N-methyl-D-aspartic Acid Receptor Agonist Research Articles

Effect of electrical and chemical (activation versus inactivation) stimulation of the infralimbic division of the medial prefrontal cortex in rats with chronic neuropathic pain.

Neuropathic pain (NP) represents a complex disorder with sensory, cognitive, and emotional symptoms. The medial prefrontal cortex (mPFC) takes critical regulatory roles and may change functionally and morphologically during chronic NP. There needs to be a complete understanding of the neurophysiological and psychopharmacological bases of the NP phenomenon. This study aimed to investigate the participation of the infralimbic division (IFL) of the mPFC in chronic NP, as well as the role of the N-methyl-D-aspartic acid receptor (NMDAr) in the elaboration of chronic NP. Male Wistar rats were submitted to the von Frey and acetone tests to assess mechanical and cold allodynia after 21days of chronic constriction injury (CCI) of the sciatic nerve or Sham-procedure ("false operated"). Electrical neurostimulation of the IFL/mPFC was performed by low-frequency stimuli (20 μA, 100Hz) applied for 15s by deep brain stimulation (DBS) device 21days after CCI. Either cobalt chloride (CoCl2 at 1.0mM/200 nL), NMDAr agonist (at 0.25, 1.0, and 2.0nmol/200 nL) or physiological saline (200 nL) was administered into the IFL/mPFC. CoCl2 administrationin the IFL cortex did not alter either mechanical or cold allodynia. DBS stimulation of the IFL cortex decreased mechanical allodynia in CCI rats. Chemical stimulation of the IFL cortex by an NMDA agonist (at 2.0nmol) decreased mechanical allodynia. NMDA at any dose (0.25, 1.0, and 2.0nmol) reduced the flicking/licking duration in the cold test. These findings suggest that the IFL/mPFC and the NMDAr of the neocortex are involved in attenuating chronic NP in rats.

Erythropoietin ameliorates cognitive deficits by improving hippocampal and synaptic damage in streptozotocin-induced diabetic mice

Recent studies have shown that erythropoietin (EPO) is an effective neuroprotective and neurotrophic agent for neurological disorders, such as traumatic brain injury and Alzheimer's disease. However, the effectiveness of EPO administration against diabetic cognitive impairments has rarely been examined. In this study, we investigated the effects of EPO on streptozotocin (STZ)-induced male C57BL/6 J mice. Then, we sought to clarify the mechanisms of EPO-mediated neuroprotection in high-glucose (HG)-stimulated HT22 cells. In vivo, we found that STZ-induced diabetic mice showed impaired spatial learning and memory, which was alleviated by EPO treatment. EPO also significantly lowered elevated fasting blood glucose levels, improved pancreatic and hippocampal damage, and restored oxidative stress in the STZ-induced diabetic mice. In vitro, EPO markedly increased cell viability, restrained the expression of pro-apoptotic Bax, enhanced the expression of pro-caspase 3, anti-apoptotic Bcl-2, brain-derived neurotrophic factor (BDNF) and postsynaptic density 95 (PSD-95), and attenuated the upregulation of N-methyl-d-aspartic acid (NMDA) receptor subunits NR1, NR2A and NR2B in HG-induced HT22 cells. The protective effects of EPO was obviously abolished by treatment with an NMDA receptor agonist. Our findings revealed that EPO impedes hippocampal and synaptic damage and neuronal apoptosis by regulating BDNF and PSD-95 expression through NMDA receptors, thereby ameliorating cognitive impairments in mice with T1DM.

Network Pharmacology and Experimental Verification to Unveil the Mechanism of N-Methyl-D-Aspartic Acid Rescue Humantenirine-Induced Excitotoxicity

