N-methyl-D-aspartic Acid Receptor Research Articles

Isoliquiritigenin Alleviates Semen Strychni-Induced Neurotoxicity by Restoring the Metabolic Pathway of Neurotransmitters in Rats.

Acute neurotoxicity of Semen Strychni can result in sudden death in epilepsy. The detoxification method and mechanism of Semen Strychni acute poisoning have not been clarified. This experiment focused on the mechanism of Semen Strychni neurotoxicity and the alleviation effects of isoliquiritigenin. The rats were intraperitoneally injected with Semen Strychni extract (125 mg/kg), followed by oral administration of isoliquiritigenin (50 mg/kg) for 7 days. FJ-B staining was used to evaluate the degree of injury on hippocampus neurons. The concentration of monoamines, amino acids, and choline neurotransmitters, the Dopamine (DA) and 5-hydroxytryptamine (5-HT) metabolic pathway in the hippocampus, cerebellum, striatum, prefrontal cortex, hypothalamus, serum, and plasma were detected by LC-MS/MS. The expression of neurotransmitter metabolic enzymes [catechol-O-methyl transferase (COMT) and monoamine oxidase (MAO)] and neurotransmitter receptors [glutamate N-methyl-D-aspartic acid receptors (NMDARs) and gamma-aminobutyric acid type A receptor (GABRs)] were, respectively determined using ELISA and qRT-PCR. The results indicated that Semen Strychni induced neuronal degeneration in the hippocampal CA1 region. Meanwhile, Semen Strychni inhibited the mRNA expression of NMDAR1, NMDAR2A, NMDAR2B, GABRa1, GABRb2 and reduced the level of MAO, which disrupted the DA and 5-HT metabolic pathway. However, isoliquiritigenin reversed these effects. In summary, isoliquiritigenin showed alleviation effects on Semen Strychni-induced neurotoxicity, which could be attributed to restoring neurotransmitters metabolic pathway, most likely through the activation of NMDA receptors.

Autism-Like Behavioral Phenotypes in Mice Treated with Systemic N-Methyl-D-Aspartate.

Autism spectrum disorder (ASD) having core characteristics of social interaction problems and repetitive behaviors and interests affects individuals at varying degrees and comorbidities, making it difficult to determine the precise etiology underlying the symptoms. Given its heterogeneity, ASD is difficult to treat and the development of therapeutics is slow due to the scarcity of animal models that are easy to produce and screen with. Based on the theory of excitation/inhibition imbalance in the brain with ASD which involves glutamatergic and/or GABAergic neurotransmission, a pharmacologic agent to modulate these receptors might be a good starting point for modeling. N-methyl-D-aspartic acid (NMDA) is an amino acid derivative acting as a specific agonist at the NMDA receptor and therefore imitates the action of the neurotransmitter glutamate on that receptor. In contrast to glutamate, NMDA selectively binds to and regulates the NMDA receptor, but not other glutamate receptors such as AMPA and kainite receptors. Given this role, we aimed to determine whether NMDA administration could result in autistic-like behavior in adolescent mice. Both male and female mice were treated with saline or NMDA (50 and 75 mg/kg) and were tested on various behavior experiments. Interestingly, acute NMDA-treated mice showed social deficits and repetitive behavior similar to ASD phenotypes. These results support the excitation/inhibition imbalance theory of ASD and that NMDA injection can be used as a pharmacologic model of ASD-like behaviors.

Tongluo Huatan capsule improves cognitive function by regulating the endocytosis of N-methyl-D-aspartic acid receptors mediated by clathrin in a rat model of vascular dementia.

To explore the neuroprotective mechanisms of Tongluo Huatan capsule (THC) in a rat model of vascular dementia (VD). A rat model of VD was established by repeated clamping of bilateral common carotid arteries with the intraperitoneal injection of sodium nitroprusside solution. VD rats were administered THC, memantine hydrochloride, or distilled water daily for 14 d after operation. Learning and memory abilities were assessed using the step-down passive avoidance test, novel object recognition (NOR) test, and Morris water maze (MWM) test. Pathological changes in the hippocampus were observed through hematoxylin and eosin and Nissl staining. The expression levels of clathrin, RAB5B, and N-methyl-D-aspartic acid receptor 1 (NMDAR1) were measured by immunohistochemistry staining, real-time quantitative polymerase chain reaction and Western blot. Rats in VD group showed impaired learning and memory abilities (step-down passive avoidance, NOR, and MWM) and abnormalities in neuronal morphology (light microscopy) in the hippocampus. The mRNA or protein expression levels of clathrin and RAB5B were decreased, and NMDAR1 was increased in hippocampal tissues (P < 0.05). Administration of THC promoted the learning and memory abilities and the morphological structure of hippocampal neurons in VD rats. Besides, THC enhanced mRNA or protein expression levels of clathrin and RAB5B, and decreased NMDAR1 (P < 0.05). THC may improve cognitive functions by regulating the endocytosis of NMDA receptors mediated by clathrin.

