N-methyl-D-aspartate Stimulation Research Articles

Activation of innate immune receptor TLR9 by mitochondrial DNA plays essential roles in the chemical long-term depression of hippocampal neurons

Synaptic plasticity is believed to be the cellular basis for experience-dependent learning and memory. Although long-term depression (LTD), a form of synaptic plasticity, is caused by the activity-dependent reduction of cell surface α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPA receptors) at postsynaptic sites, its regulation by neuronal activity is not completely understood. In this study, we showed that the inhibition of toll-like receptor-9 (TLR9), an innate immune receptor, suppresses N-methyl-d-aspartate (NMDA)-induced reduction of cell surface AMPA receptors in cultured hippocampal neurons. We found that inhibition of TLR9 also blocked NMDA-induced activation of caspase-3, which plays an essential role in the induction of LTD. siRNA-based knockdown of TLR9 also suppressed the NMDA-induced reduction of cell surface AMPA receptors, although the scrambled RNA had no effect on the NMDA-induced trafficking of AMPA receptors. Overexpression of the siRNA-resistant form of TLR9 rescued the AMPA receptor trafficking abolished by siRNA. Furthermore, NMDA stimulation induced rapid mitochondrial morphological changes, mitophagy, and the binding of mitochondrial DNA (mtDNA) to TLR9. Treatment with dideoxycytidine and mitochondrial division inhibitor-1, which block mtDNA replication and mitophagy, respectively, inhibited NMDA-dependent AMPA receptor internalization. These results suggest that mitophagy induced by NMDA receptor activation releases mtDNA and activates TLR9, which plays an essential role in the trafficking of AMPA receptors during the induction of LTD.

α-Synuclein-induced internalization of NMDA receptors in hippocampal neurons is associated with reduced inward current and Ca2+ influx upon NMDA stimulation

Abnormal α-synuclein (α-syn) expression and aggregation have been implicated in the pathogenesis of Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and Alzheimer’s disease (AD). These neurodegenerative disorders, collectively known as synucleinopathies, are usually associated with cognitive impairment that could be caused by impaired hippocampal function. Although abnormal expressions of α-syn and N-methyl-d-aspartate (NMDA) receptor are frequently observed in the hippocampus of patients with synucleinopathies, how these proteins interact with each other in hippocampal neurons remains poorly understood. In the present study, primary cultures of hippocampal neurons and α-syn transgenic mice were used to investigate the effect of α-syn on NMDA receptors. Neurons were treated either by direct addition of recombinant human α-syn (hα-syn) to the medium, or by infection with the hα-syn gene, to increase intracellular levels of α-syn. In both cases, NMDA receptor NR1 subunits on the cell surface were reduced while the total amount of NR1 was unchanged, indicating an internalization of NR1 subunits. Neurons with elevated α-syn also showed a profound increase in Rab5B, an isoform of the small GTPases essential for NMDA receptor endocytosis. Knockdown of Rab5B expression by siRNA inhibited the α-syn-induced reduction in surface NR1. The in vitro findings were confirmed in α-syn transgenic mice, showing that increased α-syn expression was accompanied by reduced levels of surface NR1 and increased expression of Rab5B. Due to the essential role of NR1 subunits for assembling a complete NMDA receptor, its reduction on the cell surface indicated impaired receptor function. This was demonstrated by observations that neurons with elevated α-syn showed profound reductions in NMDA-elicited Ca2+ influx and inward current, which were also inhibited by knockdown of Rab5B expression. Our data suggest that increased α-syn expression may impair NMDA receptor function in the hippocampus by reducing the density of NR1 subunits on the cell surface.

Changes in hypothalamic neurotransmitter and prostanoid levels in response to NMDA, CRF, and GLP-1 stimulation.