Gelsemium is a medicinal plant that has been used to treat various diseases, but it is also well-known for its high toxicity. Complex alkaloids are considered the main poisonous components in Gelsemium. However, the toxic mechanism of Gelsemium remains ambiguous. In this work, network pharmacology and experimental verification were combined to systematically explore the specific mechanism of Gelsemium toxicity. The alkaloid compounds and candidate targets of Gelsemium, as well as related targets of excitotoxicity, were collected from public databases. The crucial targets were determined by constructing a protein-protein interaction (PPI) network. Subsequently, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to explore the bioprocesses and signaling pathways involved in the excitotoxicity corresponding to alkaloids in Gelsemium. Then, the binding affinity between the main poisonous alkaloids and key targets was verified by molecular docking. Finally, animal experiments were conducted to further evaluate the potential mechanisms of Gelsemium toxicity. A total of 85 alkaloids in Gelsemium associated with 214 excitotoxicity-related targets were predicted by network pharmacology. Functional analysis showed that the toxicity of Gelsemium was mainly related to the protein phosphorylation reaction and plasma membrane function. There were also 164 pathways involved in the toxic mechanism, such as the calcium signaling pathway and MAPK signaling pathway. Molecular docking showed that alkaloids have high affinity with core targets, including MAPK3, SRC, MAPK1, NMDAR2B and NMDAR2A. In addition, the difference of binding affinity may be the basis of toxicity differences among different alkaloids. Humantenirine showed significant sex differences, and the LD50 values of female and male mice were 0.071 mg·kg-1 and 0.149 mg·kg-1, respectively. Furthermore, we found that N-methyl-D-aspartic acid (NMDA), a specific NMDA receptor agonist, could significantly increase the survival rate of acute humantenirine-poisoned mice. The results also show that humantenirine could upregulate the phosphorylation level of MAPK3/1 and decrease ATP content and mitochondrial membrane potential in hippocampal tissue, while NMDA could rescue humantenirine-induced excitotoxicity by restoring the function of mitochondria. This study revealed the toxic components and potential toxic mechanism of Gelsemium. These findings provide a theoretical basis for further study of the toxic mechanism of Gelsemium and potential therapeutic strategies for Gelsemium poisoning.

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.

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.

Induction of analgesia using atorvastatin in experimental diabetic neuropathy through NMDA receptor and inflammatory cytokines inhibition

Background: Diabetic neuropathy is a complication of diabetes causing damage to the nerves. Objectives: Considering the neuroprotective anti-inflammatory antioxidant characteristics of statins, the current study aimed at determining the effects of atorvastatin on diabetic neuropathy through assessing the involvement of N-methyl-D-aspartic acid (NMDA) receptor, factors of oxidative stress, and inflammatory cytokines in rats with diabetes. Methods: Male rats were randomly assigned into six groups of the saline- and atorvastatin-treated controls, and streptozotocin (STZ)-induced diabetic animals treated with vehicle, diabetic animals treated with morphine (5 mg/kg), and rats treated with atorvastatin (10 mg/kg/day for 10 weeks) alone or in combination with NMDA receptor agonist. The hot plate and formalin tests were carried out on the rats. Moreover, malondialdehyde level, catalase and superoxide dismutase activities, levels of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in the dorsal root ganglia (DRG) of the animals were measured. Finally, the expression of the NMDA receptor in DRG was investigated in the current study. Results: Diabetes resulted in analgesia in all tests, and pretreatment with atorvastatin exacerbated diabetes-induced analgesic effects in the hot plate and early phase of the formalin test (P≤0.01 and P≤0.05 in comparison to those reported for the diabetic vehicle-treated group, respectively). The injection of NMDA could reverse the atorvastatin-induced analgesia in the hot plate test (P≤0.05). Diabetes caused to increase the levels of IL-1β, IL-6, and TNF-α in DRG in comparison to those reported for the control group (P≤0.05). Furthermore, the pretreatment of rats using atorvastatin could significantly reverse the increase in TNF-α level (P≤0.05). Conclusions: Atorvastatin showed analgesic properties, which might be acting through NMDA receptors and reduction of inflammatory cytokines.

Excessive activation of NMDA receptor inhibits the protective effect of endogenous bone marrow mesenchymal stem cells on promoting alveolarization in bronchopulmonary dysplasia.

We studied the role of bone marrow mesenchymal stem cells (MSCs) in our established model of bronchopulmonary dysplasia (BPD) induced by intrauterine hypoxia in the rat. First, we found that intrauterine hypoxia can reduce the number of MSCs in lungs and bone marrow of rat neonates, whereas the administration of granulocyte colony-stimulating factor or busulfan to either motivate or inhibit bone marrow MSCs to lungs altered lung development. Next, in vivo experiments, we confirmed that intrauterine hypoxia also impaired bone marrow MSC proliferation and decreased cell cycling activity. In vitro, by using the cultured bone marrow MSCs, the proliferation and the cell cycling activity of MSCs were also reduced when N-methyl-d-aspartic acid (NMDA) was used as an NMDA receptor (NMDAR) agonist. When MK-801 or memantine as NMDAR antagonists in vitro or in vivo was used, the reduction of cell cycling activity and proliferation were partially reversed. Furthermore, we found that intrauterine hypoxia could enhance the concentration of glutamate, an amino acid that can activate NMDAR, in the bone marrow of neonates. Finally, we confirmed that the increased concentration of TNF-ɑ in the bone marrow of neonatal rats after intrauterine hypoxia induced the release of glutamate and reduced the cell cycling activity of MSCs, and the latter could be partially reversed by MK-801. In summary, intrauterine hypoxia could decrease the number of bone marrow MSCs that could affect lung development and lung function through excessive activation of NMDAR that is partially caused by TNF-ɑ.

