Trolox aids coenzyme Q10 in neuroprotection against NMDA induced damage via upregulation of VEGF in rat model of glutamate excitotoxicity.

Glutamate induced damage to retinal ganglion cells (RGCs) requires tight physiological regulation of the N-methyl-D-aspartate (NMDA) receptors. Previously, studies have demonstrated the neuroprotective abilities of antioxidants like coenzyme Q10 (CoQ10) and vitamin E analogs like α-tocopherol against neuropathies resulting from NMDA insult, but have failed to shed light on the effect of CoQ10 and trolox, a hydrophilic analog of vitamin E, on glaucomatous neurodegeneration. In the current study, we wanted to investigate whether the combined effect of trolox with CoQ10 could alleviate NMDA-induced death of retinal cells while also trying to elucidate the underlying mechanism in relation to the yet unexplained role of vascular endothelial growth factor (VEGF) in NMDA-mediated excitotoxicity. After successful NMDA-induced degeneration, we followed it up with the treatment of combination of Trolox and CoQ10. The structural damage by NMDA was repaired significantly and retina retained structural integrity comparable to levels of control in the treatment group of Trolox and CoQ10. Detection of ROS generation after NMDA insult showed that together, Trolox and CoQ10 could significantly bring down the high levels of free radicals while also rescuing mitochondrial membrane potential (MMP). A significant increase in NMDA receptor Grin2A by CoQ10 alone as well as by CoQ10 and trolox was accompanied by a lowered Grin2B receptor expression, suggesting neuroprotective action of Trolox and CoQ10. Subsequently, lowered VEGFR1 and VEGFR2 receptor expression by NMDA treatment also recovered when subjected to combined treatment of Trolox and CoQ10. Western blot analyses also indicated the same whereby Trolox and CoQ10 could increase the diminished levels of phosphorylated VEGFR2. Immunofluorescence studies also indicated a positive correlation between recovered VEGFR2 and NMDAR2A levels and diminished levels of NMDAR2D, confirming the results obtained by RT-PCR analysis. This is the first report in our knowledge that demonstrates the efficacy of trolox in combination with CoQ10 highlighting the importance of maintaining VEGF levels that are lowered in ocular diseases due to NMDA-related toxicities.

Methyl Palmitate Modulated NMDA-Induced Cerebral Hyperemia in Hypertensive Rats.

N-methyl-D-aspartate (NMDA) receptors were found to be dysfunctional in hypertensive rats. Methyl palmitate (MP) has been shown to diminish the nicotine-induced increase in blood flow in the brainstem. The aim of this study was to determine how MP modulated NMDA-induced increased regional cerebral blood flow (rCBF) in normotensive (WKY), spontaneously hypertensive (SHR), and renovascular hypertensive (RHR) rats. The increase in rCBF after the topical application of experimental drugs was measured using laser Doppler flowmetry. Topical NMDA application induced an MK-801-sensitive increase in rCBF in anesthetized WKY rats, which was inhibited by MP pretreatments. This inhibition was prevented by pretreatment with chelerythrine (a PKC inhibitor). The NMDA-induced increase in rCBF was also inhibited by the PKC activator in a concentration-dependent manner. Neither MP nor MK-801 affected the increase in rCBF induced by the topical application of acetylcholine or sodium nitroprusside. Topical application of MP to the parietal cortex of SHRs, on the other hand, increased basal rCBF slightly but significantly. MP enhanced the NMDA-induced increase in rCBF in SHRs and RHRs. These results suggested that MP had a dual effect on the modulation of rCBF. MP appears to play a significant physiological role in CBF regulation.

Delays to Reward Delivery Enhance the Preference for an Initially Less Desirable Option: Role for the Basolateral Amygdala and Retrosplenial Cortex.

