Epilepsy is a chronic brain disease affecting millions of individuals. Kainate receptors, especially kainate‐type of ionotropic glutamate receptor 2 (GluK2), play an important role in epileptogenesis. Recent data showed that GluK2 could undergo post‐translational modifications in terms of S‐nitrosylation (SNO ), and affect the signaling pathway of cell death in cerebral ischemia‐reperfusion. However, it is unclear whether S‐nitrosylation of GluK2 (SNO ‐GluK2) contributes to cell death induced by epilepsy. Here, we report that kainic acid‐induced SNO ‐GluK2 is mediated by GluK2 itself, regulated by neuronal nitric oxide synthase (nNOS ) and the level of cytoplasmic calcium in vivo and in vitro hippocampus neurons. The whole‐cell patch clamp recordings showed the influence of SNO ‐GluK2 on ion channel characterization of GluK2‐Kainate receptors. Moreover, immunohistochemistry staining results showed that inhibition of SNO ‐GluK2 by blocking nNOS or GluK2 or by reducing the level of cytoplasmic calcium‐protected hippocampal neurons from kainic acid‐induced injury. Finally, immunoprecipitation and western blotting data revealed the involvement of assembly of a GluK2‐PSD 95‐nNOS signaling complex in epilepsy. Taken together, our results showed that the SNO ‐GluK2 plays an important role in neuronal injury of epileptic rats by forming GluK2‐PSD 95‐nNOS signaling module in a cytoplasmic calcium‐dependent way, suggesting a potential therapeutic target site for epilepsy.
The attribution of incentive salience to reward‐predictive stimuli has been shown to be associated with substance abuse‐like behavior such as increased drug taking. Evidence suggests that glutamate neurotransmission and sequential N‐methyl‐D‐aspartate (NMDA) activation are involved in the attribution of incentive salience. Here, we further explore the role of second‐by‐second glutamate neurotransmission in the attribution of incentive salience to reward‐predictive stimuli by measuring sign‐tracking behavior during a Pavlovian conditioned approach procedure using ceramic‐based microelectrode arrays configured for sensitive measures of extracellular glutamate in awake behaving Sprague‐Dawley rats. Specifically, we show that there is an increase in extracellular glutamate levels in the prelimbic cortex (PrL) and the nucleus accumbens core (NAcC) during sign‐tracking behavior to a food‐predictive conditioned stimulus (CS+) compared to the presentation of a non‐predictive conditioned stimulus (CS?). Furthermore, the results indicate greater increases in extracellular glutamate levels in the PrL compared to NAcC in response to the CS+, including differences in glutamate release and signal decay. Taken together, the present research suggests that there is differential glutamate signaling in the NAcC and PrL during sign‐tracking behavior to a food‐predictive CS+.
Airborne particulate matter (PM) from urban vehicular aerosols altered glutamate receptor functions and induced glial inflammatory responses in rodent models after chronic exposure. Potential neurotoxic mechanisms were analyzed in vitro. In hippocampal slices, 2 h exposure to aqueous nanosized PM (nPM) selectively altered post‐synaptic proteins in cornu ammonis area 1 (CA1) neurons: increased GluA1, GluN2A, and GluN2B, but not GluA2, GluN1, or mGlur5; increased post synaptic density 95 and spinophilin, but not synaptophysin, while dentate gyrus (DG) neurons were unresponsive. In hippocampal slices and neurons, MitoSOX red fluorescence was increased by nPM, implying free radical production. Specifically, N? production by slices was increased within 15 min of exposure to nPM with dose dependence, 1–10 μg/mL. Correspondingly, CA1 neurons exhibited increased nitrosylation of the GluN2A receptor and dephosphorylation of GluN2B (S1303) and of GluA1 (S831 & S845). Again, DG neurons were unresponsive to nPM. The induction of N? and nitrosylation were inhibited by AP5, an NMDA receptor antagonist, which also protects neurite outgrowth in vitro from inhibition by nPM. Membrane injury (EthidiumD‐1 uptake) showed parallel specificity. Finally, nPM decreased evoked excitatory post‐synaptic currents of CA1 neurons. These findings further document the selective impact of nPM on glutamatergic functions and identify novel responses of NMDA receptor‐stimulated N? production and nitrosylation reactions during nPM‐mediated neurotoxicity.