Enriched environment remedies cognitive dysfunctions and synaptic plasticity through NMDAR-Ca2+-Activin A circuit in chronic cerebral hypoperfusion rats.

Chronic cerebral ischemia (CCI) is one of the critical factors in the occurrence and development of vascular cognitive impairment (VCI). Apoptosis of nerve cells and changes in synaptic activity after CCI are the key factors to induce VCI. Synaptic stimulation up-regulates intraneuronal Ca2+ level through N-methyl-D-aspartic acid receptor (NMDAR) via induction of the activity-regulated inhibitor of death (AID) expression to produce active-dependent neuroprotection. Moreover, the regulation of synaptic plasticity could improve cognition and learning ability. Activin A (ActA), an exocrine protein of AID, can promote NMDAR phosphorylation and participate in the regulation of synaptic plasticity. We previously found that exogenous ActA can improve the cognitive function of rats with chronic cerebral ischemia and enhance the oxygenated glucose deprivation of intracellular Ca2+ level. In addition to NMDAR, the Wnt pathway is critical in the positive regulation of LTP through activation or inhibition. It plays an essential role in synaptic transmission and activity-dependent synaptic plasticity. The enriched environment can increase ActA expression during CCI injury. We speculated that the NMDAR-Ca2+-ActA signal pathway has a loop-acting mode, and the environmental enrichment could improve chronic cerebral ischemia cognitive impairment via NMDAR-Ca2+-ActA, Wnt/β-catenin pathway is involved in this process. For the hypothesis verification, this study intends to establish chronic cerebral hypoperfusion (CCH) rat model, explore the improvement effect of enriched environment on VCI, detect the changes in plasticity of synaptic morphology and investigate the regulatory mechanism NMDAR-Ca2+-ActA-Wnt/β-catenin signaling loop, providing a therapeutic method for the treatment of CCH.

Blocking N-Methyl-D-aspartic Acid (NMDA) Receptor Inhibits Heat-Sensitization Response of Moxibustion in Stroke Rats.

Our previous studies demonstrated that effects of moxibustion heavily relied on heat-sensitization response, a specific sensation induced by moxibustion in the ill body. On the sensation, long-term potentiation (LTP) of prelimbic cortex was attributed to heat-sensitization responses. The N-methyl-D-aspartic acid (NMDA) receptor plays a key role in LTP induction; however, little is known about the role of NMDA receptor in heat-sensitization response. The present study investigated the role of NMDA receptor in heat-sensitization response, specifically, NMDA receptor was inhibited by competitive glutamatergic antagonist, (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), observing the frequency of heat-sensitization response in moxibustion treatment and evaluating the conducive outcomes to cerebral infarct rats for rehabilitation. Heat-sensitization response in cerebral infarct rats was regularly measured for all the samples when exposed to moxibustion. Intraperitoneal injection of CPP was conducted, and soon afterwards, a significant drop of heat-sensitization response in all the samples was measured. Moreover, moxibustion efficiency on rehabilitation was unfavourably affected in cerebral infarct rats when compared to vehicle injection control. This indicated that NMDA receptor antagonist made a negative impact on induction of heat-sensitization response and consequently affected cerebral infarct rats to rehabilitate under moxibustion treatment. It also suggested that activating NMDA receptor played a positive part in ischemic stroke rehabilitation, and regulating its activity could be a feasible way to increase heat-sensitization response, improving the effect of moxibustion.