Determination of neuroactive substances, such as neurotransmitters and prostanoids, in the extracellular space of the living brain is a very important technique in neuroscience. The hypothalamic paraventricular nucleus (PVN) is one of the most important autonomic control centers in the brain. Recently, we demonstrated that thromboxane (Tx) A2 in the PVN may play an important role in adrenomedullary outflow evoked by N-methyl-D-aspartate (NMDA), corticotrophin-releasing factor (CRF), and glucagon-like peptide-1 (GLP-1) stimulation using microdialysis sampling and liquid chromatography-ion trap tandem mass spectrometry (LC-ITMS(n)). In the present study, we investigated whether centrally administered NMDA, CRF, and GLP-1 can release five neurotransmitters, acetylcholine (ACh), histamine, glutamate (Glu), γ-aminobutyric acid (GABA), and serotonin (5-HT), along with six prostanoids, TxB2, prostaglandin (PG) E2, PGD2, 15-deoxy-∆(12,14) (15d)-PGJ2, 6-keto-PGF1α, and PGF2α in rat PVN microdialysates after optimization of LC-ITMS(n) conditions . All stimulations increased the levels of 5-HT, TxB2, PGE2, and PGF2α (except for 5-HT stimulated with GLP-1). Only NMDA increased the levels of ACh, Glu, and GABA. Treatment with dizocilpine maleate (MK-801), a noncompetitive NMDA receptor antagonist, attenuated the NMDA-induced increase in the levels of neuroactive substances except for Glu. This is the first study to use LC-ITMS(n) to analyze neurotransmitters and prostanoids in the same microdialysates from rat PVN.

Characterization of Human Hippocampal Neural Stem/Progenitor Cells and Their Application to Physiologically Relevant Assays for Multiple Ionotropic Glutamate Receptors

The hippocampus is an important brain region that is involved in neurological disorders such as Alzheimer disease, schizophrenia, and epilepsy. Ionotropic glutamate receptors—namely, N-methyl-D-aspartate (NMDA) receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs), and kainic acid (KA) receptors (KARs)—are well known to be involved in these diseases by mediating long-term potentiation, excitotoxicity, or both. To predict the therapeutic efficacy and neuronal toxicity of drug candidates acting on these receptors, physiologically relevant systems for assaying brain region–specific human neural cells are necessary. Here, we characterized the functional differentiation of human fetal hippocampus–derived neural stem/progenitor cells—namely, HIP-009 cells. Calcium rise assay demonstrated that, after a 4-week differentiation, the cells responded to NMDA (EC50 = 7.5 ± 0.4 µM; n = 4), AMPA (EC50 = 2.5 ± 0.1 µM; n = 3), or KA (EC50 = 33.5 ± 1.1 µM; n = 3) in a concentration-dependent manner. An AMPA-evoked calcium rise was observed in the absence of the desensitization inhibitor cyclothiazide. In addition, the calcium rise induced by these agonists was inhibited by antagonists for each receptor—namely, MK-801 for NMDA stimulation (IC50 = 0.6 ± 0.1 µM; n = 4) and NBQX for AMPA and KA stimulation (IC50 = 0.7 ± 0.1 and 0.7 ± 0.03 µM, respectively; n = 3). The gene expression profile of differentiated HIP-009 cells was distinct from that of undifferentiated cells and closely resembled that of the human adult hippocampus. Our results show that HIP-009 cells are a unique tool for obtaining human hippocampal neural cells and are applicable to systems for assay of ionotropic glutamate receptors as a physiologically relevant in vitro model.

Why sigma-1 receptor dysfunction might confer vulnerability to cannabis-induced psychosis.

The N -methyl- D -aspartate (NMDA) receptor has long been implicated in the pathophysiology of schizophrenia. NMDA receptor antagonists induce psychotic states in humans and when administered to rodents promote lasting behavioural phenotypes relevant to schizophrenia (Javitt and Zukin, 1991; Abi-Saab et al., 1998; Javitt, 2007). Further, schizophrenia brain tissue contains reduced expression and function of NMDA receptors (Errico et al., 2013). It is well established that the use of cannabis increases the risk of psychosis in vulnerable individuals; however, our understanding of the molecular basis of this susceptibility is limited. In this issue of the International Journal of Neuropsychopharmacology Javier Garzon and colleagues delineate a mechanism of relevance to cannabis-induced psychosis by uncovering that the sigma-1 receptor prevents cannabinoids from provoking NMDA receptor hypofunction. Garzon and colleagues have recently shown that CB1 receptors regulate NMDA receptors at the postsynaptic membrane (Sanchez-Blazquez et al., 2013, 2014; Vicente-Sanchez et al., 2013). This provides an additional CB1 receptor-mediated homoeostatic mechanism against neurotoxicity provoked by excessive glutamate activation of NMDA receptors. Most cannabinoid CB1 receptors are expressed presynaptically and excessive NMDA stimulation by glutamate triggers the synthesis of endocannabinoids like anandamide. Anandamide then behaves as a retrograde transmitter; travelling from the postsynaptic membrane to presynaptic CB1 receptors, which when activated reduce glutamate release (Brown et al., 2003; Melis et al., 2004). Garzon and colleagues’ research has shown that CB1 receptors may also offset excessive NMDA receptor activation via a postsynaptic mechanism. CB1 receptor activation inhibits NMDA receptor function at the postsynapse by CB1 and NMDA receptors forming a complex that is moved from the cell membrane to the cytosol (Sanchez-Blazquez et al., 2013; Vicente-Sanchez et al., 2013). Thus, CB1 receptor …

