Natural product Kaji-ichigoside F1 exhibits rapid antidepression via activating the AMPA-BDNF-mTOR pathway and inhibiting the NMDAR-CaMKIIα pathway.

BACKGROUND: Depression is a common and recurrent neuropsychiatric disorder. Recent studies have shown that the N-methyl-d-aspartate (NMDA) receptor (NMDAR) is involved in the pathophysiology of depression. Previous studies have found that Kaji-ichigoside F1 (KF1) has a protective effect against NMDA-induced neurotoxicity. However, the antidepressant mechanism of KF1 has not been confirmed yet. PURPOSE: In the present study, we aimed to evaluate the rapid antidepressant activity of KF1 and explore the underlying mechanism. STUDY DESIGN: First, we explored the effect of KF1 on NMDA-induced hippocampal neurons and the underlying mechanism. Second, depression was induced in C57BL/6 mice via chronic unpredictable mild stress (CUMS), and the immediate and persistent depression-like behavior was evaluated using the forced swimming test (FST) after a single administration of KF1. Third, the contributions of NMDA signaling to the antidepressant effect of KF1 were investigated using pharmacological interventions. Fourth, CUMS mice were treated with KF1 for 21 days, and then their depression-like behaviors and the underlying mechanism were further explored. METHODS: The FST was used to evaluate immediate and persistent depression-like behavior after a single administration of KF1 with or without NMDA pretreatment. The effect of KF1 on depressive-like behavior was investigated in CUMS mice by treating them with KF1 once daily for 21 days through the sucrose preference test, FST, open field test, and tail suspension test. Then, the effects of KF1 on the morphology and molecular and functional phenotypes of primary neuronal cells and hippocampus of mice were investigated by hematoxylin-eosin staining, Nissl staining, propidium iodide staining, TUNEL staining, Ca2+ imaging, JC-1 staining, ELISA, immunofluorescence analysis, RT-PCR, and Western blot. RESULTS: KF1 could effectively improve cellular viability, reduce apoptosis, inhibit the release of LDH and Ca2+, and increase the mitochondrial membrane potential and the number of dendritic spines numbers in hippocampal neurons. Moreover, behavioral tests showed that KF1 exerted acute and sustained antidepressant-like effects by reducing Glu-levels and ameliorating neuronal damage in the hippocampus. Additionally, in vivo and in vitro experiments revealed that PSD95, Syn1, α-amino-3­hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and brain-derived neurotrophic factor (BDNF) were upregulated at the protein level, and BDNF and AMPA were upregulated at the mRNA level. NR1 and NR2A showed the opposite trend. CONCLUSION: These results confirm that KF1 exerts rapid antidepressant effects mainly by activating the AMPA-BDNF-mTOR pathway and inhibiting the NMDAR-CaMKIIα pathway. This study serves as a new reference for discovering rapid antidepressants.

Neuropharmacological Evidence Implicating Drug-Induced Glutamate Receptor Dysfunction in Affective and Cognitive Sequelae of Subchronic Methamphetamine Self-Administration in Mice.

Methamphetamine (MA) is a highly addictive drug, and MA use disorder is often comorbid with anxiety and cognitive impairment. These comorbid conditions are theorized to reflect glutamate-related neurotoxicity within the frontal cortical regions. However, our prior studies of MA-sensitized mice indicate that subchronic, behaviorally non-contingent MA treatment is sufficient to dysregulate glutamate transmission in mouse brain. Here, we extend this prior work to a mouse model of high-dose oral MA self-administration (0.8, 1.6, or 3.2 g/L; 1 h sessions × 7 days) and show that while female C57BL/6J mice consumed more MA than males, MA-experienced mice of both sexes exhibited some signs of anxiety-like behavior in a behavioral test battery, although not all effects were concentration-dependent. No MA effects were detected for our measures of visually cued spatial navigation, spatial learning, or memory in the Morris water maze; however, females with a history of 3.2 g/L MA exhibited reversal-learning deficits in this task, and mice with a history of 1.6 g/L MA committed more working-memory incorrect errors and relied upon a non-spatial navigation strategy during the radial-arm maze testing. Relative to naïve controls, MA-experienced mice exhibited several changes in the expression of certain glutamate receptor-related proteins and their downstream effectors within the ventral and dorsal areas of the prefrontal cortex, the hippocampus, and the amygdala, many of which were sex-selective. Systemic pretreatment with the mGlu1-negative allosteric modulator JNJ 162596858 reversed the anxiety-like behavior expressed by MA-experienced mice in the marble-burying test, while systemic pretreatment with NMDA or the NMDA antagonist MK-801 bi-directionally affected the MA-induced reversal-learning deficit. Taken together, these data indicate that a relatively brief history of oral MA is sufficient to induce some signs of anxiety-like behavior and cognitive dysfunction during early withdrawal that reflect, at least in part, MA-induced changes in the corticolimbic expression of certain glutamate receptor subtypes of potential relevance to treating symptoms of MA use disorder.