The primary motor cortex electrical and chemical stimulation attenuates the chronic neuropathic pain by activation of the periaqueductal grey matter: The role of NMDA receptors

BackgroundMotor cortex stimulation (MCS) is proper as a non-pharmacological therapy for patients with chronic and neuropathic pain (NP). AimsThis work aims to investigate if the MCS in the primary motor cortex (M1) produces analgesia and how the MCS could interfere in the MCS-induced analgesia. Also, to elucidate if the persistent activation of N-methyl-d-aspartic acid receptor (NMDAr) in the periaqueductal grey matter (PAG) can contribute to central sensitisation of the NP. MethodsMale Wistar rats were submitted to the von Frey test to evaluate the mechanical allodynia after 21 days of chronic constriction injury (CCI) of the sciatic nerve. The MCS was performed with low-frequency (20 μA, 100 Hz) currents during 15 s by a deep brain stimulation (DBS) device. Moreover, the effect of M1-treatment with an NMDAr agonist (at 2, 4, and 8 nmol) was investigated in CCI rats. The PAG dorsomedial column (dmPAG) was pretreated with the NMDAr antagonist LY 235959 (at 8 nmol), followed by MCS. ResultsThe MCS decreased the mechanical allodynia in rats with chronic NP. The M1-treatment with an NMDA agonist at 2 and 8 nmol reduced the mechanical allodynia in CCI rats. In addition, dmPAG-pretreatment with LY 235959 at 8 nmol attenuated the mechanical allodynia evoked by MCS. ConclusionThe M1 cortex glutamatergic system is involved in the modulation of chronic NP. The analgesic effect of MCS may depend on glutamate signaling recruitting NMDAr located on PAG neurons in rodents with chronic NP.

Phosphoproteomics reveals NMDA receptor-mediated excitotoxicity as a key signaling pathway in the toxicity of gelsenicine

Gelsenicine is one of the most toxic compounds in the genus Gelsemium, but the mechanism of toxicity is not clear. In this paper, tandem mass tag quantitative phosphoproteomics was used to study the changes in protein phosphorylation in different brain regions at different time points after gelsenicine poisoning in mice. The correlation between neurotransmitter receptors and the toxicity of gelsenicine was analyzed by molecular docking and rescue experiments. Parallel reaction monitoring (PRM) was used to verify the related proteins. A total of 17877 unique phosphosites were quantified and mapped to 4170 brain proteins to understand the signaling pathways. Phosphoproteomics revealed gelsenicine poisoning mainly affected protein phosphorylation levels in the hippocampus, and through bioinformatics analysis, it was found gelsenicine poisoning significantly affected neurotransmitter synaptic pathway. The molecular docking results showed that gelsenicine could bind to the N-methyl-D-aspartic acid receptor (NMDAR). In addition, we found that NMDA was effective in improving the survival rate of the animals tested, and this effect was associated with reduced protein phosphorylation by PRM validation. The results revealed that gelsenicine affects neurotransmitter release and receptor function. This is the first demonstration that NMDA receptor-mediated excitotoxicity is a key signaling pathway in the toxicity of gelsenicine.

From Menopause to Neurodegeneration-Molecular Basis and Potential Therapy.

The impacts of menopause on neurodegenerative diseases, especially the changes in steroid hormones, have been well described in cell models, animal models, and humans. However, the therapeutic effects of hormone replacement therapy on postmenopausal women with neurodegenerative diseases remain controversial. The steroid hormones, steroid hormone receptors, and downstream signal pathways in the brain change with aging and contribute to disease progression. Estrogen and progesterone are two steroid hormones which decline in circulation and the brain during menopause. Insulin-like growth factor 1 (IGF-1), which plays an import role in neuroprotection, is rapidly decreased in serum after menopause. Here, we summarize the actions of estrogen, progesterone, and IGF-1 and their signaling pathways in the brain. Since the incidence of Alzheimer’s disease (AD) is higher in women than in men, the associations of steroid hormone changes and AD are emphasized. The signaling pathways and cellular mechanisms for how steroid hormones and IGF-1 provide neuroprotection are also addressed. Finally, the molecular mechanisms of potential estrogen modulation on N-methyl-d-aspartic acid receptors (NMDARs) are also addressed. We provide the viewpoint of why hormone therapy has inconclusive results based on signaling pathways considering their complex response to aging and hormone treatments. Nonetheless, while diagnosable AD may not be treatable by hormone therapy, its preceding stage of mild cognitive impairment may very well be treatable by hormone therapy.