Our recent studies have shown that endogenous zinc, co‐released with glutamate from the synaptic terminals of vertebrate retinal photoreceptors, provides a feedback mechanism that reduces calcium entry and the concomitant vesicular release of glutamate. We hypothesized that zinc feedback may serve to protect the retina from glutamate excitotoxicity, and conducted in vivo experiments on the retina of the skate (Raja erinacea) to determine the effects of removing endogenous zinc by chelation. These studies showed that removal of zinc by injecting the zinc chelator histidine results in inner retinal damage similar to that induced by the glutamate receptor agonist kainic acid. In contrast, when an equimolar quantity of zinc followed the injection of histidine, the retinal cells were unaffected. Our results are a good indication that zinc, co‐released with glutamate by photoreceptors, provides an auto‐feedback system that plays an important cytoprotective role in the retina.
Both dopamine and glutamate are critically involved in cognitive processes such as working memory. Astrocytes, which express dopamine receptors, are essential elements in the termination of glutamatergic signaling: the astrocytic glutamate transporter GLT‐1 is responsible for > 90% of cortical glutamate uptake. The effect of dopamine depletion on glutamate transporters in the prefrontal cortex (PFC) remains unknown. In an effort to determine if astrocytes are a locus of cortical dopamine–glutamate interactions, we examined the effects of chronic dopamine denervation on PFC protein and mRNA levels of glutamate transporters. PFC dopamine denervation elicited a marked increase in GLT‐1 protein levels, but had no effect on levels of other glutamate transporters; high‐affinity glutamate transport was positively correlated with the extent of dopamine depletion. GLT‐1 gene expression was not altered. Our data suggest that dopamine depletion may lead to post‐translational modifications that result in increased expression and activity of GLT‐1 in PFC astrocytes.
Recent studies have highlighted the role of mitochondria in dendritic protrusion growth and plasticity. However, the detailed mechanisms that mitochondria regulate dendritic filopodia morphogenesis remain elusive. Cyclophilin D (CypD, gene name: Ppif ) controls the opening of mitochondrial permeability transition pore. Although the pathological relevance of CypD has been intensively investigated, little is known about its physiological function in neurons. Here, we have found that genetic depletion of or pharmaceutical inhibition of CypD blunts the outgrowth of dendritic filopodia in response to KC l‐stimulated neuronal depolarization. Further cell biological studies suggest that such inhibitory effect of CypD loss‐of‐function is closely associated with compromised flexibility of dendritic mitochondrial calcium regulation during neuronal depolarization, as well as the resultant changes in intradendritic calcium homeostasis, calcium signaling activation, dendritic mitochondrial motility and redistribution. Interestingly, loss of CypD attenuates oxidative stress‐induced mitochondrial calcium perturbations and dendritic protrusion injury. Therefore, our study has revealed the physiological function of CypD in dendritic plasticity by acting as a fine‐tuner of mitochondrial calcium homeostasis. Moreover, CypD plays distinct roles in neuronal physiology and pathology.
Cover Image for this issue: doi: 10.1111/jnc.14189 . 相似文献
Intracellular protein trafficking is tightly regulated, and improper trafficking might be the fundamental provocateur for human diseases including neurodegeneration. In neurons, protein trafficking to and from the plasma membrane affects synaptic plasticity. Voltage‐gated potassium channel 2.1 (Kv2.1) is a predominant delayed rectifier potassium (K+) current, and electrical activity patterns of dopamine (DA) neurons within the substantia nigra are generated and modulated by the orchestrated function of different ion channels. The pathological hallmark of Parkinson's disease (PD) is the progressive loss of these DA neurons, resulting in the degeneration of striatal dopaminergic terminals. However, whether trafficking of Kv2.1 channels contributes to PD remains unclear. In this study, we demonstrated that MPTP/MPP+ increases the surface expression of the Kv2.1 channel and causes nigrostriatal degeneration by using a subchronic MPTP mouse model. The inhibition of the Kv2.1 channel by using a specific blocker, guangxitoxin‐1E, protected nigrostriatal projections against MPTP/MPP+ insult and thus facilitated the recovery of motor coordination. These findings highlight the importance of trafficking of Kv2.1 channels in the pathogenesis of PD.