Anti-N-Methyl-D-Aspartic Acid Receptor 2 (Anti-NR2) Antibody in Neuropsychiatric Lupus Serum Damages the Blood-Brain Barrier and Enters the Brain.

BackgroundBrain microvessel endothelial cells constitute an important component in the blood-brain barrier. Cell-culture-based models of the blood-brain barrier (BBB) have been extensively applied in pharmacology, pathology and physiology. This study investigated effects of anti-N-methyl-D-aspartic acid receptor 2 (anti-NR2), N-methyl-D-aspartic acid (NMDA) receptor antibodies, NMDA receptor antagonists, and NMDA receptor agonists on brain microvessel endothelial cell models, and verified the effect of anti-NR2 antibody on the BBB as a receptor agonist.Material/MethodsThe primary brain microvessel endothelial cells were isolated and cultured, and an in vitro BBB model was established based on microvessel endothelial cells. Anti-NR2 antibody, glutamic acid, ifenprodil, and memantine were added in the BBB model to analyze changes in transepithelial electrical resistance (TEER) and to examine the permeability of the brain microvessel endothelial cell model.ResultsThe results showed that TEER values were significantly decreased by the addition of anti-NR2 antibody and glutamate, but were significantly increased by the addition of ifenprodil and memantine. TEER values showed no changes when treated by anti-NR2 antibody and ifenprodil, as well as anti-NR2 antibody and memantine. When dexamethasone was added, the TEER values increased by 23.8%, 39.4%, and 29.6% by treating with anti-NR2 antibody, positive cerebrospinal fluid, and positive serum, respectively.ConclusionsOur findings show that anti-NR2 antibody in neuropsychiatric lupus serum can damage the BBB and enter the brain.

Activation of Sigma-1 Receptor Alleviates Postpartum Estrogen Withdrawal-Induced "Depression" Through Restoring Hippocampal nNOS-NO-CREB Activities in Mice.

Postpartum depression affects approximately 15% of mothers; however, its pathological mechanisms still remain unclear. Ovariectomized adult mice received the administration of estrogen (E2) and progesterone with a subsequent alone E2, termed hormone-simulated pregnancy (HSP). Affective behaviors as assessed by forced swim and tail suspension tests, hippocampal neuronal nitric oxide synthase (nNOS), nitric oxide (NO), cyclic AMP (cAMP) response element binding protein (CREB) phosphorylation (phosphor-CREB), and neurosteroidogenesis were examined before E2 withdrawal (EW; HSP mice) and on days 2-4 (early-EW mice) and days 8-10 (late-EW mice) after EW. Depressive-like behaviors were observed in early-EW mice but not in late-EW mice. Levels of nNOS, NO, and phosphor-CREB were increased in HSP mice followed by a significant decline in early-EW mice with a subsequent restoration in late-EW mice. The treatment of early-EW mice with NO donor alleviated depressive-like behaviors and decline of phosphor-CREB. The nNOS inhibitor and NO scavenger caused depressive-like behaviors and reduced phosphor-CREB in HSP mice and late-EW mice. Notably, the levels of steroidogenic enzymes StAR and P450scc were elevated in late-EW mice. The sigma-1 receptor (σ1R) agonist could alleviate depressive-like behaviors and decline of nNOS-NO-CREB in early-EW mice. The pharmacological blockade or deficiency of σ1R in late-EW mice caused depressive-like behaviors with decline of nNOS-NO-CREB. The reduction of hippocampal brain-derived neurotrophic factor (BDNF) or N-methyl-D-aspartic acid (NMDA) receptor NR2B phosphorylation in early-EW mice could recover in late-EW mice, which was sensitive to the blockade of σ1R. The NMDA receptor agonist, but not TrkB receptor activator, could correct the decline of nNOS-NO-CREB in early-EW mice. The findings indicate that the activation of σ1R can alleviate postpartum "depression" through increasing nNOS-NO-CREB activities.

