential and a longer latency (∼10 ms) CT-mediated negative potential. Following a single subcutaneous injection of ketamine (2.5 mg/kg), spontaneously occurring and sensory-evoked thalamic gamma oscillations increased and decreased in power, respectively. The power of the sensory-related gamma oscillations was positively correlated with both the amplitude and the area under the curve of the corresponding CT potential but not with the prethalamic potential. The present results show that the layer VI CT pathway significantly contributes in thalamic gamma oscillations, and they support the hypothesis that reduced NMDAR activation disturbs the functional state of CT and corticocortical networks.

Alleviation of chronic pain following rat spinal cord compression injury with multimodal actions of huperzine A

Diverse mechanisms including activation of NMDA receptors, microglial activation, reactive astrogliosis, loss of descending inhibition, and spasticity are responsible for ∼40% of cases of intractable neuropathic pain after spinal cord injury (SCI). Because conventional treatments blocking individual mechanisms elicit only short-term effectiveness, a multimodal approach with simultaneous actions against major pain-related pathways may have value for clinical management of chronic pain. We hypothesize that [-]-huperzine A (HUP-A), an alkaloid isolated from the club moss Huperzia serrata, that is a potent reversible inhibitor of acetylcholinesterase and NMDA receptors, could mitigate pain without invoking drug tolerance or dependence by stimulating cholinergic interneurons to impede pain signaling, inhibiting inflammation via microglial cholinergic activation, and blocking NMDA-mediated central hypersensitization. We tested our hypothesis by administering HUP-A i.p. or intrathecally to female Sprague-Dawley rats (200-235 g body weight) after moderate static compression (35 g for 5 min) of T10 spinal cord. Compared with controls, HUP-A treatment demonstrates significant analgesic effects in both regimens. SCI rats manifested no drug tolerance following repeated bolus i.p. or chronic intrathecal HUP-A dosing. The pain-ameliorating effect of HUP-A is cholinergic dependent. Relative to vehicle treatment, HUP-A administration also reduced neural inflammation, retained higher numbers of calcium-impermeable GluR2-containing AMPA receptors, and prevented Homer1a up-regulation in dorsal horn sensory neurons. Therefore, HUP-A may provide safe and effective management for chronic postneurotrauma pain by reestablishing homeostasis of sensory circuits.

NR2B-deficient mice are more sensitive to the locomotor stimulant and depressant effects of ethanol

The NR2B subunit of N-methyl d-aspartate glutamate receptors influences pharmacological properties and confers greater sensitivity to the modulatory effects of ethanol. This study examined behavioral responses to acute ethanol in a conditional knockout mouse model that allowed for a delayed genetic deletion of the NR2B subunit to avoid mouse lethality. Mice lacking the NR2B gene (knockout) were produced by mating NR2B[f/f] mice with CAMKIIa-driven tTA transgenic mice and the tetO-CRE transgenic mice. Adult male and female offspring representing each of the resultant genotypes (knockout, CAM, CRE and wildtype mice) were tested for open-field locomotor activity following acute low- and high-dose ethanol challenge as well as loss of righting reflex. Findings indicate that male and female mice lacking the NR2B subunit exhibited greater overall activity in comparison to other genotypes during the baseline locomotor activity test. NR2B knockout mice exhibited an exaggerated stimulant response to 1.5 g/kg (i.p.) and an exaggerated depressant response to 3.0 g/kg (i.p.) ethanol challenge. In addition, NR2B knockout mice slept longer following a high dose of ethanol (4.0 g/kg, i.p.). To evaluate pharmacokinetics, clearance rates of ethanol (1.5, 4.0 g/kg, i.p.) were measured and showed that female NR2B knockouts had a faster rate of metabolism only at the higher ethanol dose. Western blot analyses confirmed significant reduction in NR2B expression in the forebrain of knockout mice. Collectively, these data indicate that the NR2B subunit of the N-methyl d-aspartate glutamate receptor is involved in regulating low-dose stimulant effects of ethanol and the depressant/hypnotic effects of ethanol.

The clinically available NMDA receptor antagonist, memantine, exhibits relative safety in the developing rat brain

The N-methyl- d-aspartate glutamate receptor (NMDAR) has been implicated in preterm brain injury (periventricular leukomalacia (PVL)) and represents a potential therapeutic target. However, the antagonist dizocilpine (MK-801) has been reported to increase constitutive neuronal apoptosis in the developing rat brain, limiting its clinical use in the developing brain. Memantine is another use-dependent NMDAR antagonist with shorter binding kinetics and has been demonstrated to be protective in a rat model of PVL, without effects on normal myelination or cortical growth. To further evaluate the safety of memantine in the developing brain, we demonstrate here that, in contrast to MK-801, memantine at neuroprotective doses does not increase neuronal constitutive apoptosis. In addition, there are no long-term alterations in the expression of NMDAR subunits, AMPAR subunits, and two markers of synaptogenesis, Synapsin-1 and PSD95. Evaluating clinically approved drugs in preclinical neonatal animal models of early brain development is an important prerequisite to considering them for clinical trial in preterm infants and early childhood.