The Dorsolateral Periaqueductal Gray and Its Role in Mediating Fear Learning to Life Threatening Events

The dorsolateral column of the periaqueductal gray (dlPAG) integrates aversive emotional experiences and represents an important site responding to life threatening situations, such as hypoxia, cardiac pain and predator threats. Previous studies have shown that the dorsal PAG also supports fear learning; and we have currently explored how the dlPAG influences associative learning. We have first shown that N-methyl-D-aspartate (NMDA) 100 pmol injection in the dlPAG works as a valuable unconditioned stimulus (US) for the acquisition of olfactory fear conditioning (OFC) using amyl acetate odor as conditioned stimulus (CS). Next, we revisited the ascending projections of the dlPAG to the thalamus and hypothalamus to reveal potential paths that could mediate associative learning during OFC. Accordingly, the most important ascending target of the dlPAG is the hypothalamic defensive circuit, and we were able to show that pharmacological inactivation using β-adrenoceptor blockade of the dorsal premammillary nucleus, the main exit way for the hypothalamic defensive circuit to thalamo-cortical circuits involved in fear learning, impaired the acquisition of the OFC promoted by NMDA stimulation of the dlPAG. Moreover, our tracing study revealed multiple parallel paths from the dlPAG to several thalamic targets linked to cortical-hippocampal-amygdalar circuits involved in fear learning. Overall, the results point to a major role of the dlPAG in the mediation of aversive associative learning via ascending projections to the medial hypothalamic defensive circuit, and perhaps, to other thalamic targets, as well. These results provide interesting perspectives to understand how life threatening events impact on fear learning, and should be useful to understand pathological fear memory encoding in anxiety disorders.

Neuroprotection of Interleukin-6 Against NMDA-induced Neurotoxicity is Mediated by JAK/STAT3, MAPK/ERK, and PI3K/AKT Signaling Pathways

We have previously shown that interleukin-6 (IL-6) has neuroprotective effect against N-methyl-D-aspartate (NMDA)-induced excitotoxicity. The current study aimed to reveal signal transduction pathways involved in the IL-6 neuroprotection. Cerebellar granule neurons (CGNs) from postnatal 8-day infant rats were exposed to IL-6 (120 ng/ml) for 8 days and stimulated with NMDA (100 μM) for 15 or 30 min. Dynamic intracellular Ca(2+) fluorescence intensity, cytosolic Ca(2+)-dependent phospholipase A2 (cPLA2) expression, and apoptosis and necrosis in cultured CGNs were measured by laser scanning confocal microscope, real-time PCR and Western blot, and annexin V-FITC/propidium iodide staining, respectively. NMDA stimulation of neurons evoked an intracellular Ca(2+) overload, an upregulated expression of cPLA2, and an increase in cell death. Chronic IL-6 exposure prevented the NMDA-evoked neuronal Ca(2+) overload, cPLA2 expression upregulation, and apoptosis and necrosis. Anti-gp130 monoclonal antibody (mAb), a blocker of gp130 that is a 130-kDa signal-transducing β-subunit of IL-6 receptor complex, blocked these effects of IL-6 preventing NMDA neurotoxicity. AG490, PD98059, or LY294002, inhibitors specific for the intracellular signals, JAK, MAPK, and PI3K, respectively, partially blocked these IL-6 neuroprotective effects. Phosphorylation levels of STAT3, ERK1/2, and AKT, the downstream proteins for these enzymes of JAK, MAPK, and PI3K, respectively, were elevated by IL-6 pretreatment. The enhanced activation of STAT3, ERK1/2, and AKT by IL-6 was abolished by AG490, PD98059, and LY294002, respectively. Anti-gp130 mAb attenuated the activation of all the three detected signaling molecules. The present findings suggest that IL-6 neuroprotection is jointly mediated by the cellular signal transduction pathways, gp130-JAK-STAT3, gp130-MAPK-ERK, and gp130-PI3K-AKT.