Tonic NMDAR Currents of NR2A-Containing NMDARs Represent Altered Ambient Glutamate Concentration in the Supraoptic Nucleus.

NMDA receptors (NMDARs) modulate glutamatergic excitatory tone in the brain via two complementary modalities: a phasic excitatory postsynaptic current and a tonic extrasynaptic modality. Here, we demonstrated that the tonic NMDAR-current (I NMDA) mediated by NR2A-containing NMDARs is an efficient biosensor detecting the altered ambient glutamate level in the supraoptic nucleus (SON). I NMDA of magnocellular neurosecretory cells (MNCs) measured by nonselective NMDARs antagonist, AP5, at holding potential (V holding) -70 mV in low concentration of ECF Mg2+ ([Mg2+]o) was transiently but significantly increased 1-week post induction of a DOCA salt hypertensive model rat which was compatible with that induced by a NR2A-selective antagonist, PEAQX (I PEAQX) in both DOCA-H2O and DOCA-salt groups. In agreement, NR2B antagonist, ifenprodil, or NR2C/D antagonist, PPDA, did not affect the holding current (I holding) at V holding -70 mV. Increased ambient glutamate by exogenous glutamate (10 mM) or excitatory amino acid transporters (EAATs) antagonist (TBOA, 50 mM) abolished the I PEAQX difference between two groups, suggesting that attenuated EAATs activity increased ambient glutamate concentration, leading to the larger I PEAQX in DOCA-salt rats. In contrast, only ifenprodil but not PEAQX and PPDA uncovered I NMDA at V holding +40 mV under 1.2 mM [Mg2+]o condition. I ifenprodil was not different in DOCA-H2O and DOCA-salt groups. Finally, NR2A, NR2B, and NR2D protein expression were not different in the SON of the two groups. Taken together, NR2A-containing NMDARs efficiently detected the increased ambient glutamate concentration in the SON of DOCA-salt hypertensive rats due to attenuated EAATs activity.

Multi-faceted Anti-obesity Effects of N-Methyl-D-Aspartate (NMDA) Receptor Modulators: Central-Peripheral Crosstalk.

Hypothalamus is central to food intake and satiety. Recent data unveiled the expression of N-methyl-D-aspartate receptors (NMDAR) on hypothalamic neurons and their interaction with GABAA and serotoninergic neuronal circuits. However, the precise mechanisms governing energy homeostasis remain elusive. Notably, in females, the consumption of progesterone-containing preparations, such as hormonal replacement therapy and birth control pills, has been associated with hyperphagia and obesity-effects mediated through the hypothalamus. To elucidate this phenomenon, we employed the progesterone-induced obesity model in female Swiss albino mice. Four NMDAR modulators were selected viz. dextromethorphan (Dxt), minocycline, d-aspartate, and cycloserine. Obesity was induced in female mice by progesterone administration for 4 weeks. Mice were allocated into 7 groups, group-1 as vehicle control (arachis oil), group-2 (progesterone + arachis oil), and group-3 as positive-control (progesterone + sibutramine); other groups were treated with test drugs + progesterone. Various parameters were recorded like food intake, thermogenesis, serum lipids, insulin, AST and ALT levels, organ-to-body weight ratio, total body fat, adiposity index, brain serotonin levels, histology of liver, kidney, and sizing of fat cells. Dxt-treated group has shown a significant downturn in body weight (p < 0.05) by a decline in food intake (p < 0.01), organ-to-liver ratio (p < 0.001), adiposity index (p < 0.01), and a rise in body temperature and brain serotonin level (p < 0.001). Dxt demonstrated anti-obesity effects by multiple mechanisms including interaction with hypothalamic GABAA channels and anti-inflammatory and free radical scavenging effects, improving the brain serotonin levels, and increasing insulin release from the pancreatic ß-cells.

Ginger (Zingiber Officinale Roscoe) ameliorates ethanol-induced cognitive impairment by modulating NMDA and GABA-A receptors in rat hippocampus.