Cocaine is a recreational drug of abuse that binds to the dopamine transporter, preventing reuptake of dopamine into pre‐synaptic terminals. The increased presence of synaptic dopamine results in stimulation of both pre‐ and post‐synaptic dopamine receptors, considered an important mechanism by which cocaine elicits its reinforcing properties. However, the effects of acute cocaine administration on pre‐synaptic dopamine function remain unclear. Non‐invasive imaging techniques such as positron emission tomography have revealed impaired pre‐synaptic dopamine function in chronic cocaine users. Similar impairments have been seen in animal studies, with microdialysis experiments indicating decreased basal dopamine release. Here we use micro positron emission tomography imaging techniques in mice to measure dopamine synthesis capacity and determine the effect of acute cocaine administration of pre‐synaptic dopamine function. We show that a dose of 20 mg/kg cocaine is sufficient to elicit hyperlocomotor activity, peaking 15–20 min post treatment (p < 0.001). However, dopamine synthesis capacity in the striatum was not significantly altered by acute cocaine treatment (: 0.0097 per min vs. 0.0112 per min in vehicle controls, p > 0.05). Furthermore, expression levels of two key enzymes related to dopamine synthesis, tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase, within the striatum of scanned mice were not significantly affected by acute cocaine pre‐treatment (p > 0.05). Our findings suggest that while the regulation of dopamine synthesis and release in the striatum have been shown to change with chronic cocaine use, leading to a reduced basal tone, these adaptations to pre‐synaptic dopaminergic neurons are not initiated following a single exposure to the drug.
We are very sad that the ISN lost its President Kazuhiro Ikenaka, Professor and Chairman at National Institute for Physiological Sciences (NIPS), Director of Okazaki Institute of Integrative Biology. JNeurochem published an Obituary to value his outstanding achievements: Akio Wanaka et al. (2019) OBITUARY Kazuhiro Ikenaka (1952‐2018). https://doi.org/10.1111/jnc.14679
The aim of the present report was to analyze the involvement of glutamate neurotoxicity in retinal ganglion cell loss and optic nerve damage induced by experimental optic neuritis. For this purpose, the authors used an optic neuritis model induced by immunisation with myelin oligodendrocyte glycoprotein (AON). The authors describe a correlation in the timing of retinal ganglion cell (RGC) loss with alterations in the optic nerve actin cytoskeleton dynamic, and visual dysfunction. In addition, they show that an intravitreal injection of glutamate mimics, and an NMDA receptor antagonist avoids the effect of pre-clinical AON on visual functions and RGC number, as well as on optic nerve actin cytoskeleton. Taken together, their results support that avoiding glutamate neurotoxicity could become a new therapeutic approach for optic neuritis treatment.
Precise quantification of extracellular glutamate concentrations upon neuronal activation is crucial for the understanding of brain function and neurological disorders. While optogenetics is an outstanding method for the correlation between distinct neurons and their role in circuitry and behavior, the electrochemically inactive nature of glutamate has proven challenging for recording upon optogenetic stimulations. This difficulty is due to the necessity for using enzyme‐coated microelectrodes and the risk for light‐induced artifacts. In this study, we establish a method for the combination of in vivo optogenetic stimulation with selective measurement of glutamate concentrations using enzyme‐coated multielectrode arrays and amperometry. The glutamatergic subthalamic nucleus (STN ), which is the main electrode target site in deep brain stimulation treatment of advanced Parkinson′s disease, has recently proven opotogenetically targetable in Pitx2‐Cre‐transgenic mice and was here used as model system. Upon stereotactic injection of viral Channelrhodopsin2‐eYFP constructs into the STN , amperometric recordings were performed at a range of optogenetic stimulation frequencies in the globus pallidus, the main STN target area, in anesthetized mice. Accurate quantification was enabled through a multi‐step analysis approach based on self‐referencing microelectrodes and repetition of the experimental protocol at two holding potentials, which allowed for the identification, isolation and removal of photoelectric and photoelectrochemical artifacts. This study advances the field of in vivo glutamate detection with combined optogenetics and amperometric recordings by providing a validated analysis framework for application in a wide variety of glutamate‐based approaches in neuroscience.