Temporal costs influence reward-based decisions. This is commonly studied in temporal discounting tasks that involve choosing between cues signaling an imminent reward option or a delayed reward option. However, it is unclear whether the temporal delay before a reward can alter the value of that option. To address this, we identified the relative preference between different flavored rewards during a free-feeding test using male and female rats. Animals underwent training where either the initial preferred or the initial less preferred reward was delivered noncontingently. By manipulating the intertrial interval during training sessions, we could determine whether temporal delays impact reward preference in a subsequent free-feeding test. Rats maintained their initial preference if the same delays were used across all training sessions. When the initial less preferred option was delivered after short delays (high reward rate) and the initial preferred option was delivered after long delays (low reward rate), rats expectedly increased their preference for the initial less desirable option. However, rats also increased their preference for the initial less desirable option under the opposite training contingencies: delivering the initial less preferred reward after long delays and the initial preferred reward after short delays. These data suggest that sunk temporal costs enhance the preference for a less desirable reward option. Pharmacological and lesion experiments were performed to identify the neural systems responsible for this behavioral phenomenon. Our findings demonstrate the basolateral amygdala and retrosplenial cortex are required for temporal delays to enhance the preference for an initially less desirable reward.SIGNIFICANCE STATEMENT The goal of this study was to determine how temporal delays influence reward preference. We demonstrate that delivering an initially less desirable reward after long delays subsequently increases the consumption and preference for that reward. Furthermore, we identified the basolateral amygdala and the retrosplenial cortex as essential nuclei for mediating the change in reward preference elicited by sunk temporal costs.

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.

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.

Synthesis of novel 1-phenyl-benzopyrrolizidin-3-one derivatives and evaluation of their cytoneuroprotective effects against NMDA-induced injury in PC12 cells.

A range of novel 1-phenyl-benzopyrrolizidin-3-one derivatives were synthesized and evaluated for neuroprotective effects against N-methyl-ᴅ-aspartate (NMDA)-induced injury in PC12 cells. Interestingly, derivatives that 1-phenyl moiety bearing electron-donating group, especially benzyloxy, and the trans-forms exhibited better protective activity against NMDA-induced neurotoxicity. Compound 11 m demonstrated the best neuroprotective potency and shown a dose-dependent prevention. The increased intracellular calcium (Ca2+) influx caused by NMDA in PC12 cells was reversed in the case of compound 11 m pretreatment at 15 µM. These results suggested that the synthesized 1-phenyl-benzopyrrolizidin-3-one derivatives exerted neuroprotective effect on NMDA-induced excitotoxicity in PC12 cells associated with inhibition of Ca2+ overload and can be further optimized for the development of neuroprotective agents.

MST1 mediates the N-methyl-D-aspartate-induced excitotoxicity in mouse cortical neurons.

Excessive activation of the ionotropic N-methyl-D-aspartate (NMDA) receptor has been shown to cause abnormally high levels of Ca2+ influx, thereby leading to excitotoxic neuronal death. In this study, exposure of mouse primary cortical neurons to NMDA resulted in the cleavage and activation of mammalian sterile 20-like kinase-1 (MST1), both of which were mediated by calpain 1. In vitro cleavage assay data indicated that calpain 1 cleaves out the autoinhibitory domain of MST1 to generate an active form of the kinase. Furthermore, calpain 1 mediated the cleavage and activation of wild-type MST1, but not of MST1 (G339A). Intriguingly, NMDA/calpain-induced MST1 activation promoted the nuclear translocation of the kinase and the phosphorylation of histone H2B in mouse cortical neurons, leading to excitotoxicity. Thus, we propose a previously unrecognized mechanism of MST1 activation associated with NMDA-induced excitotoxic neuronal death.

DZNep protects against retinal ganglion cell death in an NMDA-induced mouse model of retinal degeneration.

Epigenetic gene enhancer of zeste homolog-2 (Ezh2) is reported to be associated with ocular neurodegenerative diseases; however, its underlying mechanism is poorly understood. The present study aimed to determine the role of 3-deazaneplanocin A (DZNep), which inhibits the transcription of Ezh2 by reducing the trimethylation of histone 3 lysine 27 (H3K27me3), in a retinal ganglion cell (RGC) degeneration model. Retinal damage was caused by intravitreal injection of N-methyl-D-aspartate (NMDA). DZNep and the vehicle control were intravitreally applied immediately post-NMDA injection. The severity of retinal damage was evaluated by immunofluorescence and terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining, and retinal function was determined by electroretinogram (ERG). The transcriptome was examined by RNA sequencing and quantitative PCR (qPCR). Microglial cells were detected by immunohistochemistry. DZNep significantly prevented the cell death in the ganglion cell layer (GCL) and inner nuclear layer (INL) induced by NMDA. DZNep preserved the ERG b- and a-wave amplitudes and the b/a ratio in NMDA-treated mice. Moreover, RNA sequencing and qPCR revealed that neuroprotective genes were upregulated and played an important role in preserving retinal cells. In addition, DZNep inhibited the NMDA-induced activation of microglial cells. Our results suggest that H3K27me3 controls RGC survival at the transcriptional and epigenetic levels. The absence of H3K27me3 deposition upregulates neuroprotective genes to protect RGCs. Therefore, DZNep, which inhibits Ezh2 activity, could be a novel therapeutic treatment for ocular neurodegenerative diseases.