Brain damage caused by ethanol abuse may lead to permanent damage, including severe dementia. The aim of this study was to investigate the effects of ginger powder on ethanol-induced cognitive disorders by examining oxidative damage and inflammation status, and the gene expression of N-methyl-D-aspartate (NMDA) and γ-Aminobutyric acid (GABA)-A receptors in the hippocampus of male rats. 24 adult male Sprague-Dawley rats were allocated randomly to four groups as follows control, ethanol (4g/kg/day, by gavage), ginger (1g/kg/day, by gavage), and ginger-ethanol. At the end of the study, memory and learning were evaluated by the shuttle box test. Moreover, to explore mechanisms involved in ethanol-induced cognitive impairment and the protective effect of ginger, the expression of Nuclear factor kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), NMDA receptor, and GABA-A receptor was measured along with inflammatory and oxidative biomarkers in the hippocampus tissue. The results showed that ethanol could induce cognitive impairment in the ethanol group, while pretreatment with ginger could reverse it. The gene expression of the NF-κB/ Tumor necrosis factor (TNF)-α/Interleukin (IL)-1ß pathway and NMDA and GABA-A receptors significantly increased in the ethanol group compared to the control group. While pretreatment with ginger could significantly improve ethanol-induced cognitive impairment through these pathways in the ginger-ethanol group compared to the ethanol group (P < 0.05). It can be concluded that ginger powder could ameliorate ethanol-induced cognitive impairment by modulating the expression of NMDA and GABA-A receptors and inhibiting oxidative damage and the NF-κB/TNF-α/IL-1ß pathway in the rat hippocampus.

The role of glutamate NMDA receptors of the mediodorsal thalamus in scopolamine-induced amnesia in rats.

The current study was designed to examine the role of glutamate NMDA receptors of the mediodorsal thalamus (MD) in scopolamine-induced memory impairment. Adult male rats were bilaterally cannulated into the MD. According to the results, intraperitoneal (i.p.) administration of scopolamine (1.5 mg/kg) immediately after the training phase (post-training) impaired memory consolidation. Bilateral microinjection of the glutamate NMDA receptors agonist, N-Methyl-D-aspartic acid (NMDA; 0.05 µg/rat), into the MD significantly improved scopolamine-induced memory consolidation impairment. Co-administration of D-AP5, a glutamate NMDA receptor antagonist (0.001-0.005 µg/rat, intra-MD) potentiated the response of an ineffective dose of scopolamine (0.5 mg/kg, i.p.) to impair memory consolidation, mimicking the response of a higher dose of scopolamine. Noteworthy, post-training intra-MD microinjections of the same doses of NMDA or D-AP5 alone had no effect on memory consolidation. Moreover, the blockade of the glutamate NMDA receptors by 0.003 ng/rat of D-AP5 prevented the improving effect of NMDA on scopolamine-induced amnesia. Thus, it can be concluded that the MD glutamatergic system may be involved in scopolamine-induced memory impairment via the NMDA receptor signaling pathway.

Inhibitory effect of berberine on morphine tolerance and hyperalgesia in mice.

OBJECTIVE: To evaluate the effect of berberine on morphine analgesia, tolerance, and hyperalgesia. METHODS: Morphine-induced acute tolerance model: mice received intraperitoneal berberine at doses of 2.5, 5.0, and 10 mg/kg; 30 min later, subcutaneous morphine 10 mg/kg was injected every hour for nine continuous h. Morphine 10 mg/kg alone was administered at 24 and 48 h. Morphine-induced chronic tolerance model: mice received intraperitoneal berberine 2.5, 5.0, and 10 mg/kg; 30 min later, 10 mg/kg morphine was injected subcutaneously for eight consecutive days. On the ninth day, morphine 10 mg/kg was given alone. Morphine-induced established tolerance model: mice were injected subcutaneously with morphine 10 mg/kg once a day for eight consecutive days. Berberine 2.5 mg/kg was administered on day one, four, and seven and morphine 10 mg/kg alone on day nine. The baseline latency (T0) and post-treatment latency (T1) were determined by the hot plate test, and the maximum possible analgesic effect (MPAE) was calculated. Nitric oxide synthase (NOS) activity and nitric oxide (NO) content in the spinal cord were measured by spectrophotometer. Verification of berberine analgesic effect by blocking N-methyl-D-aspartate (NMDA) receptor: HT-22 and HEK-293 cells transfected with NMDA plasmid were randomly divided into five groups: control group, NMDA group, berberine low-dose, medium-dose, and high-dose groups (5, 10, 20 µmol/L, respectively). Except for the control group, cells were treated with NMDA (HT-22 cells: 20 mmol/L; HEK-293 cells: 50 µmol/L). After 24 h, cell viability was detected by cell counting kit-8. The molecular mechanism between berberine and the NMDA receptor was studied by molecular docking. RESULTS: Berberine 2.5 and 5.0 mg/kg could prolong the analgesic time of morphine. In acute and chronic morphine tolerance models, berberine could inhibit the decrease of MPAE and baseline latency (0.05). In the established tolerance model, berberine could rapidly reverse the decreased MPAE (0.05). The combination of berberine and morphine on day one could effectively inhibit the morphine-induced increase of NOS activity and NO content in the spinal cord (0.05). Berberine significantly increased the cell viability of NMDA-induced nerve injury in HT-22 and HEK-293 cells (0.05). Molecular docking showed that berberine binds to the receptor pocket of NMDA. CONCLUSIONS: Berberine could effectively enhance and prolong the duration of morphine analgesia and inhibit the development of morphine-induced tolerance and hyperalgesia. Furthermore, berberine has a certain neuroprotective effect, which may be related to the inhibition of NMDA activity.