Shenzhi Jiannao formula ameliorates vascular dementia in vivo and in vitro by inhibition glutamate neurotoxicity via promoting clathrin-mediated endocytosis

BackgroundSynaptic damage and glutamate excitotoxicity have been implicated in the pathogenesis of vascular dementia (VD). Clathrin, RAB5B and N-methyl-d-aspartic acid receptor 1 (NMDAR1) proteins play a vital role in endocytosis of synaptic vesicles in neurons and glutamate over accumulation. Previous researches have been confirmed that Shenzhi Jiannao (SZJN) formula has an anti-apoptotic and neuroprotective effect in VD, but the underlying mechanisms are still unclear. In this study, we aimed to explore the effect of SZJN formula on cognitive impairment and glutamate excitotoxicity via clathrin-mediated endocytosis (CME) in vivo and in vitro.MethodsSZJN formula consists of Panax ginseng C.A.Mey., Anemarrhena asphodeloides Bunge, and Paeonia anomala subsp. veitchii (Lynch) D.Y.Hong & K.Y.Pan. All herbs were prepared into granules. Both common carotid arteries were permanent occluded (2‐vessel occlusion, 2VO) in male Sprague Dawley (SD) rats to model VD. One day after operation, the rats began daily treatment with SZJN formula for 2 weeks. The neuroprotective effects of SZJN formula was subsequently assessed by the novel object recognition test, Morris water maze, hematoxylin–eosin (HE) staining and Nissl staining. Glutamate cytotoxicity was assessed by detecting cell viability and cell death of PC12 cells. Immunohistochemistry, immunofluorescence, Western blot, and quantitative real‐time PCR were used to detect the expression levels of clathrin, RAB5B, and NMDAR1.ResultsAdministration of SZJN formula effectively improved short-term memory and spatial memory. SZJN formula treatment significantly reduced hippocampal neuronal loss, and recovered the arrangement and morphology of neurons and Nissl bodies. Moreover, SZJN formula promoted the proliferation of PC12 cells and inhibited glutamate-induced cell death. The down-regulation of clathrin and RAB5B, as well as the upregulation of NMDAR1 in the brain induced by 2VO or glutamate was also notably reversed by SZJN formula at both the protein and mRNA levels, which may contribute to SZJN formula induced improved neurological function.ConclusionsTaken together, our findings provide evidence that the neuroprotective effects of SZJN formula in experimental VD maybe mediated through promoting the expression of clathrin-mediated endocytosis and reducing NMDARs‐associated glutamate excitotoxicity. SZJN formula serves as a promising alternative therapy and may be a useful herbal medicine for preventing progression of VD.Graphic abstract

Exercise prevents the impairment of learning and memory in prenatally phthalate-exposed male rats by improving the expression of plasticity-related proteins

Regular exercise has been identified to facilitate neuroplasticity that maximize functional outcome after brain injuries. Brain-derived neurotrophic factor (BDNF) has emerged as a key facilitator of neuroplasticity after exercise. The activity-regulated cytoskeleton associated protein (Arc) is induced by BDNF and N-methyl-d-aspartic acid receptor (NMDAR), contributing to functional modification of neuroplasticity in the hippocampus. Meanwhile, early-life exposure to neuroendocrine disruptor di-(2-ethylhexyl)-phthalate (DEHP) is a risk factor for behavioral deficits, but the mechanisms responsible for DEHP-induced neurotoxicity are not well understood. The purpose of this study is to investigate whether hippocampal Arc expression is impaired by DEHP exposure and to examine the protective role of exercise in the prenatally DEHP-exposed male rats. Sprague Dawley dams were fed with vehicle or DEHP during gestation. The male offspring were trained to treadmill running for 5 weeks followed by examination of behavioral and biochemical outcomes. The results showed that DEHP-exposed rats exhibited impairment of spatial learning and memory as well as down-regulations of BDNF, NMDAR, Arc, and synaptophysin. Importantly, aerobic exercise during childhood-adolescence prevented the impairment of learning and memory by recovering the expressions of BDNF, NMDAR, Arc, and synaptophysin. These findings suggest that exercise may provide beneficial effects on ameliorating the impairment of neuroplasticity in the prenatally DEHP-exposed male rats at late adolescence.

Effect of Lenalidomide on Pentylenetetrazole-Induced Clonic Seizure Threshold in Mice: A Role for N-Methyl-D-Aspartic Acid Receptor/Nitric Oxide Pathway.