CHOP deletion and anti-neuroinflammation treatment with hesperidin synergistically attenuate NMDA retinal injury in mice.

Glaucoma is a leading cause of blindness worldwide and is characterized by degeneration associated with the death of retinal ganglion cells (RGCs). It is believed that glaucoma is a group of heterogeneous diseases with multifactorial pathomechanisms. Here, we investigate whether anti-inflammation treatment with an ER stress blockade can selectively promote neuroprotection against NMDA injury in the RGCs. Retinal excitotoxicity was induced with an intravitreal NMDA injection. Microglial activation and neuroinflammation were evaluated with Iba1 immunostaining and cytokine gene expression. A stable HT22 cell line transfected with an NF-kB reporter was used to assess NF-kB activity after hesperidin treatment. CHOP-deficient mice were used as a model of ER stress blockade. Retinal cell death was evaluated with a TUNEL assay. As results, in the NMDA injury group, Iba1-positive microglia increased 6 h after NMDA injection. Also at 6 h, pro-inflammatory cytokines and chemokine increased, including TNFα, IL-1b, IL-6 and MCP-1. In addition, the MCP-1 promoter-driven EGFP signal, which we previously identified as a stress signal in injured RGCs, also increased; hesperidin treatment suppressed this inflammatory response and reduced stressed RGCs. In CHOP-deficient mice that received an NMDA injection, the gene expression of pro-inflammatory cytokines, chemokines, markers of active microglia, and inflammatory regulators was greater than in WT mice. In WT mice, hesperidin treatment partially prevented retinal cell death after NMDA injury; this neuroprotective effect was enhanced in CHOP-deficient mice. These findings demonstrate that ER stress blockade is not enough by itself to prevent RGC loss due to neuroinflammation in the retina, but it has a synergistic neuroprotective effect after NMDA injury when combined with an anti-inflammatory treatment based on hesperidin.

Maintenance of the Neuroprotective Function of the Amino Group Blocked Fluorescence-Agmatine.

Agmatine, an endogenous derivative of arginine, has been found to be effective in treating idiopathic pain, convulsion, stress-mediated behavior, and attenuate the withdrawal symptoms of drugs like morphine. In the early stages of ischemic brain injury in animals, exogenous agmatine treatment was found to be neuroprotective. Agmatine is also considered as a putative neurotransmitter and is still an experimental drug. Chemically, agmatine is called agmatine 1-(4-aminobutyl guanidine). Crystallographic study data show that positively-charged guanidine can bind to the protein containing Gly and Asp residues, and the amino group can interact with the complimentary sites of Glu and Ser. In this study, we blocked the amino end of the agmatine by conjugating it with FITC, but the guanidine end was unchanged. We compared the neuroprotective function of the agmatine and agmatine-FITC by treating them in neurons after excitotoxic stimulation. We found that even the amino end blocked neuronal viability in the excitotoxic condition, by NMDA treatment for 1 h, was increased by agmatine-FITC, which was similar to that of agmatine. We also found that the agmatine-FITC treatment reduced the expression of nitric oxide production in NMDA-treated cells. This study suggests that even if the amino end of agmatine is blocked, it can perform its neuroprotective function.

Metformin Protects against NMDA-Induced Retinal Injury through the MEK/ERK Signaling Pathway in Rats.