Two types of GLR channels cooperate differently in light and dark growth of Arabidopsis seedlings.

BACKGROUND: GLutamate Receptor-like (GLR) channels are multimeric, ionotropic, ligand-gated plant transmembrane receptors. They are homologous to mammalian glutamate receptors, iGLuRs, which are critical to neuronal function. GLRs have been reported several times to play a role in photomorphogenesis. However, to date, no study has looked at the mechanism of their involvement in this process. Here we focused on examining the impact of GLRs on the regulation of early seedling growth in blue light, red light, and in the dark. RESULTS: Wild type and six photoreceptor mutant seedlings were grown on media supplemented with known iGLuR/GLR channel antagonists: MK-801, which non-competitively blocks NMDA channels in mammalian cells, and CNQX, known for competitive blocking of AMPA channels in mammalian cells. The lengths of hypocotyls and roots were measured in seedlings of phyA, phyB, phot1, phot2, cry1, and cry2 mutants after 7 days of in vitro culture. Changes in growth parameters, both in light and in darkness upon application of chemical antagonists, show that both types of GLR channels, NMDA-like and AMPA-like, are involved in the regulation of seedling growth irrespective of light conditions. Analysis of seedling growth of photoreceptor mutants indicates that the channels are influenced by signaling from phot1, phot2, and cry1. To extend our analysis, we also evaluated the elicitation of a calcium wave, which is likely to be partially driven by GLRs, in Arabidopsis seedlings. The changes in cellobiose-induced calcium waves observed after applying GLR inhibitors suggest that both types of channels likely cooperate in shaping Arabidopsis seedling growth and development. CONCLUSIONS: Our work provides the first experimental evidence that two types of GLR channels function in plants: NMDA-like and AMPA-like. We also demonstrate that the channels are involved in seedling growth and development, at least partially through modulation of calcium signaling, but they are unlikely to play a major role in photomorphogenesis.

Alzheimer's disease: Molecular aspects and treatment opportunities using herbal drugs.

Alzheimer's disease (AD), also called senile dementia, is the most common neurological disorder. Around 50 million people, mostly of advanced age, are suffering from dementia worldwide and this is expected to reach 100-130 million between 2040 and 2050. AD is characterized by impaired glutamatergic and cholinergic neurotransmission, which is associated with clinical and pathological symptoms. AD is characterized clinically by loss of cognition and memory impairment and pathologically by senile plaques formed by Amyloid ß deposits or neurofibrillary tangles (NFT) consisting of aggregated tau proteins. Amyloid ß deposits are responsible for glutamatergic dysfunction that develops NMDA dependent Ca2+ influx into postsynaptic neurons generating slow excitotoxicity process leading to oxidative stress and finally impaired cognition and neuronal loss. Amyloid decreases acetylcholine release, synthesis and neuronal transport. The decreased levels of neurotransmitter acetylcholine, neuronal loss, tau aggregation, amyloid ß plaques, increased oxidative stress, neuroinflammation, bio-metal dyshomeostasis, autophagy, cell cycle dysregulation, mitochondrial dysfunction, and endoplasmic reticulum dysfunction are the factors responsible for the pathogenesis of AD. Acetylcholinesterase, NMDA, Glutamate, BACE1, 5HT6, and RAGE (Receptors for Advanced Glycation End products) are receptors targeted in treatment of AD. The FDA approved acetylcholinesterase inhibitors Donepezil, Galantamine and Rivastigmine and N-methyl-D-aspartate antagonist Memantine provide symptomatic relief. Different therapies such as amyloid ß therapies, tau-based therapies, neurotransmitter-based therapies, autophagy-based therapies, multi-target therapeutic strategies, and gene therapy modify the natural course of the disease. Herbal and food intake is also important as preventive strategy and recently focus has also been placed on herbal drugs for treatment. This review focuses on the molecular aspects, pathogenesis and recent studies that signifies the potential of medicinal plants and their extracts or chemical constituents for the treatment of degenerative symptoms related to AD.