Background and PurposeAccumulating evidence suggest that lenalidomide, a structural analog of thalidomide, has neuro-modulatory and neuroprotective properties. In the present study, we investigated effects of acute administration of lenalidomide on clonic seizure threshold in mice induced by pentylenetetrazole (PTZ) and possible role of N-methyl-D-aspartic acid receptor (NMDAR) and nitric oxide (NO) pathway.MethodsWe have utilized a clonic model of seizure in NMRI mice induced by PTZ to evaluate the potential effect of lenalidomide on seizure threshold. Different doses of lenalidomide (5, 10, 20, and 50 mg/kg, intraperitoneal [i.p.]) were administered 1 hour before PTZ. To evaluate probable role of NMDAR/NO signaling, the non-selective NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 10 mg/kg, i.p.), neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI; 30 mg/kg, i.p.), selective inducible NOS inhibitor aminoguanidine (AG; 100 mg/kg, i.p.), selective NMDAR antagonist MK-801 (0.01 mg/kg, i.p.), and selective NMDAR agonist D-serine (30 mg/kg, i.p.) were injected 15 minutes before lenalidomide.ResultsLenalidomide at 10 and 20 mg/kg significantly elevated the PTZ-induced seizure thresholds. Interestingly, L-NAME (10 mg/kg, i.p), 7-NI (30 mg/kg, i.p), and AG (100 mg/kg, i.p) reversed the anticonvulsive effect of lenalidomide (10 mg/kg). Moreover, treatment with the NMDAR agonist D-serine (30 mg/kg, i.p.) did not alter the anticonvulsive properties of lenalidomide (10 mg/kg, i.p). However, the NMDAR antagonist MK-801 (0.01 mg/kg, i.p) significantly reversed the anticonvulsive effects of lenalidomide (10 mg/kg).ConclusionsOur study demonstrated a role for the NMDAR/NO pathway in the anticonvulsive effects of lenalidomide on the PTZ-induced clonic seizures in mice.

Forebrain GluN2A overexpression impairs fear extinction and NMDAR-dependent long-term depression in the lateral amygdala

N-methyl-d-aspartic acid receptor (NMDAR)-dependent synaptic plasticity at the thalamus-lateral amygdala (T-LA) synapses is related to acquisition and extinction of auditory fear memory. However, the roles of the NMDAR GluN2A subunit in acquisition and extinction of auditory fear memory as well as synaptic plasticity at T-LA synapses remain unclear. Here, using electrophysiologic, molecular biological techniques and behavioral methods, we found that the forebrain specific GluN2A overexpression transgenic (TG) mice exhibited normal acquisition but impaired extinction of auditory fear memory. In addition, in vitro electrophysiological data showed normal basal synaptic transmission and NMDAR-dependent long-term potentiation (LTP) at T-LA synapses, but deficit in NMDAR-dependent long-term depression (LTD) at T-LA synapses in GluN2A TG mice. Consistent with the reduced NMDAR-dependent LTD, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization was also weakened during NMDAR-dependent LTD in GluN2A TG mice. Taken together, our findings for the first time indicate that GluN2A overexpression impairs extinction of auditory fear memory and NMDAR-dependent LTD at T-LA synapses, which further confirms the close relationship between NMDAR-dependent LTD and fear extinction.

REL-1017 (Esmethadone) Increases Circulating BDNF Levels in Healthy Subjects of a Phase 1 Clinical Study.

Brain-derived neurotrophic factor (BDNF), a neurotrophin widely expressed in the central nervous system, exhibits important effects on neural plasticity. BDNF has been implicated in the mechanism of action of ketamine, a N-methyl-d-aspartic acid receptor (NMDAR) antagonist with rapid anti-depressant effects in humans. REL-1017 (esmethadone), the d-optical isomer of the racemic mixture d-l-methadone, is devoid of clinically relevant opioid activity at doses expected to exert therapeutic NMDAR antagonistic activity in humans. The present study was conducted to ascertain the effects of oral administration of 25 mg of REL-1017 for 10 days on plasma BDNF in healthy subjects confined to an inpatient unit for a phase 1 clinical trial. We observed an increase in post-treatment BDNF plasma levels compared to pre-treatment levels. Post-treatment, Day 10 BDNF plasma levels ranged from 2 to 17 times pre-treatment levels in the 25 mg REL-1017 treatment group, whereas in the placebo group, BDNF plasma levels remained unchanged (p = 0.028). Diastolic blood pressure decreased significantly in subjects treated with REL-1017, while no effect could be observed in the placebo group. In conclusion, the administration of 25 mg REL-1017 significantly increased BDNF plasma levels and significantly decreased diastolic blood pressure in healthy subjects confined to an inpatient unit for a phase 1 clinical trial.

Memantine Alleviates Acute Lung Injury Via Inhibiting Macrophage Pyroptosis.