Metformin, an anti-hyperglycemic drug of the biguanide class, exerts positive effects in several non-diabetes-related diseases. In this study, we aimed to examine the protective effects of metformin against N-methyl-D-aspartic acid (NMDA)-induced excitotoxic retinal damage in rats and determine the mechanisms of its protective effects. Male Sprague-Dawley rats (7 to 9 weeks old) were used in this study. Following intravitreal injection of NMDA (200 nmol/eye), the number of neuronal cells in the ganglion cell layer and parvalbumin-positive amacrine cells decreased, whereas the number of CD45-positive leukocytes and Iba1-positive microglia increased. Metformin attenuated these NMDA-induced responses. The neuroprotective effect of metformin was abolished by compound C, an inhibitor of AMP-activated protein kinase (AMPK). The AMPK activator, AICAR, exerted a neuroprotective effect in NMDA-induced retinal injury. The MEK1/2 inhibitor, U0126, reduced the neuroprotective effect of metformin. These results suggest that metformin protects against NMDA-induced retinal neurotoxicity through activation of the AMPK and MEK/extracellular signal-regulated kinase (ERK) signaling pathways. This neuroprotective effect could be partially attributable to the inhibitory effects on inflammatory responses.

Tyrosine triple mutated AAV2-BDNF gene therapy in an inner retinal injury model induced by intravitreal injection of N-methyl-D-aspartate (NMDA).

Purpose: Glaucoma is a group of chronic optic neuropathies characterized by the degeneration of retinal ganglion cells (RGCs) and their axons, and they ultimately cause blindness. Because neuroprotection using neurotrophic factors against RGC loss has been proven a beneficial strategy, extensive attempts have been made to perform gene transfer of neurotrophic proteins. This study used the inner retinal injury mouse model to evaluate the neuroprotective effect of tyrosine triple mutated and self-complementary adeno-associated virus (AAV) encoding brain-derived neurotrophic factor (BDNF; tm-scAAV2-BDNF). Methods: C57BL/6J mice were intravitreally injected with 1 µl of tm-scAAV2-BDNF and its control AAV at a titer of 6.6 E+13 genome copies/ml. Three weeks later, 1 µl of 2 mM N-methyl-D-aspartate (NMDA) was administered in the same way as the viral injection. Six days after the NMDA injection, we assessed the dark-adapted electroretinography (ERG). Mice were sacrificed at one week after the NMDA injection, followed by RNA quantification, protein detection, and histopathological analysis. Results: The RNA expression of BDNF in retinas treated with tm-scAAV2-BDNF was about 300-fold higher than that of its control AAV. Meanwhile, the expression of recombinant BDNF protein increased in retinas treated with tm-scAAV2-BDNF. In addition, histological analysis revealed that tm-scAAV2-BDNF prevented thinning of the inner retina. Furthermore, b-wave amplitudes of the tm-scAAV2-BDNF group were significantly higher than those of the control vector group. Histopathological and electrophysiological evaluations showed that tm-scAAV2-BDNF treatment offered significant protection against NMDA toxicity. Conclusions: Results showed that tm-scAAV2-BDNF-treated retinas were resistant to NMDA injury, while retinas treated with the control AAV exhibited histopathological and functional changes after the administration of NMDA. These results suggest that tm-scAAV2-BDNF is potentially effective against inner retinal injury, including normal tension glaucoma.

Endogenous Wnt/ß-catenin signaling in Müller cells protects retinal ganglion cells from excitotoxic damage.

Purpose: To analyze whether activation of endogenous wingless (Wnt)/ß-catenin signaling in Müller cells is involved in protection of retinal ganglion cells (RGCs) following excitotoxic damage. Methods: Transgenic mice with a tamoxifen-dependent ß-catenin deficiency in Müller cells were injected with N-methyl-D-aspartate (NMDA) into the vitreous cavity of one eye to induce excitotoxic damage of the RGCs, while the contralateral eye received PBS only. Retinal damage was quantified by counting the total number of RGC axons in cross sections of optic nerves and measuring the thickness of the retinal layers on meridional sections. Then, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to identify apoptotic cells in retinas of both genotypes. Western blot analyses to assess the level of retinal ß-catenin and real-time RT-PCR to quantify the retinal expression of neuroprotective factors were performed. Results: Following NMDA injection of wild-type mice, a statistically significant increase in retinal ß-catenin protein levels was observed compared to PBS-injected controls, an effect that was blocked in mice with a Müller cell-specific ß-catenin deficiency. Furthermore, in mice with a ß-catenin deficiency in Müller cells, NMDA injection led to a statistically significant decrease in RGC axons as well as a substantial increase in TUNEL-positive cells in the RGC layer compared to the NMDA-treated controls. Moreover, in the retinas of the control mice a NMDA-mediated statistically significant induction of leukemia inhibitory factor (Lif) mRNA was detected, an effect that was substantially reduced in mice with a ß-catenin deficiency in Müller cells. Conclusions: Endogenous Wnt/ß-catenin signaling in Müller cells protects RGCs against excitotoxic damage, an effect that is most likely mediated via the induction of neuroprotective factors, such as Lif.