Dual Effect of 17β-Estradiol on NMDA-Induced Neuronal Death: Involvement of Metabotropic Glutamate Receptor 1

Pretreatment with 10 nm 17β-estradiol (17βE2) or 100 μm of the metabotropic glutamate 1 receptor (mGlu1R) agonist, dihydroxyphenylglycine (DHPG), protected neurons against N-methyl-d-aspartate (NMDA) toxicity. This effect was sensitive to blockade of both estrogen receptors and mGlu1R by their respective antagonists. In contrast, 17βE2 and/or DHPG, added after a low-concentration NMDA pulse (45 μm), produced an opposite effect, i.e. an exacerbation of NMDA toxicity. Again this effect was prevented by both receptor antagonists. In support of an interaction of estrogen receptors and mGlu1R in mediating a neurotoxic response, exacerbation of NMDA toxicity by 17βE2 disappeared when cultures were treated with DHPG prior to NMDA challenge, and conversely, potentiation of NMDA-induced cell death by DHPG was prevented by pretreatment with 17βE2. Addition of calpain III inhibitor (10 μm), 2 h before NMDA, prevented the increased damage induced by the two agonists, an affect that can be secondary to cleavage of mGlu1R by calpain. Accordingly, NMDA stimulation reduced expression of the full-length (140 kDa) mGluR1, an effect partially reversed by calpain inhibitor. Finally, in the presence of NMDA, the ability of 17βE2 to stimulate phosphorylation of AKT and ERK was impaired. Pretreatment with calpain inhibitor prevented the reduction of phosphorylated ERK but had no significant effect on phosphorylated AKT. Accordingly, the inhibition of ERK signaling by U0126 (1 μm) counteracted the effect of calpain inhibition on 17βE2-induced exacerbation of NMDA toxicity. The present data confirm the dual role of estrogens in neurotoxicity/neuroprotection and highlight the role of the timing of exposure to estrogens.

P38 MAPK is involved in enhanced NMDA receptor-dependent excitotoxicity in YAC transgenic mouse model of Huntington disease

Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the protein huntingtin (htt). Previous studies have shown enhanced N-methyl-d-aspartate (NMDA)-induced excitotoxicity in neuronal models of HD, mediated in part by increased NMDA receptor (NMDAR) GluN2B subunit binding with the postsynaptic density protein-95 (PSD-95). In cultured hippocampal neurons, the NMDAR-activated p38 Mitogen-activated Protein Kinase (MAPK) death pathway is disrupted by a peptide (Tat-NR2B9c) that uncouples GluN2B from PSD-95, whereas NMDAR-mediated activation of c-Jun N-terminal Kinase (JNK) MAPK is PSD-95-independent. To investigate the mechanism by which Tat-NR2B9c protects striatal medium spiny neurons (MSNs) from mutant htt (mhtt)-enhanced NMDAR toxicity, we compared striatal tissue and cultured MSNs from presymptomatic yeast artificial chromosome (YAC) mice expressing htt with 128 polyQ (YAC128) to those from YAC18 and/or WT mice as controls. Similar to the previously published shift of GluN2B-containing NMDARs to extrasynaptic sites, we found increased PSD-95 localization as well as elevated PSD-95-GluN2B interactions in the striatal non-PSD (extrasynaptic) fraction from YAC128 mice. Notably, basal levels of both activated p38 and JNK MAPKs were elevated in the YAC128 striatum. NMDA stimulation of acute slices increased activation of p38 and JNK in WT and YAC128 striatum, but Tat-NR2B9c pretreatment reduced only the p38 activation in YAC128. In cultured MSNs, p38 MAPK inhibition reduced YAC128 NMDAR-mediated cell death to WT levels, and occluded the Tat-NR2B9c peptide protective effect; in contrast, inhibition of JNK had a similar protective effect in cultured MSNs from both WT and YAC128 mice. Our results suggest that altered activation of p38 MAPK contributes to mhtt enhancement of GluN2B/PSD-95 toxic signaling.