N-Methyl- d-aspartate glutamate receptors in the hypothalamic paraventricular nucleus modulate cardiac component of the baroreflex in unanesthetized rats

In the present study, we investigated the role played by the hypothalamic paraventricular nucleus (PVN) in the modulation of cardiac baroreflex activity in unanesthetized rats. Bilateral microinjections of the nonselective neurotransmission blocker CoCl 2 into the PVN decreased the reflex bradycardic response evoked by blood pressure increases, but had no effect on reflex tachycardia evoked by blood pressure decreases. Bilateral microinjections of the selective NMDA glutamate receptor antagonist LY235959 into the PVN caused effects that were similar to those observed after microinjections of CoCl 2, decreasing reflex bradycardia without affecting tachycardic response. The microinjection of the selective non-NMDA glutamate receptor antagonist NBQX into the PVN did not affect the baroreflex activity. Also, the microinjection of L-glutamate into the PVN increased the reflex bradycardia, an effect opposed to that observed after PVN treatment with CoCl 2 or LY235959, and this effect of L-glutamate was blocked by PVN pretreatment with LY235959. LY235959 injected into the PVN after i.v. treatment with the selective β 1-adrenoceptor antagonist atenolol still decreased the reflex bradycardia. Taken together, our results suggest a facilitatory influence of the PVN on the bradycardic response of the baroreflex through activation of local NMDA glutamate receptors and a modulation of the cardiac parasympathetic activity.

Fibroblast growth factor-1 within the ventral tegmental area participates in motor sensitizing effects of morphine

Drug addiction is viewed as a form of neural plasticity, and neurotrophic factors have been implicated in many forms of plasticity in the adult nervous system. Here we show that the fibroblast growth factor-1 (FGF-1), that is expressed on dopamine and GABA neurons of the ventral tegmental area (VTA), is involved in the sensitizing effects of morphine. The receptor FGFR-1 is expressed on VTA astrocytes, as well as dopamine and GABA neurons. FGF-1 or anti-FGF-1 infusions into the VTA during the induction (not expression) phase of sensitization advanced or blocked morphine's activating motor effects respectively, in a dose–dependent manner. Infusions into the adjacent substantia nigra, whose neurons also express FGF-1 and FGFR-1, did not modify normal morphine-induced sensitization. Biochemical traits related to morphine's sensitizing effects were altered by intra-VTA anti-FGF-1 because morphine-induced upregulation of both tyrosine hydroxylase (TH) and N-methyl d-aspartate glutamate receptor 1 (NMDAR1) in the VTA was blocked after anti-FGF-1. Changes in the activation state of VTA calcium/calmodulin-dependent kinase type II seem to participate in FGF-1-induced effects as well. We conclude that the FGF-1 system of the ventral tegmental area is required for biochemical and behavioral sensitization to this drug.

MRNA Expression is a Relevant Tool to Identify Developmental Neurotoxicants Using an In Vitro Approach

So far, only a few industrial chemicals have been identified as developmental neurotoxicants. Because the current developmental neurotoxicity (DNT) guideline (Organisation for Economic Co-operation and Development TG 426) is based entirely on in vivo studies that are both time consuming and costly, there is a need to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. In this study, gene expression at the mRNA level was evaluated to determine whether this could be a suitable endpoint to detect potential developmental neurotoxicants. Primary cultures of rat cerebellar granule cells (CGCs) were exposed to well known (developmental) neurotoxicants (methyl mercury chloride, lead chloride, valproic acid, and tri-methyl tin chloride) for different time periods. A significant downregulation of the mRNA level for the neuronal markers (NF-68, NF-200, N-methyl D-aspartate glutamate receptor, and gamma-amino butyric acid receptor) was observed after exposure to methyl mercury chloride, valproic acid, and tri-methyl tin chloride. Moreover, a significant increase of the neural precursor marker nestin mRNA was also observed. The mRNA expression of the astrocytic markers (glial fibrillary acidic protein [GFAP] and S100beta) was unchanged. In contrast, exposure to lead chloride significantly decreased the mRNA level of the astrocytic marker GFAP, whereas the neuronal markers were less affected. These results suggest that gene expression could be used as a sensitive tool for the initial identification of DNT effects induced by different mechanisms of toxicity in both cell types (neuronal and glial) and at various stages of cell development and maturation.