Involvement of Hippocampal NMDA Receptor and Nitric Oxide Synthase in Depression Induced by Chronic Unpredicted Mild Stress

In order to investigate whether N-methyl-D-aspartic acid (NMDA) receptor and nitric oxide synthase (NOS) made contributions to the depression induced by chronic unpredicted mild stress (CUMS)，the depression model was established by using the CUMS. Using the stereotaxic and intra-hippocampal microinjection observing rat emotion and behaviors by the change in body weight, sucrose preference test, open field test and forced swimming test, and immunohistochemistry, and the expression of NOS in hippocampus were tested. The results showed that depression-like behavior and the expression of NOS increased in hippocampus induced by rats receiving CUMS for 21 days or intra-hippocampal injections of NMDA receptor agonist. Intra-hippocampal injection of noncompetitive NMDA receptor antagonist MK-801 significantly prevented CUMS-induced depression-like behavioral changes, and decreased the expression of NOS. These results provide evidence that CUMS resulting in development of depression may induce neurotoxicity in hippocampal neurons by stimulating Glu release, inducing excessive activation of NMDA receptor, and increasing expression of NOS.

Ionotropic NMDA receptor evokes an excitatory response in superior salivatory nucleus neurons in anaesthetized rats

Extracellular recordings were taken from preganglionic superior salivatory nucleus (SSN) neurons projecting to submandibular and intra-lingual ganglia, in order to study the action of SSN neurons resulting from ionophoretic application of ionotropic NMDA receptor agonist in urethane-chloralose anaesthetized rats. Single SSN neurons were identified by their antidromic spike responses following stimulation of the chorda–lingual nerve (CLN), chorda tympani branches (CTBs) and the lingual nerve (LN). About one-third (33%, 10/30) of the identified SSN neurons were induced to fire by ionophoretic application of the NMDA receptor agonists used, dl-homocysteic acid (DLH) and N-methyl- d-aspartic acid (NMDA). More than half exhibited firing at high frequencies, often exceeding 40 Hz. About one-fifth (20%; 6/30) of the identified SSN neurons exhibited orthodromic spike responses to the combination of NMDA receptor agonist application and sensory nerve (CLN or LN) stimulus. These excitatory responses evoked by application of NMDA receptor agonist were attenuated ( n=4) by ionophoretic application of dl-2-amino-5-phosphonovaleric acid (AP5; NMDA receptor antagonist). About half (47%) of the neurons did not respond to any combination of NMDA receptor agonist and sensory nerve stimuli. No differences were observed between SSN neurons with B fibre axons and those with C fibre axons in response to ionophoresis of the NMDA receptor agonists. The NMDA-sensitive neurons, which exhibited high frequency firing, were predominantly found in the rostral part of the SSN. In summary, activation of ionotropic NMDA receptors exerts an excitatory effect on about half of the SSN neurons. These data support the view that NMDA receptors are involved in information processing and transmission on SSN neurons.

Behavioral effects of NMDA receptor agonists and antagonists in combination with nitric oxide-related compounds

Responding of rats was maintained under a 120-response fixed ratio (FR) schedule of food delivery, and animals received individual and combined injections of N-methyl- d-aspartic acid (NMDA), phencyclidine hydrochloride, (+)-MK-801 hydrogen maleate (MK-801), (±)-2-amino-5-phosphonopentanoic acid (AP5), 7-chlorokynurenic acid (7CK), ifenprodil tartrate, N G-nitro- l-arginine methyl ester hydorchloride ( l-NAME), 7-nitroindazole, aminoguanidine hemisulfate, l-arginine, molsidomine, sodium nitroprusside, and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8). Behavioral suppression after NMDA was completely and dose-dependently reversed by MK-801, phencyclidine, AP5, and aminoguanidine; partially and dose-dependently attenuated by molsidomine, ifenprodil, and 7CK; and not attenuated at all by l-NAME, 7-nitroindazole, or TMB-8. These findings suggested that behavioral suppression after NMDA was associated with nitric oxide from the inducible synthase. In a second series of experiments, comparable behavioral suppression by 0.1 mg/kg MK-801, but not 3 mg/kg phencyclidine, was attenuated by nitroprusside, molsidomine, and l-arginine, suggesting that suppressions from MK-801 and phencyclidine were mediated by different final common pathways, and that behavioral suppression from MK-801, but not phencyclidine, may be associated with Ca 2+-dependent nitric oxide.