Interleukin-6 Reduces NMDA-Induced Ca2+ Overload via Prevention of Ca2+ Release From Intracellular Store

ABSTRACTA mechanism for neuroprotection of interleukin-6 (IL-6) via reduction of intracellular Ca2+ overload induced by N-methyl-D-aspartate (NMDA) was explored. Cerebellar granule neurons (CGNs) from postnatal 8-day infant rats were chronically exposed to IL-6 for 8 days. Confocal laser scanning microscope was used to measure dynamic changes of intracellular Ca2+ fluorescence intensity. NMDA triggered an acute and sustaining enhancement of intracellular Ca2+ fluorescence intensity in the cultured CGNs, whereas NMDA stimulation of the neurons that had been exposed to IL-6 reduced the intracellular Ca2+ fluorescence intensity relative to that of non-IL-6-pretreated neurons. Ethylene glycol bis(aminoethylether) tetraacetate (EGTA), a chelator of extracellular Ca2+, decreased the intracellular Ca2+ overload triggered by NMDA. The component of intracellular Ca2+ overload after EGTA treatment was prevented by IL-6 chronic exposure. Cotreatment with dantrolene sodium (DAN) and 2-aminoethoxydiphenylborate (2-APB), blockers of ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP3R), respectively, also decreased the intracellular Ca2+ overload triggered by NMDA. However, the component of intracellular Ca2+ overload after DAN and 2-APB treatment was only slightly but not significantly diminished by IL-6. These results suggest that IL-6 has a neuroprotective effect against NMDA-induced intracellular Ca2+ overload, and that the effect is implemented primarily via a suppression of Ca2+ release from the intracellular Ca2+ store.

An interlocked oscillator model for high-frequency firing of the midbrain dopaminergic neuron

Dopamine neurotransmission has been found to play a role in addictive behavior and is impaired in psychiatric disorders. Dopaminergic (DA) neurons display two functionally distinct modes of electrophysiological activity: low- and high-frequency firing. The puzzling feature of the DA neuron is the combination of its high-frequency response to N-methyl-D-aspartate (NMDA) receptor activation coupled with the inability of other treatments to elevate its frequency effectively. We suggest a new computational model that reproduces this combination of responses and accounts for recent experimental data. The model is presented in two morphologies: (1) a reconstruction of a DA neuron and (2) a single compartment that ignores the spatial structure of the neuron. We show that these two model morphologies display very similar patterns. Therefore, an equipotential representation of the DA neuron is sufficient for combining its high- and low-frequency firing. Our comparison of the reconstructed morphology and the one-compartment model suggests that different regions of the neuron contribute differently to the high- and low frequencies. The model suggests how NMDA current restricted to the soma evokes high-frequency oscillations (Fig. ​(Fig.1A)1A) - a recent experimental result. Alternatively, distal NMDA stimulation must span an extensive part of the dendritic tree to evoke the burst. The two distinct patterns of stimulation suggest that the burst may report different cue types, such as saliency and reward. In both cases, the voltage dependence of the NMDA current is central for this capability. Additionally, we introduced a putative potassium current that allows for sustained oscillations under blockade of the calcium-dependent (SK-type) potassium current. Given multiple de- and repolarizing currents that sustain pacemaking, the neuron has two interlocked mechanisms (calcium-dependent and independent; Fig. ​Fig.1B)1B) for producing oscillatory activity. Figure 1 (A) The model switches to high-frequency oscillations at the onset of NMDA receptor stimulation at 500 msec. Dashed is the Ca2+ concentration; Solid is the voltage. The increase in the frequency is based on the reduction in the amplitude of Ca2+ oscillations. ...

Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity

Excitotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-D-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca(2+)-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca(2+)](i)) increase. However, the basal level of [Ca(2+)](i) is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca(2+)](i) with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron.