Systemic Administration of an Apelin Receptor Agonist Prevents NMDA-Induced Loss of Retinal Neuronal Cells in Mice.

Glutamate excitotoxicity via N-methyl-D-aspartate (NMDA) receptors is thought to be a factor involved in the loss of retinal neuronal cells, including retinal ganglion cells, in retinal diseases such as diabetic retinopathy and acute angle closure glaucoma. Herein we report the protective effect of systemic administration of ML233, an apelin receptor agonist, against retinal neuronal cell death induced by the intravitreal injection of NMDA into mice. Intraperitoneal administration of ML233 prevented the NMDA-induced reduction in the amplitude of scotopic threshold responses (STR), which mainly reflect the activity of the retinal ganglion cells. Immunohistochemical staining showed that ML233 inhibited the NMDA-induced loss of retinal ganglion cells and amacrine cells. In addition, ML233 suppressed the breakdown of spectrin αII, a neuronal cytoskeleton protein cleaved by calpain activation, in the retina after intravitreal injection of NMDA. Intraperitoneal administration of ML233 increased the phosphorylation of Akt, a potent anti-apoptotic protein in neurons, in the retina. Furthermore, oral administration of ML233 protected against the decrease in the STR amplitudes and the loss of retinal ganglion cells caused by NMDA. These results suggest that systemic administration of ML233 protected retinal neurons from NMDA receptor-mediated excitotoxicity and that drugs activating the apelin receptor may be a new candidate for preventing the progression of these retinal diseases.

Kukoamine A Protects against NMDA-Induced Neurotoxicity Accompanied with Down-Regulation of GluN2B-Containing NMDA Receptors and Phosphorylation of PI3K/Akt/GSK-3ß Signaling Pathway in Cultured Primary Cortical Neurons.

Kukoamine (KuA) is a spermine alkaloid present in traditional Chinese medicine Cortex Lycii radices, which possesses various pharmacological properties. Our previous studies have demonstrated that KuA exerts neuroprotective effects against H2O2-induced oxidative stress, radiation-induced neuroinflammation, oxidative stress and neuronal apoptosis, as well as neurotoxin-induced Parkinson's disease through apoptosis inhibition and autophagy enhancement. The present study aimed to investigate the neuroprotective effects of KuA against NMDA-induced neuronal injury in cultured primary cortical neurons and explore the underlying mechanism. Incubation with 200 µM NMDA for 30 min induced excitotoxicity in primary cultured cortical neurons. The results demonstrated that pretreatment with KuA attenuated NMDA induced cell injury, LDH leakage and neuronal apoptosis. KuA also regulated apoptosis-related proteins. Thus, incubation with the alkaloid decreased the ratio of Bax/Bcl-2, and inhibited the release of cytochrome C, the expression of p53 and the cleavage of caspase-3. Moreover, KuA prevented the upregulation of GluN2B-containing NMDA receptors (NMDAR). Additionally, pretreatment with KuA reversed NMDA-induced dephosphorylation of Akt and GSK-3ß and the protective effect of KuA on NMDA-induced cytotoxicity was abolished by wortmannin, a PI3K inhibitor. Taken together, these results indicated that KuA exerted neuroprotective effects against NMDA-induced neurotoxicity in cultural primary cortical neurons and caused the down-regulation of GluN2B-containing NMDARs as well as the phosphorylation of proteins belonging to the PI3K/Akt/GSK-3ß signaling pathway.

miR-382-3p Overexpression attenuates N-methyl-D-aspartate-induced HT22 cell apoptosis via the regulation of the RhoC/ROCK1 signaling pathway.