Interaction of stress and strain on glutamatergic neurotransmission: relevance to schizophrenia

Psychosis caused by phencyclidine (PCP) stimulated interest in characterizing rodent behaviors elicited by PCP and its analogues. We have shown that MK-801 antagonizes electrically precipitated seizures (defined as tonic hindlimb extension) and elicits episodes of intense jumping behavior, referred to as “popping,” in mice. Moreover, 24 h after stress, MK-801's ability to antagonize electrically precipitated seizures is reduced in outbred NIH Swiss mice. Inbred BALBc mice are more resistant to electrically precipitated seizures than the NIH Swiss strain, and are more sensitive to both MK-801's anticonvulsant effect and ability to elicit popping. In the current experiments, we examined the influence of stress and genetic mouse strain on both MK-801's ability to antagonize electrically precipitated seizures and elicit popping. Stress significantly reduced the threshold voltage for precipitation of seizures in BALBc mice and the anticonvulsant properties of MK-801 in both strains. These data show that factors relevant to schizophrenia and its exacerbation (i.e., acute stress and genetics) influence N-methyl- d-aspartic acid (NMDA) receptor-mediated neurotransmission in intact mice. The BALBc inbred strain of mouse may possess advantages in preclinical screening paradigms designed to assess NMDA receptor agonist interventions for disorders such as schizophrenia. Specifically, stressed BALBc mice showed the greatest behavioral sensitivity to MK-801 with regard to electrically precipitated seizures in the incremental electroconvulsive shock (IECS) paradigm, whereas unstressed BALBc showed the greatest behavioral sensitivity to MK-801 in the “popping” paradigm, relative to BALBc and NIH Swiss mice in the appropriate comparison conditions.

Sexual differences in the role of kainate receptors in controlling gonadotrophin secretion in prepubertal rats.

Glutamate stimulates LH secretion in adult female rats after activation of N-methyl-D-aspartic acid (NMDA), kainate, and 2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid receptors. In contrast to the positive role of kainate receptors in the control of LH secretion in adult females, neither activation nor antagonization of kainate receptors in immature rats modified the onset of puberty. The present experiments were carried out to establish why, if kainate stimulates LH release in adult rats, it fails to advance puberty in immature rats, and to determine whether the role of kainate receptors is sexually dimorphic around puberty. In Expt 1, 4-, 8-, 12-, 16-, 20- and 30-day-old females were investigated 15 min after administration of vehicle or kainic acid, a kainate receptor agonist (2.5 mg kg-1). In Expt 2, 30-day-old female rats were studied 2, 5 and 10 min after administration of vehicle, 2.5 mg kainic acid kg-1 or NMDA, an NMDA receptor agonist (15 mg kg-1). In Expt 3, female and male rats were gonadectomized or sham-gonadectomized on day 23 and investigated on day 30 after injection of vehicle, kainic acid (2.5 mg kg-1 at -15 min) or 6,7 dinitroquinoxaline-2,3 dione (DNQX), a kainate antagonist (2 mg kg-1 in two injections at -120 and -60 min). Finally, 30-day-old female and male rats were investigated 15 min after injection of vehicle or NMDA (15 mg kg-1) or 60 min after administration of different doses of 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), an NMDA antagonist (0.1, 0.25 or 0.50 mg kg-1). The results indicate that the role of kainate receptors in the control of gonadotrophin secretion is sexually dimorphic around puberty, since: (a) LH secretion was stimulated by kainic acid in male rats but inhibited in females; (b) FSH secretion was inhibited by kainic acid in ovariectomized females, but not in orchidectomized males; and (c) DNQX inhibited LH secretion in males but not in females. These differences were specific for kainate receptors since, in both sexes, NMDA stimulated and MK-801 inhibited LH secretion. It may be concluded that the secretion of gonadotrophins is modulated differently by kainate receptors in prepubertal male and female rats.