Acute lung injury (ALI) is caused by direct pulmonary insults and indirect systemic inflammatory responses that result from conditions such as sepsis and trauma. Alveolar macrophages are the main and critical leukocytes in the airspace, and through the synthesis and release of various inflammatory mediators critically influence the development of ALI following infection and non-infectious stimuli. There is increasing recognition that inflammation and cell death reciprocally affect each other, which forms an auto-amplification loop of these two factors, and in turn, exaggerates inflammation. Therefore, pharmacological manipulation of alveolar macrophage death signals may serve as a logical therapeutic strategy for ALI. In this study, we demonstrate that memantine, a N-methyl-D-aspartic acid receptor (NMDAR) antagonist, through suppressing Ca2+ influx and subsequent ASC oligomerization inhibits macrophage Nlrp3 inflammasome activation and pyroptosis, therefore, alleviates ALI in septic mice. This finding explores a novel application of memantine, an FDA already approved medication, in the treatment of ALI, which is currently lacking effective therapy.

A preliminary study on the mechanism of the neurosteroid-mediated ionotropic receptor dysfunction in neurodevelopmental toxicity induced by decabromodiphenyl ether

The mechanism of neurodevelopmental toxicity of decabromodiphenyl ether (BDE209) remains unclear. Recent evidence suggests that neurosteroids disorders play a vital role in BDE209 induced-neurodevelopmental toxicity. To explore the mechanism of it, pregnant ICR mice were orally gavaged with 0, 225, and 900 mg kg−1 BDE209 for about 42 days. Spatial learning and memory abilities of offspring were tested on postnatal day (PND) 21. Offspring were euthanized at PND26, the neuronal structure, neurosteroids level, and related proteins including neurosteroids synthase, ionotropic receptors and cAMP-response element binding protein (CREB) pathway were evaluated, as well as Ca2+ concentration and the mitochondrial membrane potential (Mmp). Our results showed that BDE209 impaired learning and memory abilities and disrupted neuronal structure. Meanwhile, BDE209 decreased the pregnenolone (PREG), dehydroepiandrosterone (DHEA), progesterone (PROG) and allopregnanolone (ALLO) levels in the serum and brain, as well as the mRNA and protein levels of cholesterol-side-chain cleavage enzyme (P450scc), steroid 17α-hy-droxylase (P450C17), 3β-hydroxysteroid dehydrogenase (3β-HSD) and steroid 5α-reductase of type I (5α-R) in the hippocampi. Also, BDE209 suppressed mRNA and protein levels of NR1, NR2A and NR2B subunits of the N-methyl-D-aspartic acid receptor (NMDAR) and α1 subunit of the Gamma-amino butyric acid A receptor (GABAAR), but increased the levels of β2 and γ2 subunits of the GABAAR in the hippocampi. Moreover, BDE209 increased the Ca2+ concentration and phosphorylation extracellular regulated protein kinases (P-ERK) 1/2 level, but decreased the P-CREB and Mmp level in the hippocampi. These results indicate that BDE209 exposure during pregnancy and lactation is possible to affect learning and memory formation of offspring by the neurosteroid-mediated ionotropic receptors dysfunction.

Slack K+ channels attenuate NMDA-induced excitotoxic brain damage and neuronal cell death.

The neuronal Na+ -activated K+ channel Slack (aka Slo2.2, KNa 1.1, or Kcnt1) has been implicated in setting and maintaining the resting membrane potential and defining excitability and firing patterns, as well as in the generation of the slow afterhyperpolarization following bursts of action potentials. Slack activity increases significantly under conditions of high intracellular Na+ levels, suggesting this channel may exert important pathophysiological functions. To address these putative roles, we studied whether Slack K+ channels contribute to pathological changes and excitotoxic cell death caused by glutamatergic overstimulation of Ca2+ - and Na+ -permeable N-methyl-D-aspartic acid receptors (NMDAR). Slack-deficient (Slack KO) and wild-type (WT) mice were subjected to intrastriatal microinjections of the NMDAR agonist NMDA. NMDA-induced brain lesions were significantly increased in Slack KO vs WT mice, suggesting that the lack of Slack renders neurons particularly susceptible to excitotoxicity. Accordingly, excessive neuronal cell death was seen in Slack-deficient primary cerebellar granule cell (CGC) cultures exposed to glutamate and NMDA. Differences in neuronal survival between WT and Slack KO CGCs were largely abolished by the NMDAR antagonist MK-801, but not by NBQX, a potent and highly selective competitive antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, NMDAR-evoked Ca2+ signals did not differ with regard to Slack genotype in CGCs. However, real-time monitoring of K+ following NMDAR activation revealed a significant contribution of this channel to the intracellular drop in K+ . Finally, TrkB and TrkC neurotrophin receptor transcript levels were elevated in NMDA-exposed Slack-proficient CGCs, suggesting a mechanism by which this K+ channel contributes to the activation of the extracellular-signal-regulated kinase (Erk) pathway and thereby to neuroprotection. Combined, our findings suggest that Slack-dependent K+ signals oppose the NMDAR-mediated excitotoxic neuronal injury by promoting pro-survival signaling via the BDNF/TrkB and Erk axis.