miR-382-3p can regulate apoptosis through multiple pathways, but the mechanism remains unknown. In this experiment, we explored whether miR-382-3p can modulate the N-methyL-D-aspartate (NMDA)- induced HT22 cell apoptosis by regulating the RhoC/ROCK1 signaling pathway. An excitatory neurotoxicity model of HT22 cells was induced in vitro with 2 mmol/L NMDA. The cells were divided into normal control, NMDA-induced, NMDA + miR-382-3p mimic, and NMDA + miR-382-3p inhibitor groups. The 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method, Real-time PCR, Western blot, and flow cytometry were performed to investigate the mechanisms. The results found that NMDA can increase the oxidative stress of HT22 cells in a dose-dependent manner, downregulate the expression of miR-382-3p, upregulate the expression of mRNA and protein abundance of ROCK1 and RhoC, increase the expression levels of proapoptotic proteins Bax, Caspase-3, and Caspase-9, increase the apoptosis of HT22 cells, and reduce the activity and survival rate of HT22 cells. Compared with the NMDA-induced group, the miR-382-3p mimic-transfected HT22 cells increased the expression of miR- 382-3p, reduced the expression of the mRNA and protein abundance of ROCK1 and RhoC, inhibited the expression of proapoptotic proteins Bax, Caspase-3, and Caspase-9, reduced the apoptosis of HT22 cells, and increased the activity and survival rate of HT22 cells. The results suggest that increasing the expression of miR-382-3p can inhibit the activity of the RhoC/ROCK1 signaling pathway, reduce the expression of proapoptotic proteins, reduce the oxidative stress and apoptosis of HT22 cells, and increase the activity and survival rate of HT22 cells.

Interaction of morphine tolerance with pentylenetetrazole-induced seizure threshold in mice: The role of NMDA-receptor/NO pathway.

N-methyl-d-aspartate receptor (NMDA-R)/nitric oxide (NO) pathway is involved in the intensification of the analgesic effect of opioids and the reduction of the intensity of opioids tolerance and dependence. In the current study, we investigated the involvement of NMDA-R/NO pathway in chronic morphine-treated mice in both the development of tolerance to the analgesic effect of morphine and in pentylenetetrazole (PTZ)-induced seizure threshold. Chronic treatment with morphine (30 mg/kg) exhibited increased seizure resistance in morphine-induced tolerant mice. The development of morphine tolerance was withdrawn when used concomitantly with NOS inhibitors and NMDA-R antagonist, suggesting that the development of tolerance to the anticonvulsant effect of morphine (30 mg/kg) is mediated through the NMDA-R/NO pathway. A dose-dependent biphasic seizure modulation of morphine was demonstrated in the acute treatment with morphine; acute treatment at a dose of 0.5 mg/kg shows the anticonvulsant effect and at a dose of 30 mg/kg shows proconvulsant effect. However, a different pattern was observed in the mice treated chronically with morphine: they demonstrated tolerance in the tail-flick test; five consecutive days of chronic treatment with a high dose of morphine (30 mg/kg) showed anticonvulsant effect while a low dose of morphine (0.5 mg/kg) showed a proconvulsant effect. The anticonvulsant effect of morphine was inhibited completely by the concomitant administration of NO synthase (NOS) inhibitors including nonspecific NOS inhibitor (L-NAME, 10 mg/kg), inducible NOS inhibitor (aminoguanidine, 50 mg/kg), and neuronal NOS inhibitor (7-nitroindazole (7-NI), 15 mg/kg) for five consecutive days. Besides, five days injection of NMDA-R antagonist (MK-801, 0.05 mg/kg) significantly inhibited the anticonvulsant effect of morphine on the PTZ-induced clonic seizures. The results revealed that chronic treatment with morphine leads to the development of tolerance in mice, which in turn may cause an anticonvulsant effect in a high dose of morphine via the NMDA-R/NO pathway.

Exposing immature hippocampal neurons to excitotoxins reveals distinct transcriptome and protein regulation with induction of common survival signaling pathways.