An Approach to Experimental Synaptic Pathology Using Green Fluorescent Protein-Transgenic Mice and Gene Knockout Mice to Show Mitochondrial Permeability Transition Pore-Driven Excitotoxicity in Interneurons and Motoneurons

Researchers used transgenic mice expressing enhanced-green fluorescent protein (eGFP) driven by either the glycine transporter-2 gene promoter to specifically visualize glycinergic interneurons or the homeobox-9 (Hb9) gene promoter to visualize motoneurons for assessing their vulnerabilities to excitotoxins in vivo. Stereotaxic excitotoxic lesions were made in adult male and female mouse lumbar spinal cord with the specific N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA) and the non-NMDA ion channel glutamate receptor agonist kainic acid (KA). QA and KA induced large-scale degeneration of glycinergic interneurons in spinal cord. Glycinergic interneurons were more sensitive than motoneurons to NMDA receptor-mediated and non-NMDA glutamate receptor-mediated excitotoxicity. Outcome after spinal cord excitotoxicity was gender-dependent, with males showing greater sensitivity than females. Excitotoxic degeneration of spinal interneurons resembled apoptosis, while motoneuron degeneration appeared non-apoptotic. Perikaryal mitochondrial accumulation was antecedent to both NMDA and non-NMDA receptor-mediated excitotoxic stimulation of interneurons and motoneurons. Genetic ablation of cyclophilin D, a regulator of the mitochondrial permeability transition pore (mPTP), protected both interneurons and motoneurons from excitotoxicity. The results demonstrate in adult mouse spinal cord that glycinergic interneurons are more sensitive than motoneurons to excitotoxicity that stimulates mitochondrial accumulation, and that the mPTP has pro-death functions mediating apoptotic and non-apoptotic neuronal degeneration in vivo.

Neuroprotection of Interleukin-6 Against NMDA-Induced Apoptosis and Its Signal-Transduction Mechanisms

We have previously shown that interleukin-6 (IL-6)-protected neurons against the suppression of neuronal vitality and overload of intracellular Ca(2+) induced by glutamate or N-methyl-D: -aspartate (NMDA). Herein we provide further evidence for IL-6 neuroprotection against NMDA-induced apoptosis and explore the signal-transduction mechanisms underlying the anti-apoptotic action of IL-6. Cerebellar granule neurons (CGNs) from postnatal 8-day infant rats were chronically exposed to IL-6 (40 or 120ng/ml) for 8days, and stimulated with NMDA (100μM) for 30min. To observe the signaling pathways, we employed AG490 (5 or 10μM), an inhibitor of Janus kinases (JAKs), or LY294002 (5 or 10μM), an inhibitor of phosphatidylinositol 3-kinase (PI3K), to pretreat the CGNS together with IL-6. The levels of phosphorylation for the downstream effectors of JAKs and PI3K, i.e., phosphorylated STAT3 and Akt, were quantified by Western blot assay. In the cultured CGNs with various drug exposures, the expressions of Bcl-2, Bax, and caspase-3 were measured by real-time PCR and Western blot, and the percentage of apoptotic nuclei was tested by Hoechst 33342 staining. After the CGNs were chronically exposed to IL-6, NMDA stimulation led to an increase in the expression of Bcl-2 mRNA and a decrease in the expression of Bax and caspase-3 mRNAs and proteins when compared with those neurons lacking IL-6 exposure. IL-6 pretreatment of the neurons without NMDA stimulation concentration-dependently enhanced the expressions of Bcl-2 mRNA and protein while attenuating the expressions of Bax and caspase-3 mRNAs and proteins in comparison with control lacking any treatment. Furthermore, IL-6 prevented the increase in the percentage of apoptotic neurons induced by NMDA. The combined pretreatment of the CGNs with AG490 and IL-6 or with LY294002 and IL-6 reduced these anti-apoptotic effects of IL-6. Neither AG490 nor LY294002 exposure alone altered the expressions of Bcl-2, Bax, and cleaved caspase-3 proteins. IL-6 up-regulated the levels of phosphorylated STAT3 and Akt, and this was blocked by AG490 and LY294002, respectively. These results suggest that IL-6 protects neurons against NMDA-induced apoptosis, and that the IL-6 neuroprotection is jointly mediated by JAK-STAT3 and PI3K-Akt signaling pathways.