Non-NMDA and NMDA receptor-mediated excitotoxic neuronal deaths in adult brain are morphologically distinct: Further evidence for an apoptosis-necrosis continuum

Apoptosis and necrosis are generally recognized as two distinct pathways of cell death, based on biochemical and morphological characteristics. Despite rapid advances in elucidating molecular mechanisms of cell death, little is known about the morphological progression of death in neurons and the relationship between different mechanisms of neuronal death and the resulting subcellular alterations. With excitotoxicity, a clinically relevant model of neuronal death, apoptotic DNA laddering and morphologic evidence of necrosis can occur simultaneously in the same region of adult brain. Here, we tested the hypothesis that activation of N-methyl-D-aspartic acid (NMDA) and non-NMDA glutamate receptors (GluR) results in a spectrum of morphologically distinct phenotypes of neuronal death, with apoptosis and necrosis as its endpoints. The ultrastructural morphologies of newborn and adult neurons at different times following intrastriatal injections of non-NMDA and NMDA GluR agonists were compared to apoptosis, as established during naturally occurring neuronal death in the developing rat brain. Excitotoxic neuronal death in newborn striatum was morphologically indistinguishable from developmental apoptosis. In the adult, non-NMDA receptor agonist-induced neuronal death was characterized by extensive chromatin condensation that was reminiscent of, but not identical to, apoptosis during normal development. In contrast, quinolinate, an NMDA receptor agonist, produced only minor chromatin clumping and rapid cytoplasmic disintegration, which is suggestive of necrosis. These findings support the concept that degenerative phenotypes of excitotoxically injured neurons are influenced by the degree of brain maturity and GluR subtype stimulation, independent of the severity of excitotoxic insult, along a morphological continuum or gradient ranging from apoptosis to necrosis.

An in vitro sciatic nerve preparation for the evaluation of pyrethroid neurotoxicity and the possible assessment of organophosphate-induced neuropathy

In this study, we examined the effects of a novel water-soluble, putative AMPA receptor antagonist, (−)(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid monohydrate (LY326325), on glutamate-, N-methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, and kainic acid (KA)-induced elevations of intracellular Ca2+ concentrations ([Ca2+]i) and 45Ca2+ uptake, as well as glutamate agonist-induced neurotoxicity in primary cultures of intact rat cortical and cerebellar granule neurons. In some experiments, the actions of LY326325 were tested in the presence of cyclothiazide, a compound that is known to block glutamate-induced desensitization of AMPA-preferring subtypes of glutamate receptors, thereby largely potentiating the functional effects of AMPA. LY326325 fully blocked the elevations of [Ca2+]i induced by NMDA and non-NMDA glutamate receptor agonists in both cortical and cerebellar granule neurons. The application of increasing concentrations of cyclothiazide was not able to reverse the LY326325-induced blockade of glutamate receptors in cortical neurons. In contrast, the same cyclothiazide treatment fully reversed the blockade produced by the noncompetitive AMPA/KA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine HCl (GYKI 52466). In 45Ca2+ uptake studies. LY325325 inhibited the NMDA-, AMPA-, and KA-induced enhancement of 45Ca2+ uptake in a concentration-dependent fashion in both cortical and cerebellar granule cells. In analogy to the results obtained with [Ca2+]i recordings, cyclothiazide failed to counteract the LY326325-induced blockade of KA-stimulated 45Ca2+ uptake in cerebellar granule neurons, whereas the blockade induced by the noncompetitive AMPA/KA receptor blocking agent GYKI 52466 was fully reversed by cyclothiazide. Because a similar, although not identical pattern of actions was seen following the application of the competitive AMPA/KA receptor antagonist 6-nitro-7-sulphamoyl-benzo(f)quinoxaline-2–3-dione (NBQX), it is suggested that the inhibitory actions of LY326325 are similar to those produced by NBQX but clearly differ from those caused by the noncompetitive AMPA/KA receptor antagonist GYKI 52466. Finally, when the neuroprotective actions of LY326325 on glutamate agonist-induced neurotoxicity were examined in cerebellar granule neurons, we found that LY326325 almost completely blocked the neurotoxic actions of NMDA, AMPA, and KA, respectively, whereas it produced only a partial blockade of glutamate-induced neurotoxicity. Taken together, our current results suggest that although LY326325 blocked both nonNMDA and NMDA-induced Ca2+ responses, it still displayed a preferential affinity for nonNMDA receptors as compared to NMDA receptors. However, LY326325 appears to be a less selective AMPA/KA receptor antagonist than NBQX and GYKI52466, respectively.