Early life traumas lead to neuroprotection by preconditioning mechanisms. To determine which genes and pathways are most likely involved in specific adaptive effects, immature hippocampal cultures were exposed to a single high dose of glutamate (250 µM), NMDA (100 µM), or KA (300 µM) for 48 h (5-7 DIV) based on our prior "two hit" in vitro model of preconditioning. Transcriptome profiling and immunocytochemistry of gene candidates were performed 7 days later when cultured neurons mature (14 DIV). Many genes were up- and down- regulated involving distinct Ca2+-binding protein families, G-coupled proteins, various growth factors, synaptic vesicle docking factors, certain neurotransmitter receptors, heat shock, oxidative stress, and certain anti-apoptotic Bcl-2 gene members that influence neuronal survival. Immunohistochemistry showed a marked decrease in the number of Calb1 and Calm2 positive neurons following NMDA but not after glutamate exposure whereas ryanodine and Cav1.2 voltage gated channel expression was less affected. Survivors had marked increases in Calm2 immunostaining; however, high-density neural clusters observed in controls, were depleted after NMDA and partly diminished after glutamate. While NR1 mRNA expression was decreased in the microarray, specific antibodies revealed selective loss of the NR1C1 splice variant. Calm2 which can inactivate NMDA receptors by binding to C1 but not C2 regions of its NR1 subunit suggests that loss of the C1 splice variant will reduce co-regulation with Calm2 and alter NR1 trafficking, phosphorylation, and NMDA currents following early life NMDA exposure. A dramatic reduction in the density of GABAAα5 and GABAB receptor expressing neurons was observed after NMDA exposure but immunodensity measurements were unchanged as was the expression of the GABA synthesizing enzyme, GAD, suggesting that fast inhibitory neurotransmission and response to benzodiazepines and GABAB-mediated IPSPs may be preserved in matured survivors. Selective upregulation of Chat and CNRIP was detected after glutamate treatment suggesting this condition would decrease cholinergic and excitatory neurotransmission by decreasing Ach content and CB1 interacting protein function. This decrease likely contributes to memory and attention tasks deficits that follow a single early neurological insult. Diverse changes that follow overactivation of excitatory networks of immature neurons appear long-lasting or permanent and are expected to have profound effects on network function and adaptive responses to further insult.

Astaxanthin Counteracts Excitotoxicity and Reduces the Ensuing Increases in Calcium Levels and Mitochondrial Reactive Oxygen Species Generation.

Astaxanthin (ASX) is a carotenoid pigment with strong antioxidant properties. We have reported previously that ASX protects neurons from the noxious effects of amyloid-ß peptide oligomers, which promote excessive mitochondrial reactive oxygen species (mROS) production and induce a sustained increase in cytoplasmic Ca2+ concentration. These properties make ASX a promising therapeutic agent against pathological conditions that entail oxidative and Ca2+ dysregulation. Here, we studied whether ASX protects neurons from N-methyl-D-aspartate (NMDA)-induced excitotoxicity, a noxious process which decreases cellular viability, alters gene expression and promotes excessive mROS production. Incubation of the neuronal cell line SH-SY5Y with NMDA decreased cellular viability and increased mitochondrial superoxide production; pre-incubation with ASX prevented these effects. Additionally, incubation of SH-SY5Y cells with ASX effectively reduced the basal mROS production and prevented hydrogen peroxide-induced cell death. In primary hippocampal neurons, transfected with a genetically encoded cytoplasmic Ca2+ sensor, ASX also prevented the increase in intracellular Ca2+ concentration induced by NMDA. We suggest that, by preventing the noxious mROS and Ca2+ increases that occur under excitotoxic conditions, ASX could be useful as a therapeutic agent in neurodegenerative pathologies that involve alterations in Ca2+ homeostasis and ROS generation.

Deleterious Effect of NMDA Plus Kainate on the Inner Retinal Cells and Ganglion Cell Projection of the Mouse.

Combined administration of N-Methyl-D-Aspartate (NMDA) and kainic acid (KA) on the inner retina was studied as a model of excitotoxicity. The right eye of C57BL6J mice was injected with 1 µL of PBS containing NMDA 30 mM and KA 10 mM. Only PBS was injected in the left eye. One week after intraocular injection, electroretinogram recordings and immunohistochemistry were performed on both eyes. Retinal ganglion cell (RGC) projections were studied by fluorescent-cholerotoxin anterograde labeling. A clear decrease of the retinal "b" wave amplitude, both in scotopic and photopic conditions, was observed in the eyes injected with NMDA/KA. No significant effect on the "a" wave amplitude was observed, indicating the preservation of photoreceptors. Immunocytochemical labeling showed no effects on the outer nuclear layer, but a significant thinning on the inner retinal layers, thus indicating that NMDA and KA induce a deleterious effect on bipolar, amacrine and ganglion cells. Anterograde tracing of the visual pathway after NMDA and KA injection showed the absence of RGC projections to the contralateral superior colliculus and lateral geniculate nucleus. We conclude that glutamate receptor agonists, NMDA and KA, induce a deleterious effect of the inner retina when injected together into the vitreous chamber.