A new experimental model of focal seizures in the entorhinal cortex

Despite intensive studies, our understanding of the cellular and molecular mechanisms underlying epileptogenesis remains largely unsatisfactory. Our defective knowledge derives in part from the lack of adequate experimental models of the distinct phases that characterize the epileptic event, that is, initiation, propagation, and cessation. The aim of our study is the development of a new brain slice model in which a focal seizure can be repetitively evoked at a precise and predictable site. Epileptiform activities were studied by fast Ca²(+) imaging coupled with simultaneous single and double patch-clamp or extracellular recordings from neurons of entorhinal cortex (EC) slices from Wistar rats and C57BL6J mice at postnatal days 13-17. In the presence of 4-aminopyridine (4-AP) and low Mg²(+) , activation of layer V-VI neurons by local N-methyl-d-aspartate (NMDA) applications evolved into an ictal discharge (ID) that propagated to the entire EC. NMDA-evoked IDs were similar to spontaneous events. IDs with similar pattern and duration could be repetitively triggered from the same site by successive NMDA stimulations. The high ID reproducibility is an important feature of the model that allowed testing of the effects of currently used antiepileptic drugs (AEDs) on initiation, propagation, and cessation of focal ictal events. By offering the unique opportunity to repetitively evoke an ID from the same restricted site, this model represents a powerful approach to study the cellular and molecular events at the basis of initiation, propagation, and cessation of focal seizures.

Glucocorticoid Rapidly Enhances NMDA-Evoked Neurotoxicity by Attenuating the NR2A-Containing NMDA Receptor-Mediated ERK1/2 Activation

Glucocorticoid (GC) has been shown to affect the neuronal survival/death through a genomic mechanism, but whether or not it does through a nongenomic mechanism is unknown. Using a previously identified GR-deficient primary hippocampal neuron culture, we show here that a 15-min coexposure of N-methyl-D-aspartate (NMDA) with corticosterone at a stress-induced level significantly enhances neuronal death compared to NMDA alone. This enhancing effect of GC can be mimicked by the BSA-conjugated corticosterone, which is plasma membrane impermeable and cannot be blocked by RU38486 spironolactone. Furthermore, using a calcium-imaging technique, we found that B could increase both the percentage of neurons showing a significant increment of intracellular free calcium ([Ca2+](i)) due to NMDA stimulation and the amplitude of [Ca2+](i) increment in the individual responsive cells. Interestingly, this boosting effect of GC on [Ca2+](i) increment could be blocked by the NMDA receptor subunit 2A (NR2A)-specific antagonist [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydro-quinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) but not by the NMDA receptor subunit 2B (NR2B)-specific antagonist Ro25-6981. Moreover, we also found that GC can dramatically attenuate the NMDA-induced activation of ERK1/2 without affecting that of p38; and that the NMDA-induced ERK1/2 activation and its attenuation by GC both can be occluded by the NVP-AAM077 but not by Ro25-6981. Consistently, the enhancing effect of GC on NMDA neurotoxicity can also be blocked by NVP-AAM077 and the ERK1/2 inhibitor PD98059 but not by Ro25-6981 and p38 inhibitor SB203580. Indeed, the NMDA neurotoxicity itself can be blocked by Ro25-6981 or SB203580, whereas it is increased by NVP-AAM077 and PD98059. Therefore, it is probable that NMDA triggers a prodeath signaling through the NR2B-p38 MAPK pathway, and a prosurvival signaling through the NR2A-ERK1/2 MAPK pathway, whereas the latter was negatively regulated by rapid GC action. Taken together, the present data suggest a nongenomic action by GC that enhances NMDA neurotoxicity through facilitating [Ca2+](i) increment and attenuating the NR2A-ERK1/2-mediated neuroprotective signaling, implicating a novel pathway underlying the regulatory effect of GC on neuronal survival/death.

Involvement of NMDAR2A tyrosine phosphorylation in depression-related behaviour

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression-related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression-related behaviour. The NR2A subunit of the NMDA receptor is tyrosine-phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr-1325-Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock-in mice show antidepressant-like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock-in mice, DARPP-32 phosphorylation at Thr 34, which is important for the regulation of depression-related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src-induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor-mediated downstream signalling to modulate depression-related behaviour.