N-methyl-D-aspartic acid receptor agonists: resolution, absolute stereochemistry, and pharmacology of the enantiomers of 2-amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid.

(R,S)-2-Amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid [(R,S)-AMAA, 4] is a potent and selective agonist at the N-methyl-D-aspartic acid (NMDA) subtype of excitatory amino acid receptors. Using the Ugi "four-component condensation" method, the two diastereomers (2R)- and (2S)-2-[3-(benzyloxy)-5-methyl-4-isoxazolyl]N-tert-butyl-2- [N-[(S)-1-phenylethyl]benzamido]-acetamide (16 and 17, respectively) were synthesized and separated chromatographically. The absolute stereochemistry of 16 was confirmed by an X-ray analysis. Deprotection of these intermediates did, however, provide (R)- (8) and (S)- (9) AMAA, respectively, in extensively racemized forms. N-BOC-protected (R,S)-AMAA (21) was successfully resolved via diastereomeric salt formation using cinchonidine. The stereochemical purity and stability of 8 and 9 obtained via this resolution were determined using chiral HPLC. (R)-AMAA (8) showed peak affinity for [3H]AMPA receptor sites (IC50 = 72 +/- 13 microM) and was shown to be a more potent inhibitor of [3H]CPP binding (IC50 = 3.7 +/- 1.5 microM) than (S)-AMAA (9) (IC50 = 61 +/- 6.4 microM). Neither enantiomer of AMAA affected [3H]kainic acid receptor binding significantly. In electrophysiological studies using rat brain tissue, 8 (EC50 = 7.3 +/- 0.3 microM) was 1 order of magnitude more potent than 9 (EC50 = 75 +/- 9 microM) as an NMDA receptor agonist.

Glycine modulates N-methyl-D-aspartic acid induced learning facilitation in rats

Pretraining i.p. administration of N-methyl-D-aspartic acid (NMDA) at doses of 10 and 20mg/kg dose-dependently facilitated performance in a water T-maze learning task in rats. The effect of NMDA was inhibited by the competitive NMDA receptor antagonist CGP37849 [(DL)-E(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid] (CGP) at a dose of 6mg/kg, and by the NMDA receptor complex glycine site antagonist 1-hydroxy-3-amino-2-pyrrolidone (HA-966) at a dose of 10mg/kg. The NMDA site antagonist, when given alone, did not impair learning. The glycine precursor milacemide (2-N-pentylaminoacetamide HCl), at doses of 5 and 10mg/kg accelearted learning acquisition and its effect was antagonized by HA-966. The learning rate was impaired following the administration of NMDA 10mg/kg together with milacemide 5mg/kg when compared with the effect of 10mg/kg NMDA alone.The administration of 5mg/kg NMDA was associated with an elevated tissue concentration of aspartate in the hippocampus, an effect which was antagonized by 6mg/kg of CGP. NMDA at doses of 10 and 20mg/kg elevated the concentration of glycine but decreased the concentration of aspartate, glutamate and glutamine in the cortex and aspartate in the hippocampus. The cortical effects of NMDA 10mg/kg were antagonized by 6mg/kg of CGP. Milacemide at the dose of 10mg/kg elevated glycine, aspartate, glutamate and taurine concentrations. The coadministration of 5 mg/kg NMDA with 5mg/kg milacemide elevated the concentrations of glycine, glutamate and glutamine in the cortex and taurine in the hippocampus. These amino acid levels were higher than after administration of 5mg/kg either agent alone. The results demonstrate a dose-dependent facilitation effect on learning performance by NMDA and glycine receptor agonists. Antagonists at the NMDA and glycine sites counteracted the learning improvement of NMDA, and the glycine site antagonist the effect of milacemide.

Effects of decahydroisoquinoline-3-carboxylic acid monohydrate, a novel AMPA receptor antagonist, on glutamate-induced Ca 2+ responses and neurotoxicity in rat cortical and cerebellar granule neurons