The α2δ-1-NMDAR1 interaction in the trigeminal ganglion contributes to orofacial ectopic pain following inferior alveolar nerve injury

Orofacial ectopic pain can often arise following nerve injury. However, the exact mechanism responsible for orofacial ectopic pain induced by trigeminal nerve injury remains unknown. The α2δ-1 and glutamate N-methyl-d-aspartic acid receptor (NMDAR) interactions have been demonstrated to participate in neuropathic pain regulation in the spinal cord. In this study, a rat model of inferior alveolar nerve transection (IANX) was used to investigate the role of α2δ-1-NMDAR1 interaction in the trigeminal ganglion (TG) in regard to the regulation of orofacial ectopic pain. Western blot (WB) analysis indicated that α2δ-1 and NMDAR1 in the TG were substantially higher in IANX rats than they were in sham/naive rats. Additionally, immunofluorescence (IF) results revealed that α2δ-1 and NMDAR1 were co-expressed and distributed within neurons and activated satellite glial cells in the TG. Co-immunoprecipitation (Co-IP) results indicated that α2δ-1-NMDAR1 complex levels in the TG were higher in IANX rats than they were in sham rats. Furthermore, the results of behavioral tests demonstrated that intra-TG injection of gabapentin (α2δ-1 inhibitory ligand) or memantine hydrochloride (NMDAR antagonist) reversed the decrease in mechanical head-withdrawal threshold (HWT) in IANX rats. Moreover, inhibition of α2δ-1 by intra-TG administration of gabapentin suppressed the upregulation of the NMDAR1 protein, and the inhibition of NMDAR by intra-TG administration of memantine hydrochloride inhibited the increased expression of α2δ-1 protein induced by IANX. In conclusion, the physical and functional interaction between α2δ-1 and NMDAR1 is critical for the development of orofacial ectopic pain, indicating that α2δ-1, NMDAR1, and the α2δ-1–NMDAR1 complex may represent potential targets for the treatment of orofacial ectopic pain.

Hydrogen enriched saline alleviates morphine tolerance via inhibiting neuroinflammation, GLT-1, GS nitration and NMDA receptor trafficking and functioning in the spinal cord of rats

The development and maintenance of morphine tolerance showed association with neuroinflammation and dysfunction of central glutamatergic system (such as nitration of glutamate transporter). Recent evidence indicated that hydrogen could reduce the levels of neuroinflammation and oxidative stress, but its role in morphine tolerance has not been studied. The rats were intrathecally administered with morphine (10 μg/10 μL each time, twice/day for 5 days). Hydrogen enriched saline (HS) or saline was given intraperitoneally at 1, 3 and 10 mL/kg for 10 min before each dose of morphine administration. The tail-flick latency, mechanical threshold and thermal latency were assessed one day (baseline) before and daily for up to 5 days during morphine injection. The pro-inflammatory cytokine expressions [tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6)] (by western blotting), astrocyte activation (by immunofluorescence and western blotting), and nitration of glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) (by immunoprecipitation), membrane and total expression of N-methyl-d-aspartic acid (NMDA) receptor NR1 and NR2B subunits were carried out in the spinal dorsal horns. Chronic morphine administration induced antinociceptive tolerance, and together led to increased TNF-α, IL-1β and IL-6 expression, astrocyte activation, GLT-1 and GS nitration, increased membrane and total NR1, NR2B expression. Injection of HS attenuated morphine tolerance in a dose-dependent manner, decreased proinflammatory cytokine expression, inhibited astrocyte activation, decreased GLT-1 and GS nitration, and inhibited membrane trafficking of NMDA receptor. Our result showed that hydrogen pretreatment prevented morphine tolerance by reducing neuroinflammation, GLT-1, GS nitration, NMDA receptor trafficking in the spinal dorsal horn. Pretreatment with hydrogen might be considered as a novel therapeutic strategy for the prevention of morphine tolerance.

Abnormal Rat Cortical Development Induced by Ventricular Injection of rHMGB1 Mimics the Pathophysiology of Human Cortical Dysplasia.