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.

A NMDA receptor glycine site partial agonist, GLYX-13, simultaneously enhances LTP and reduces LTD at Schaffer collateral–CA1 synapses in hippocampus

N-methyl- d-aspartate glutamate receptors (NMDARs) are a key route for Ca 2+ influx into neurons important to both activity-dependent synaptic plasticity and, when uncontrolled, triggering events that cause neuronal degeneration and death. Among regulatory binding sites on the NMDAR complex is a glycine binding site, distinct from the glutamate binding site, which must be co-activated for NMDAR channel opening. We developed a novel glycine site partial agonist, GLYX-13, which is both nootropic and neuroprotective in vivo. Here, we assessed the effects of GLYX-13 on long-term synaptic plasticity and NMDAR transmission at Schaffer collateral–CA1 synapses in hippocampal slices in vitro. GLYX-13 simultaneously enhanced the magnitude of long-term potentiation (LTP) of synaptic transmission, while reducing long-term depression (LTD). GLYX-13 reduced NMDA receptor-mediated synaptic currents in CA1 pyramidal neurons evoked by low frequency Schaffer collateral stimulation, but enhanced NMDAR currents during high frequency bursts of activity, and these actions were occluded by a saturating concentration of the glycine site agonist d-serine. Direct two-photon imaging of Schaffer collateral burst-evoked increases in [Ca 2+] in individual dendritic spines revealed that GLYX-13 selectively enhanced burst-induced NMDAR-dependent spine Ca 2+ influx. Examining the rate of MK-801 block of synaptic versus extrasynaptic NMDAR-gated channels revealed that GLYX-13 selectively enhanced activation of burst-driven extrasynaptic NMDARs, with an action that was blocked by the NR2B-selective NMDAR antagonist ifenprodil. Our data suggest that GLYX-13 may have unique therapeutic potential as a learning and memory enhancer because of its ability to simultaneously enhance LTP and suppress LTD.

Diabetes changes ionotropic glutamate receptor subunit expression level in the human retina

Early diabetic retinopathy is characterized by changes in subtle visual functions such as contrast sensitivity and dark adaptation. The outcome of several studies suggests that glutamate is involved in retinal neurodegeneration during diabetes. We hypothesized that the protein levels of ionotropic glutamate receptor subunits are altered in the retina during diabetes. Therefore, we investigated whether human diabetic patients have altered immunoreactivity of ionotropic glutamate receptor subunits in the retina. In total, 12 donor eyes from subjects with diabetes mellitus were examined and compared to 6 eyes from non-diabetic subjects without known ocular disease, serving as controls. Immunohistochemical analysis was performed using specific antibodies directed against the ionotropic α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor subunits GluR1, GluR2, GluR4, and against the N-methyl- d-aspartate glutamate receptor subunit NR1. In the inner plexiform and outer plexiform layers the immunoreactivity of GluR2 and NR1 subunits was significantly increased in subjects with diabetes when compared to the levels found in controls. No significant changes in GluR1 and GluR4 subunit expression were observed. These results suggest that early visual dysfunction in diabetic patients may be due, at least partially, to changes in glutamate receptor subunit expression or distribution.

MEK inhibition exacerbates ischemic calcium imbalance and neuronal cell death in rat cortical cultures

Interruption in the brain's blood supply leads to an ischemic condition, which is characterised by a depletion of energy phosphates and related failure of ionic pumps, increased extracellular potassium, neuronal depolarisation and release of excitatory amino acids, e.g. glutamate. The subsequent activation of N-methyl- d-aspartate glutamate receptors triggers a wide range of intracellular signals, including the mitogen-activated protein kinase (MAPK) pathway. Activation and inhibition of the MAPK/extracellular regulated kinases (ERK) pathway are both reported to be neuroprotective in conditions associated with excitotoxic injury. The present study was designed to explore the involvement of this signalling pathway in cultured rat cortical neurons subjected to chemically-induced ischemia obtained by coupling the mitochondrial toxin 3-nitropropionic acid with glucose deprivation. Loss of neuronal viability, reduced neuronal energy state (ATP level and mitochondrial membrane potential) and increased cytoplasmic mitochondrial calcium were all observed. The NMDA antagonist MK-801 counteracted these effects, suggesting a glutamate-dependent ischemic cell death. Addition of U0126, a selective inhibitor of MAPK kinase, exacerbated this neuronal cell death. However, non-significant changes in activated cAMP response element-binding protein were seen. The rise in cytoplasmic calcium under ischemic conditions was associated with neuronal cell swelling. Both swelling and increase in cytoplasmic calcium were exacerbated and prevented by U0126 and MK-801, respectively. These data suggest that in this ischemic model the MAPK/ERK pathway might exert a regulatory effect on calcium entry independent from gene expression.