In this study, we examined the effects of a novel water-soluble, putative AMPA receptor antagonist, (−)(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid monohydrate (LY326325), on glutamate-, N-methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, and kainic acid (KA)-induced elevations of intracellular Ca 2+ concentrations ([Ca 2+] i) and 45Ca 2+ uptake, as well as glutamate agonist-induced neurotoxicity in primary cultures of intact rat cortical and cerebellar granule neurons. In some experiments, the actions of LY326325 were tested in the presence of cyclothiazide, a compound that is known to block glutamate-induced desensitization of AMPA-preferring subtypes of glutamate receptors, thereby largely potentiating the functional effects of AMPA. LY326325 fully blocked the elevations of [Ca 2+] i induced by NMDA and non-NMDA glutamate receptor agonists in both cortical and cerebellar granule neurons. The application of increasing concentrations of cyclothiazide was not able to reverse the LY326325-induced blockade of glutamate receptors in cortical neurons. In contrast, the same cyclothiazide treatment fully reversed the blockade produced by the noncompetitive AMPA/KA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine HCl (GYKI 52466). In 45Ca 2+ uptake studies. LY325325 inhibited the NMDA-, AMPA-, and KA-induced enhancement of 45Ca 2+ uptake in a concentration-dependent fashion in both cortical and cerebellar granule cells. In analogy to the results obtained with [Ca 2+] i recordings, cyclothiazide failed to counteract the LY326325-induced blockade of KA-stimulated 45Ca 2+ uptake in cerebellar granule neurons, whereas the blockade induced by the noncompetitive AMPA/KA receptor blocking agent GYKI 52466 was fully reversed by cyclothiazide. Because a similar, although not identical pattern of actions was seen following the application of the competitive AMPA/KA receptor antagonist 6-nitro-7-sulphamoyl-benzo(f)quinoxaline-2–3-dione (NBQX), it is suggested that the inhibitory actions of LY326325 are similar to those produced by NBQX but clearly differ from those caused by the noncompetitive AMPA/KA receptor antagonist GYKI 52466. Finally, when the neuroprotective actions of LY326325 on glutamate agonist-induced neurotoxicity were examined in cerebellar granule neurons, we found that LY326325 almost completely blocked the neurotoxic actions of NMDA, AMPA, and KA, respectively, whereas it produced only a partial blockade of glutamate-induced neurotoxicity. Taken together, our current results suggest that although LY326325 blocked both nonNMDA and NMDA-induced Ca 2+ responses, it still displayed a preferential affinity for nonNMDA receptors as compared to NMDA receptors. However, LY326325 appears to be a less selective AMPA/KA receptor antagonist than NBQX and GYKI52466, respectively.

Differentiation of in vivo effects of AMPA and NMDA receptor ligands using drug discrimination methods and convulsant/anticonvulsant activity

The discriminative stimulus properties of the AMPA (( RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid) receptor agonist ATPA (( RS)-2-amino-3-(3-hydroxy-5- tert-butylisoxazol-4-yl)propionic acid) and NMDA ( N-methyl- d-aspartic acid) in rats have been characterized. It is suggested that the cues are mediated by separate mechanisms in the central nervous system. The ATPA cue is not mimicked by NMDA or an NMDA receptor agonist, and is inhibited by the AMPA receptor antagonist ( R)-APPA (( R)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid) but not the AMPA receptor antagonist ATOA (( RS-2-amino-3-(3-carboxymethoxy-5- tert-butylisoxazol-4-yl)propionic acid) or the NMDA receptor antagonist CPP (( RS)-3-(2-carboxypiperazin-4-yl)propyl)phosphonic acid). The ATPA cue is not mimicked by AMPA which is believed not to penetrate the blood-brain barrier. In contrast, ATPA does not generalize to the NMDA cue, which is mimicked by some NMDA receptor agonists (tetrazol-5-yl-glycine and AMAA (( RS)-2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid)) and is inhibited by the NMDA receptor antagonist CPP. Highly potent convulsant activity was demonstrated in mice with all AMPA and NMDA receptor agonists after intracerebroventricular (i.c.v.) injection, whereas weaker or no effects were found after subcutaneous (s.c.) or intravenous injection. Only ( RS)-tetrazol-5-yl-glycine had a potent effect after s.c. administration. I.c.v. ATOA and CPP inhibited convulsions induced by i.c.v. injectionof AMPA or NMDA, while ( R)-APPA was ineffective. These results indicate that there are differences in the structure-activity relations in the drug discrimination and convulsant/anticonvulsant models, even when effects after i.c.v. and s.c. injection are taken into consideration. The convulsion models are rapid tests which can give an indication of central nervous system penetration, but are less pharmacologically specific with respect to differentiation between AMPA and NMDA ligands where cue models demonstrate clear differences in effects of ligands with selectivity for receptor subtypes.