Anti-depressant-like effect of fermented Gastrodia elata Bl. by regulating monoamine levels and BDNF/NMDAR pathways in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodia elata Blume (GE) is a Chinese medicinal herb commonly used to treat central nervous system-related diseases, including headaches, dizziness, epilepsy, numbness of the limbs and depression. AIM OF THE STUDY: Microbial-based fermentation has been successfully used to increase the extract efficiency of medicinal herbs in recent years. However, no study has hitherto explored the anti-depressant-like effect of GE processed by microorganisms. Herein, this subject aimed to clarify the anti-depressant-like effect of fermented Gastrodia elata Bl. (FGE) and its active chemical constituents. MATERIALS AND METHODS: The chronic unpredictable mild stress (CUMS) model, a well-established animal model of depression, was induced in Kunming (KM) mice. The mice were administrated with FGE for 3 weeks. The sucrose preference test (SPT), open field test (OFT) and tail suspension test (TST) were conducted. Moreover, the levels of serotonin (5-HT) and dopamine (DA) in brain tissue homogenates, the concentration of Ca2+ and the activity of MAO in serum, H&E and Nissl staining in the hippocampus, and the hippocampus protein expressions of BDNF, NMDAR1, NMDAR2A and NMDAR2B relevant to depression were detected. Furthermore, chemical constituents of FGE were further isolated, and the protective activity of the obtained compounds against NMDA-induced PC-12 cell damage was assessed. RESULTS: FGE could alleviate the depression state in CUMS-induced mice and reduce apoptosis of neuronal cells in the hippocampus. Furthermore, FGE could improve the contents of 5-HT, DA and decrease the concentration of Ca2+ and MAO activity in brain tissue and serum compared with the control group. It could reverse the decreased expression of BDNF, NMDAR2A and NMDAR2B and increase NMDAR1 protein expression. Investigation of the active constituents from FGE yielded two new compounds, (4-(((4-ethoxybenzyl) oxy)methyl)-phenol 1 and 3-((4-hydroxy benzyl)oxy)propane-1,2-diol) 2, with twelve known compounds (3-14). The compounds (3-((4-hydroxybenzyl)oxy)propane-1,2-diol 2, 4, 4'-dihydroxyd iphenyl methane 3, and bungein A 4) protected against NMDA-induced PC-12 cells damage. CONCLUSION: This study demonstrated that FGE could improve the depressive behavior of CUMS-induced mice and exert a protective effect on nerve cells in the brain. Importantly, compounds 2-4 are the active components of FGE. Overall, the above findings suggest that FGE has huge prospects for application in treating depression-related diseases.

Discovery of a cysteine-rich peptide with glycation modification from Achyranthes bidentata Blume.

Cysteine-rich peptides (CRPs) are stable molecules that contain multiple disulphide bonds. Various CRPs are found in plants and animals, representing potential compounds for drug development with diverse activities. Modification of CRPs, such as glycation, has attracted increased attention due to its special structural and functional properties. Hence, this study explored a CRP isolated from the Chinese herb Achyranthes bidentata Blume, which contains a glycation modification. Herein, a reverse phase high-performance liquid chromatography system with mobile phases was used to extract and purify the peptide. The eluted peptide was detected using high resolution mass spectrometry and structurally identified using high resolution mass spectrometry and nuclear magnetic resonance. The effect of the peptide on the viability of N-methyl-D-aspartic acid (NMDA)-induced HT22 cells was determined using a cell assay. Here, a new cysteine-rich glycation peptide, termed glycation-bidentatide (Gly-BTP), with three pairs of disulphide bonds and a glycation modification at the N-terminus linked to cysteine, was discovered. Cell bioactivity assay results suggested that Gly-BTP might be a potential therapeutic and provide a neuroprotective effect in NMDA-induced HT22 murine hippocampal neuronal cells. The discovery of Gly-BTP will promote the understanding of the role of CRPs in neuroprotection.