Evidence of acute tolerance to remifentanil in intensive care but not in anesthesia

We report the case of a 19-year-old man with a drug abuse history, admitted to the intensive care unit for head and chest trauma, who experienced an acute tolerance to sedative and respiratory depression effects of remifentanil, which was given as the sole agent for sedation. He did not exhibit any signs of drug tolerance or intraoperative awareness during prolonged remifentanil-based anesthesia using propofol or sevoflurane as adjuvants. Several recent studies support the hypothesis of a possible involvement of N-methyl- d-aspartate glutamate receptors. The clinical relevance of this report is that if a patient with a previously acute tolerance to remifentanil during sedation undergoes long-term surgery, and propofol or sevoflurane is coadministered in a remifentanil-based anesthesia, the patient will not necessarily develop opioid tolerance. It is of interest for anesthesiologists, given the high frequency of patients with drug abuse history who are admitted to intensive care units, often sedated with remifentanil, who undergo anesthesia for emergency surgery.

Chronic treatment with sulbutiamine improves memory in an object recognition task and reduces some amnesic effects of dizocilpine in a spatial delayed-non-match-to-sample task

The effect of a sulbutiamine chronic treatment on memory was studied in rats with a spatial delayed-non-match-to-sample (DNMTS) task in a radial maze and a two trial object recognition task. After completion of training in the DNMTS task, animals were subjected for 9 weeks to daily injections of either saline or sulbutiamine (12.5 or 25 mg/kg). Sulbutiamine did not modify memory in the DNMTS task but improved it in the object recognition task. Dizocilpine, impaired both acquisition and retention of the DNMTS task in the saline-treated group, but not in the two sulbutiamine-treated groups, suggesting that sulbutiamine may counteract the amnesia induced by a blockade of the N-methyl- d-aspartate glutamate receptors. Taken together, these results are in favor of a beneficial effect of sulbutiamine on working and episodic memory.

Effects of N-methyl- d-aspartate glutamate receptor antagonists on oscillatory signal propagation in the guinea-pig accessory olfactory bulb slice: Characterization by optical, field potential and patch clamp recordings

To characterize the role of N-methyl- d-aspartate glutamate receptors in oscillations induced by a single electrical stimulation of the vomeronasal nerve layer, optical, field potential and patch clamp recordings were carried out in guinea-pig accessory olfactory bulb slice preparations. Bath application of the N-methyl- d-aspartate receptor antagonists, 2-amino-5-phosphonovaleric acid or MK-801, produced an increase in frequency of oscillating waves (oscillation) in external plexiform and mitral cell layers. The removal of Mg 2+ from perfusate abolished oscillations, while subsequent application of 2-amino-5-phosphonovaleric acid or MK-801 restored oscillations. Vomeronasal nerve layer-evoked postsynaptic currents were analyzed by whole-cell clamp recordings from mitral and granule cells. A long-lasting excitatory postsynaptic current and periodic inhibitory postsynaptic currents, which were superimposed on the long excitatory postsynaptic current, were observed in mitral cells. The frequency of the periodic inhibitory postsynaptic currents correlated with the frequency of oscillations observed in the optical and field potential recordings. Furthermore, periodic inhibitory postsynaptic currents were blocked by puff application of bicuculline to the external plexiform layer/mitral cell layer, where mitral cells make dendrodendritic synapses with granule cells. In addition, puff application of the non- N-methyl- d-aspartate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, to the external plexiform layer/mitral cell layer suppressed an early phase of periodic inhibitory postsynaptic currents (membrane oscillation), whereas 2-amino-5-phosphonovaleric acid suppressed the late phase of periodic inhibitory postsynaptic currents. These data indicate that periodic excitatory postsynaptic currents of granule cells induce relevantly periodic inhibitory postsynaptic currents in mitral cells via dendrodendritic synapses and suggest that feedback inhibition regulates generation of oscillation via activation of non- N-methyl- d-aspartate glutamate receptors and gradual attenuation of oscillation via activation of N-methyl- d-aspartate receptors on granule cells.

Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen–glucose deprivation of hippocampal slice cultures

Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen–glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen–glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen–glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen–glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen–glucose deprivation induced necrotic cell death only. Addition of 10μM of the N-methyl- d-aspartate glutamate receptor antagonist MK-801, and 20μM of the non- N-methyl- d-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo( F)quinoxaline to the culture medium confirmed that both N-methyl- d-aspartate and non- N-methyl- d-aspartate ionotropic glutamate receptors were involved in the oxygen–glucose deprivation-induced cell death. Glutamate is normally quickly removed, from the extracellular space by sodium-dependent glutamate transporters. Effects of blocking the transporters by addition of the dl-threo-beta-benzyloxyaspartate are reviewed in the last part of the paper. Under normal conditions addition of dl-threo-beta-benzyloxyaspartate in concentrations of 25μM or more to otherwise untreated hippocampal slice cultures induced neuronal cell death, which was prevented by addition of 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo( F)quinoxaline and MK-801. In energy failure situations, like cerebral ischemia and oxygen–glucose deprivation, the transporters are believed to reverse and release glutamate to the extracellular space. Blockade of the transporters by a subtoxic (10μM) dose of dl-threo-beta-benzyloxyaspartate during oxygen–glucose deprivation (but not during the next 48 h after oxygen–glucose deprivation) significantly reduced the oxygen–glucose deprivation-induced propidium iodide uptake, suggesting a neuroprotective inhibition of reverse transporter activity by dl-threo-beta-benzyloxyaspartate during oxygen–glucose deprivation under these conditions. Adding to this, other results from our laboratory have demonstrated that pre-treatment of the slice cultures with glial cell-line derived neurotrophic factor upregulates glutamate transporters. As a logical, but in some glial cell-line derived neurotrophic factor therapy-related conditions clearly unwanted consequence the susceptibility for oxygen–glucose deprivation-induced glutamate receptor-mediated cell death is increased after glial cell-line derived neurotrophic factor treatment. In summary, we conclude that both ionotropic glutamate receptors and glutamate transporters are involved in oxygen–glucose deprivation-induced necrotic cell death in hippocampal slice cultures, which have proven to be a feasible tool in experimental studies on this topic.

A single oral dose of geranylgeranylacetone attenuates kainic acid-induced seizures and neuronal cell death in rat hippocampus

The present study evaluated the potential effect of geranylgeranylacetone (GGA), which is known as an antiulcer agent, against kainic acid (KA)-induced neurotoxicity. Pretreatment with a single oral GGA dose (800 mg/kg, 2 days before KA) significantly attenuated KA-induced seizures and cell death in rat hippocampus. These effects of GGA were prevented by the coinjection of MK801, a noncompetitive N-methyl- d-aspartate glutamate receptor antagonist, which indicates that the protection was indeed mediated by glutamate receptor activation.

Single oral dose of geranylgeranylacetone for protection against delayed neuronal death induced by transient ischemia

The present study evaluated the potential effect of geranylgeranylacetone (GGA), which is known as an antiulcer agent, against the delayed neuronal death (DND) of hippocampal neurons an otherwise lethal ischemic insult. Pretreatment with single oral GGA (800 mg/kg, 2 days before ischemia) significantly attenuated DND in gerbil hippocampus. These effects of GGA were prevented by the coinjection of MK801, a noncompetitive N-methyl- d-aspartate glutamate receptor antagonist, which indicates that the protection was indeed glutamate receptor activation mediated.

A chemical LTP induced by co-activation of metabotropic and N-methyl- d-aspartate glutamate receptors in hippocampal CA1 neurons

In CA1 neurons of guinea pig hippocampal slices, long-term depression (LTD) was induced in the field EPSP response in the absence of test synaptic inputs (one stimulus every 20 s) by application of the metabotropic glutamate receptor (mGluR) agonist, aminocyclopentane-1S, 3R-dicarboxylic acid (ACPD). This effect was blocked and long-term potentiation (LTP) was induced by co-application of N-methyl- d-aspartate (NMDA) during ACPD perfusion (ACPD/NMDA-induced LTD). These results indicate that the state of NMDA receptor activation during ACPD perfusion determines whether LTP or LTD is induced in hippocampal CA1 neurons. Co-application of an inositol 1, 4, 5-trisphosphate (IP3) receptor inhibitor, 2-aminotheoxydiphenyl borate, during ACPD application had no effect on the ACPD/NMDA-induced LTP, but increased the magnitude of the ACPD-induced LTD, suggesting that the ACPD/NMDA-induced LTP involves NMDA receptors, but not IP3 receptors, whereas the converse applies to the ACPD-induced LTD.

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

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