N-Methyl- d-Aspartate Receptor-Mediated Chemotaxis and Ca 2+ Signaling in Tetrahymena pyriformis

Although the ciliate Tetrahymena is a good model for the study of chemotaxis, its profound motility makes it difficult to monitor intracellular calcium (Ca(2+)) changes induced by chemotactic stimuli. In this study, we report a microfluidic-based chemotaxis system generating directional chemotactic gradients under highly viscous conditions, suppressing T. pyriformis motility, and allowing for the stable confocal imaging of changes in intracellular Ca(2+) in the ciliate. Once the viscous condition was achieved, directional chemical gradients were formed inside the center chamber via the release of N-methyl-d-aspartate (NMDA), a known chemoattractant, from the surrounding chemical reservoirs into the center chamber. As a result, intracellular Ca(2+) in the ciliate increased up to three-fold, and its distribution was skewed in the direction of NMDA stimulation. However, the Ca(2+) in ciliates pretreated with phospholipase C (PLC) or phosphatidylinositol-3-kinase (PI3K) blockers did not increase even after stimulation. Additionally, the PI3K blocker induced the secretion of granules, the size of which was dependent on the concentration of the blocker. Collectively, the results indicate that both PLC and PI3K perform pivotal roles in controlling the levels of intracellular Ca(2+) in T. pyriformis during chemotaxis.

Stimulation of NMDA and AMPA glutamate receptors elicits distinct concentration dynamics of nitric oxide in rat hippocampal slices

Nitric oxide ((*)NO) is an intercellular messenger implicated in memory formation and neurodegeneration in the hippocampus. Owing to its physical and chemical properties, the concentration dynamics of (*)NO is a critical issue in determining its bioactivity as a signaling molecule. Its production is closely related to glutamate N-methyl-D-aspartate (NMDA) receptors, following a rise in intracellular calcium levels. However, that dependent on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors remains elusive and controversial, despite reports describing a role for these receptors in other brain regions, largely because of lack of quantitative and dynamic measurements of (*)NO. Using a (*)NO-selective microsensor inserted in the diffusional spread of (*)NO in the CA1 region of rat hippocampal slices, we measured its real-time endogenous production, following activation of ionotropic glutamate receptors and under tissue physiological oxygen tension. Both NMDA and AMPA stimulation resulted in a concentration-dependent (*)NO production but encompassing distinct kinetics for lag phases and slower rates of (*)NO production were observed for AMPA stimulation. Robustness of the results was achieved instrumentally and pharmacologically, by means of nitric oxide synthase (NOS) inhibitors and antagonists of NMDA (D-(-)-2-amino-5-phosphonopentanoic acid, AP5) and AMPA (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, NBQX) receptors. When using glutamate as a stimulus, (*)NO production was of lower magnitude in the presence of AP5 plus NBQX than with AP5 alone, suggesting that even when NMDA receptors are inhibited Ca(2+) rises to levels to induce a peak of (*)NO from the background. Whereas extracellular Ca(2+) was required for the (*)NO signals, Philanthotoxin-4,3,3 (PhTX-4,3,3) a toxin used to target Ca(2+)-permeable AMPA receptors, attenuated (*)NO production. These observations are interpreted on basis of a distinct coupling between the glutamate receptors and neuronal NOS. A role for Ca(2+)-permeable AMPA receptors in the Ca(2+) activation of neuronal NOS is suggested.

Kinase Networks Integrate Profiles of N-Methyl-d-aspartate Receptor-mediated Gene Expression in Hippocampus

The postsynaptic N-methyl-d-aspartate (NMDA) receptor activates multiple kinases and changes the phosphorylation of many postsynaptic proteins organized in signaling networks. Because the NMDA receptor is known to regulate gene expression, it is important to examine whether networks of kinases control signaling to gene expression. We examined the requirement of multiple kinases and NMDA receptor-interacting proteins for gene expression in mouse hippocampal slices. Protocols that induce long-term depression (LTD) and long-term potentiation (LTP) activated common kinases and overlapping gene expression profiles. Combinations of kinases were required for induction of each gene. Distinct combinations of kinases were required to up-regulate Arc, Npas4, Egr2, and Egr4 following either LTP or LTD protocols. Consistent with the combinatorial data, a mouse mutant model of the human cognition disease gene SAP102, which couples ERK kinase to the NMDA receptor, showed deregulated expression of specific genes. These data support a network model of postsynaptic integration where kinase signaling networks are recruited by differential synaptic activity and control both local synaptic events and activity-dependent gene expression.