Rutin prevents seizures in kainic acid-treated rats: evidence of glutamate levels, inflammation and neuronal loss modulation.

Rutin, a naturally derived flavonoid molecule with known neuroprotective properties, has been demonstrated to have anticonvulsive potential, but the mechanism of this effect is still unclear. The current study aimed to investigate the probable antiseizure mechanisms of rutin in rats using the kainic acid (KA) seizure model. Rutin (50 and 100 mg kg-1) and carbamazepine (100 mg kg-1) were administered daily by oral gavage for 7 days before KA (15 mg kg-1) intraperitoneal (i.p.) injection. Seizure behavior, neuronal cell death, glutamate concentration, excitatory amino acid transporters (EAATs), glutamine synthetase (GS), glutaminase, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, N-methyl-D-aspartate (NMDA) receptor subunits GluN2A and GluN2B, activated astrocytes, and inflammatory and anti-inflammatory molecules in the hippocampus were evaluated. Supplementation with rutin attenuated seizure severity in KA-treated rats and reversed KA-induced neuronal loss and glutamate elevation in the hippocampus. Decreased glutaminase and GluN2B, and increased EAATs, GS, GluA1, GluA2 and GluN2A were observed with rutin administration. Rutin pretreatment also suppressed activated astrocytes, downregulated the protein levels of inflammatory molecules [interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), high mobility group Box 1 (HMGB1), interleukin-1 receptor 1 (IL-1R1), and Toll-like receptor-4 (TLR-4)] and upregulated anti-inflammatory molecule interleukin-10 (IL-10) protein expression. Taken together, the results indicate that the preventive treatment of rats with rutin attenuated KA-induced seizures and neuronal loss by decreasing glutamatergic hyperactivity and suppressing the IL-1R1/TLR4-related neuroinflammatory cascade.

Short- and Long-Term Effects of Suboptimal Selenium Intake and Developmental Lead Exposure on Behavior and Hippocampal Glutamate Receptors in a Rat Model.

Selenium (Se) is an essential trace element required for normal development as well as to counteract the adverse effects of environmental stressors. Conditions of low Se intake are present in some European countries. Our aim was to investigate the short- and long-term effects of early-life low Se supply on behavior and synaptic plasticity with a focus on the hippocampus, considering both suboptimal Se intake per se and its interaction with developmental exposure to lead (Pb). We established an animal model of Se restriction and low Pb exposure; female rats fed with an optimal (0.15 mg/kg) or suboptimal (0.04 mg/kg) Se diet were exposed from one month pre-mating until the end of lactation to 12.5 µg/mL Pb via drinking water. In rat offspring, the assessment of motor, emotional, and cognitive endpoints at different life stages were complemented by the evaluation of the expression and synaptic distribution of NMDA and AMPA receptor subunits at post-natal day (PND) 23 and 70 in the hippocampus. Suboptimal Se intake delayed the achievement of developmental milestones and induced early and long-term alterations in motor and emotional abilities. Behavioral alterations were mirrored by a drop in the expression of the majority of NMDA and AMPA receptor subunits analyzed at PND 23. The suboptimal Se status co-occurring with Pb exposure induced a transient body weight increase and persistent anxiety-like behavior. From the molecular point of view, we observed hippocampal alterations in NMDA (Glun2B and GluN1) and AMPA receptor subunit trafficking to the post-synapse in male rats only. Our study provides evidence of potential Se interactions with Pb in the developing brain.

Calcineurin Controls Hypothalamic NMDA Receptor Activity and Sympathetic Outflow.