Cortical dysplasia (CD) is a common cause of drug-resistant epilepsy. Increasing studies have implicated innate immunity in CD with epilepsy. However, it is unclear whether innate immune factors induce epileptogenic CD. Here, we injected recombinant human high mobility group box 1 (rHMGB1) into embryonic rat ventricles to determine whether rHMGB1 can induce epileptogenic CD with pathophysiological characteristics similar to those of human CD. Compared with controls and 0.1 μg rHMGB1-treated rats, the cortical organization was severely disrupted in the 0.2 μg rHMGB1-treated rats, and microgyria and heterotopia also emerged; additionally, disoriented and deformed neurons were observed in the cortical lesions and heterotopias. Subcortical heterotopia appeared in the white matter and the gray–white junction of the 0.2 μg rHMGB1-treated rats. Moreover, there was decreased number of neurons in layer V–VI and an increased number of astrocytes in layer I and V of the cortical lesions. And the HMGB1 antagonist dexmedetomidine alleviated the changes induced by rHMGB1. Further, we found that TLR4 and NF-κB were increased after rHMGB1 administration. In addition, the excitatory receptors, N-methyl-D-aspartate receptor 1 (NR1), 2A (NR2A), and 2B (NR2B) immunoreactivity were increased, and immunoreactivity of excitatory amino acid transporter 1 (EAAT1) and 2 (EAAT2) were reduced in 0.2 μg rHMGB1-treated rats compared with controls. While there were no differences in the glutamic acid decarboxylase 65/67 (GAD65/67) immunoreactivity between the two groups. These results indicate that the excitation of cortical lesions was significantly increased. Furthermore, electroencephalogram (EEG) showed a shorter latency of seizure onset and a higher incidence of status epilepticus in the 0.2 μg rHMGB1-treated rats; the frequency and amplitude of EEG were higher in the treated rats than controls. Intriguingly, spontaneous electrographic seizure discharges were detected in the 0.2 μg rHMGB1-treated rats after 5 months of age, and spike-wave discharges of approximately 8 Hz were the most significantly increased synchronous propagated waves throughout the general brain cortex. Taken together, these findings indicate that rHMGB1 exposure during pregnancy could contribute to the development of epileptogenic CD, which mimicked some pathophysiological characteristics of human CD.

77822 PSD95-nNOS interaction alters the basolateral amygdala transcriptome following fear conditioning: implications for molecular mechanisms underlying PTSD

ABSTRACT IMPACT: This research takes a transcriptomic approach to parse genes and molecular pathways that underlie the fear memory circuitry and, in doing so, identifies therapeutic targets that can further be developed into treatments for fear disorders, such as post-traumatic stress disorder. OBJECTIVES/GOALS: Normal fear learning produces avoidance behavior that promotes survival, but excessive and persistent fear after trauma can lead to development of phobias and post-traumatic stress disorder (PTSD). Our goal is to understand the mechanism and identify novel genetic targets underlying fear responses. METHODS/STUDY POPULATION: Involvement of the basolateral amygdala (BLA) in fear acquisition is well established and requires activation of N-methyl-D-aspartic acid receptors (NMDARs). At a cellular level, NMDAR activation leads to production of nitric oxide (NO) by a process mediated by interaction between postsynaptic density protein 95 (PSD95) and neuronal nitric oxide synthase (nNOS). To elucidate mechanisms underlying the role of the PSD95-nNOS-NO pathway in conditioned fear, here we use rodent behavioral paradigms, pharmacological treatment with a small molecule PSD95-nNOS inhibitor, RNA-sequencing, and an AAV-mediated knockdown of the nNOS gene in the BLA. RESULTS/ANTICIPATED RESULTS: We show that treatment with ZL006 attenuates rodent cued-fear consolidation. Additionally, with RNA-sequencing, expression of 516 genes was altered in the BLA following fear expression; of these genes, 83 were restored with systemic ZL006 treatment. Network data and gene ontology enrichment analyses further revealed that cGMP effects, insulin-like growth factor binding, and cognition-related pathways were significantly altered. Finally, we show that a BLA-specific knockdown of nNOS attenuates cued fear consolidation, without adverse effects on other memory and motor behaviors. DISCUSSION/SIGNIFICANCE OF FINDINGS: Via a model of NMDAR-mediated fear consolidation, our results reveal novel pathways and genetic targets that underlie plasticity of fear memory circuitry. Importantly, these results will inform future therapeutic strategies for targeting fear related disorders like PTSD.