Proper neuronal function requires essential biological cargoes to be packaged within membranous vesicles and transported, intracellularly, through the extensive outgrowth of axonal and dendritic fibers. The precise spatiotemporal movement of these cargoes is vital for neuronal survival and, thus, is highly regulated. In this study we test how the axonal movement of a neuropeptide‐containing dense‐core vesicle (DCV ) responds to alcohol stressors. We found that ethanol induces a strong anterograde bias in vesicle movement. Low doses of ethanol stimulate the anterograde movement of neuropeptide‐DCV while high doses inhibit bi‐directional movement. This process required the presence of functional kinesin‐1 motors as reduction in kinesin prevented the ethanol‐induced stimulation of the anterograde movement of neuropeptide‐DCV . Furthermore, expression of inactive glycogen synthase kinase 3 (GSK ‐3β) also prevented ethanol‐induced stimulation of neuropeptide‐DCV movement, similar to pharmacological inhibition of GSK ‐3β with lithium. Conversely, inhibition of PI 3K/AKT signaling with wortmannin led to a partial prevention of ethanol‐stimulated transport of neuropeptide‐DCV . Taken together, we conclude that GSK ‐3β signaling mediates the stimulatory effects of ethanol. Therefore, our study provides new insight into the physiological response of the axonal movement of neuropeptide‐DCV to exogenous stressors.
Cover Image for this Issue: doi: 10.1111/jnc.14165 . 相似文献
Dopaminergic neurotransmission in the nucleus accumbens is important for various reward‐related cognitive processes including reinforcement learning. Repeated cocaine enhances hippocampal synaptic plasticity, and phasic elevations of accumbal dopamine evoked by unconditioned stimuli are dependent on impulse flow from the ventral hippocampus. Therefore, sensitized hippocampal activity may be one mechanism by which drugs of abuse enhance limbic dopaminergic activity. In this study, in vivo microdialysis in freely moving adult male Sprague–Dawley rats was used to investigate the effect of repeated cocaine on ventral hippocampus‐mediated dopaminergic transmission within the medial shell of the nucleus accumbens. Following seven daily injections of saline or cocaine (20 mg/kg, ip), unilateral infusion of N‐methyl‐d ‐aspartate (NMDA, 0.5 μg) into the ventral hippocampus transiently increased both motoric activity and ipsilateral dopamine efflux in the medial shell of the nucleus accumbens, and this effect was greater in rats that received repeated cocaine compared to controls that received repeated saline. In addition, repeated cocaine altered NMDA receptor subunit expression in the ventral hippocampus, reducing the NR2A : NR2B subunit ratio. Together, these results suggest that repeated exposure to cocaine produces maladaptive ventral hippocampal‐nucleus accumbens communication, in part through changes in glutamate receptor composition.
Hemopexin (Hpx) binds heme with extraordinary affinity, and after haptoglobin may provide a second line of defense against the toxicity of extracellular hemoglobin (Hb). In this series of experiments, the hypothesis that Hpx protects neurons from Hb neurotoxicity was evaluated in murine primary cultures containing neurons and glial cells. Contrary to hypothesis, Hpx increased neuronal loss due to micromolar concentrations of Hb by 4‐ to 12‐fold, as measured by LDH release assay; conversely, the neurotoxicity of hemin was completely prevented. The endogenous fluorescence of Hpx was quenched by Hb, consistent with transfer of Hb‐bound heme to Hpx. This was associated with precipitation of globin chains, as detected by immunostaining and fluorescent Hb labeling. A portion of this precipitate attached firmly to cells and could not be removed by multiple washes. Concomitant treatment with haptoglobin (Hp) prevented globin precipitation and most of the increase in neuronal loss. Hpx weakly attenuated the increase in culture non‐heme iron produced by Hb treatment, quantified by ferrozine assay. However, Hb‐Hpx toxicity was iron‐dependent, and was blocked by deferoxamine and ferrostatin‐1. Up‐regulation of cell ferritin expression, a primary cell defense against Hb toxicity, was not observed on western blots of culture lysates that had been concomitantly treated with Hpx. These results suggest that Hpx destabilizes Hb in the absence of haptoglobin, leading to globin precipitation and exacerbation of iron‐dependent oxidative cell injury. Combined therapy with hemopexin plus haptoglobin may be preferable to hemopexin alone after CNS hemorrhage.
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