RATIONALE: Hypertension is a common and serious adverse effect of calcineurin inhibitors, including cyclosporine and tacrolimus (FK506). Although increased sympathetic nerve discharges are associated with calcineurin inhibitor-induced hypertension, the sources of excess sympathetic outflow and underlying mechanisms remain elusive. Calcineurin (protein phosphatase-2B) is broadly expressed in the brain, including the paraventricular nuclear (PVN) of the hypothalamus, which is critically involved in regulating sympathetic vasomotor tone. OBJECTIVE: We determined whether prolonged treatment with the calcineurin inhibitor causes elevated sympathetic output and persistent hypertension by potentiating synaptic N-methyl-D-aspartate (NMDA) receptor activity in the PVN. METHODS AND RESULTS: Telemetry recordings showed that systemic administration of FK506 (3 mg/kg per day) for 14 days caused a gradual and profound increase in arterial blood pressure in rats, which lasted at least 7 days after discontinuing FK506 treatment. Correspondingly, systemic treatment with FK506 markedly reduced calcineurin activity in the PVN and circumventricular organs, but not rostral ventrolateral medulla, and increased the phosphorylation level and synaptic trafficking of NMDA receptors in the PVN. Immunocytochemistry labeling showed that calcineurin was expressed in presympathetic neurons in the PVN. Whole-cell patch-clamp recordings in brain slices revealed that treatment with FK506 increased baseline firing activity of PVN presympathetic neurons; this increase was blocked by the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist. Also, treatment with FK506 markedly increased presynaptic and postsynaptic NMDA receptor activity of PVN presympathetic neurons. Furthermore, microinjection of the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist into the PVN of anesthetized rats preferentially attenuated renal sympathetic nerve discharges and blood pressure elevated by FK506 treatment. In addition, systemic administration of memantine, a clinically used NMDA receptor antagonist, effectively attenuated FK506 treatment-induced hypertension in conscious rats. CONCLUSIONS: Our findings reveal that normal calcineurin activity in the PVN constitutively restricts sympathetic vasomotor tone via suppressing NMDA receptor activity, which may be targeted for treating calcineurin inhibitor-induced hypertension.

NMDA Receptors in the Lateral Preoptic Hypothalamus Are Essential for Sustaining NREM and REM Sleep.

The lateral preoptic (LPO) hypothalamus is a center for NREM and REM sleep induction and NREM sleep homeostasis. Although LPO is needed for NREM sleep, we found that calcium signals were, surprisingly, highest in REM sleep. Furthermore, and equally surprising, NMDA receptors in LPO were the main drivers of excitation. Deleting the NMDA receptor GluN1 subunit from LPO abolished calcium signals in all cells and produced insomnia. Mice of both sexes had highly fragmented NREM sleep-wake patterns and could not generate conventionally classified REM sleep. The sleep phenotype produced by deleting NMDA receptors depended on where in the hypothalamus the receptors were deleted. Deleting receptors from the anterior hypothalamic area (AHA) did not influence sleep-wake states. The sleep fragmentation originated from NMDA receptors on GABA neurons in LPO. Sleep fragmentation could be transiently overcome with sleeping medication (zolpidem) or sedatives (dexmedetomidine; Dex). By contrast, fragmentation persisted under high sleep pressure produced by sleep deprivation (SD), mice had a high propensity to sleep but woke up. By analyzing changes in δ power, sleep homeostasis (also referred to as "sleep drive") remained intact after NMDA receptor ablation. We suggest NMDA glutamate receptor activation stabilizes firing of sleep-on neurons and that mechanisms of sleep maintenance differ from that of the sleep drive itself.SIGNIFICANCE STATEMENT Insomnia is a common affliction. Most insomniacs feel that they do not get enough sleep, but in fact, often have good amounts of sleep. Their sleep, however, is fragmented, and sufferers wake up feeling unrefreshed. It is unknown how sleep is maintained once initiated. We find that in mice, NMDA-type glutamate receptors in the hypothalamus are the main drivers of excitation and are required for a range of sleep properties: they are, in fact, needed for both sustained NREM sleep periods, and REM sleep generation. When NMDA receptors are selectively reduced from inhibitory preoptic (PO) neurons, mice have normal total amounts of sleep but high sleep-wake fragmentation, providing a model for studying intractable insomnia.

Testing the Role of Glutamate NMDA Receptors in Peripheral Trigeminal Nociception Implicated in Migraine Pain.

The pro-nociceptive role of glutamate in the CNS in migraine pathophysiology is well established. Glutamate, released from trigeminal afferents, activates second order nociceptive neurons in the brainstem. However, the function of peripheral glutamate receptors in the trigeminovascular system suggested as the origin site for migraine pain, is less known. In the current project, we used calcium imaging and patch clamp recordings from trigeminal ganglion (TG) neurons, immunolabelling, CGRP assay and direct electrophysiological recordings from rat meningeal afferents to investigate the role of glutamate in trigeminal nociception. Glutamate, aspartate, and, to a lesser extent, NMDA under free-magnesium conditions, evoked calcium transients in a fraction of isolated TG neurons, indicating functional expression of NMDA receptors. The fraction of NMDA sensitive neurons was increased by the migraine mediator CGRP. NMDA also activated slowly desensitizing currents in 37% of TG neurons. However, neither glutamate nor NMDA changed the level of extracellular CGRP. TG neurons expressed both GluN2A and GluN2B subunits of NMDA receptors. In addition, after removal of magnesium, NMDA activated persistent spiking activity in a fraction of trigeminal nerve fibers in meninges. Thus, glutamate activates NMDA receptors in somas of TG neurons and their meningeal nerve terminals in magnesium-dependent manner. These findings suggest that peripherally released glutamate can promote excitation of meningeal afferents implicated in generation of migraine pain in conditions of inherited or acquired reduced magnesium blockage of NMDA channels and support the usage of magnesium supplements in migraine.

Anxiogenic and anxiolytic effects of memantine injected into the ventral hippocampus in male stressed mice.

OBJECTIVES: The effects of intra-ventral hippocampal memantine administration in male NMRI stressed mice were studied. METHODS: Two stainless steel gauge 23 guide cannulas were placed in the middle part of the mice ventral hippocampus using stereotaxic coordination. Seven days later, the animals were undergone to the stress protocol as follows: They experience four consecutive electro-foot shock stress sessions lasting for 10 min. Five or 30 min before each stress session, the animals received intra-ventral hippocampal (0.1, 1 and, 5 µg/mouse) or intraperitoneal (1, 5, and 10 mg/kg) memantine respectively. Eight days after stress termination, the animals were tested either for the maintenance of either anxiety (elevated plus maze) or depression (forced swimming test). RESULTS: Animals show anxiety eight days after stress termination. Intra-ventral hippocampal infusion of memantine (5 µg/mouse) 5 min before stress inhibited the anxiety-like behaviors. However, other doses of the drug exacerbate the stress effect. The drug, when injected peripherally exacerbated the stress effect in all doses. The drug by itself had no effect. In addition, animals also show depression nine days after stress termination and memantine (0.1, 1, and 5 µg/mouse) reduced the stress effect. The drug (0.1 µg/mouse) by itself induced depression in the animals. However, the drug when injected peripherally reduced the stress effect in all doses. CONCLUSIONS: It could be concluded that NMDA glutamate receptors in the ventral hippocampus may play a pivotal role in the mediation of maintenance of anxiety and depression induced by stress in the mice.

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.

OBJECTIVE: To explore the neuroprotective mechanisms of Tongluo Huatan capsule (THC) in a rat model of vascular dementia (VD). METHODS: 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. RESULTS: 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). CONCLUSION: THC may improve cognitive functions by regulating the endocytosis of NMDA receptors mediated by clathrin.

The Gliopeptide ODN, a Ligand for the Benzodiazepine Site of GABAA Receptors, Boosts Functional Recovery after Stroke.

Following stroke, the survival of neurons and their ability to reestablish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAARs). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the suboptimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which makes it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that, during the subacute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.SIGNIFICANCE STATEMENT Stroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide, can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.

[Effect of wrist-ankle acupuncture on the expression of glutamate and NMDA receptor of the spinal dorsal horn in rats with neuropathic pain].

OBJECTIVE: To observe the effect of wrist-ankle acupuncture (WA) stimulation at "R4"- "R5" - "R6" on the expression of glutamate (Glu) and phosphorylated protein NMDAR1(p-NMDAR1) of the spinal dorsal horn in spared nerve injury (SNI) rats, so as to explore its mechanism underlying improvement of SNI. METHODS: A total of 36 SD rats were randomly divi-ded into sham operation, model and WA groups, with 12 rats in each group. The SNI procedure comprised an axotomy and ligation of the tibial and common peroneal nerves leaving the sural nerve intact. Rats of the WA group were treated by acupuncture at "R4"-"R5"-"R6" points from the 5th day to the 14th day after modeling. The mechanical pain thresholds were measured before and 5, 10 and 14 d after SNI, respectively. The cold allodynia was dectected by Acetone solution dropped onto the lateral plantar surface of the paw. Glu content and p-NMDAR1 expression of spinal dorsal horn were detected by 1H-MRS, ELISA and immunohistochemistry Methods. RESULTS: Compared with the sham operation group, the mechanical pain threshold of the model group was significantly decreased (P<0.01), the duration of cold stimulation foot contraction was increased (P<0.01), and the Glu content and p-NMDAR1 expression in the spinal dorsal horn were significantly increased (P<0.05, P<0.01). After WA intervention, the mechanical pain threshold was significantly increased (P<0.01), the duration of cold stimulation was significantly shortened (P<0.01), and Glu content and p-NMDAR1 protein expression of spinal dorsal horn were decreased significantly (P<0.05, P<0.01) in the WA group compared with the model group. CONCLUSION: WA can reduce pain sensitivity in rats with neuropathic pain, possibly by inhibiting the expression of Glu and p-NMDAR1 in the spinal dorsal horn.