Activation Of Ionotropic Glutamate Receptors Research Articles

Constitutive activity of ionotropic glutamate receptors via hydrophobic substitutions in the ligand-binding domain.

Neurotransmitter ligands electrically excite neurons by activating ionotropic glutamate receptor (iGluR) ion channels. Knowledge of the iGluR amino acid residues that dominate ligand-induced activation would enable the prediction of function from sequence. We therefore explored the molecular determinants of activity in rat N-methyl-D-aspartate (NMDA)-type iGluRs (NMDA receptors), complex heteromeric iGluRs comprising two glycine-binding GluN1 and two glutamate-binding GluN2 subunits, using amino acid sequence analysis, mutagenesis, and electrophysiology. We find that a broadly conserved aspartate residue controls both ligand potency and channel activity, to the extent that certain substitutions at this position bypass the need for ligand binding in GluN1 subunits, generating NMDA receptors activated solely by glutamate. Furthermore, we identify a homomeric iGluR from the placozoan Trichoplax adhaerens that has utilized native mutations of this crucial residue to evolve into a leak channel that is inhibited by neurotransmitter binding, pointing to a dominant role of this residue throughout the iGluR superfamily.

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

Molecular mechanisms underpinning deconditioning-update in fear memory.

Traumatic experiences are closely associated with some psychiatric conditions such as post-traumatic stress disorder. Deconditioning-update promotes robust and long-lasting attenuation of aversive memories. The deconditioning protocol consists of applying weak/neutral footshocks during reactivations, so that the original tone-shock association is replaced by an innocuous stimulus that does not produce significant fear response. Here, we present the molecular bases that can support this mechanism. To this end, we used pharmacological tools to inhibit the activity of ionotropic glutamate receptors (NMDA-GluN2B and CP-AMPA), the activity of proteases (calpains), and the receptors that control intracellular calcium storage (IP3 receptors), as well as the endocannabinoid system (CB1). Our results indicate that blocking these molecular targets prevents fear memory update by deconditioning. Therefore, this study uncovered the molecular substrate of deconditioning-update strategy, and, broadly, shed new light on the traumatic memory destabilization mechanisms that might be used to break the boundaries regarding reconsolidation-based approaches to deal with maladaptive memories.

Ultrasound modulates neuronal potassium currents via ionotropic glutamate receptors

BackgroundFocused ultrasound stimulation (FUS) has the potential to provide non-invasive neuromodulation of deep brain regions with unparalleled spatial precision. However, the cellular and molecular consequences of ultrasound stimulation on neurons remains poorly understood. We previously reported that ultrasound stimulation induces increases in neuronal excitability that persist for hours following stimulation in vitro. In the present study we sought to further elucidate the molecular mechanisms by which ultrasound regulates neuronal excitability and synaptic function. ObjectivesTo determine the effect of ultrasound stimulation on voltage-gated ion channel function and synaptic plasticity. MethodsPrimary rat cortical neurons were exposed to a 40 s, 200 kHz pulsed ultrasound stimulus or sham-stimulus. Whole-cell patch clamp electrophysiology, quantitative proteomics and high-resolution confocal microscopy were employed to determine the effects of ultrasound stimulation on molecular regulators of neuronal excitability and synaptic function. ResultsWe find that ultrasound exposure elicits sustained but reversible increases in whole-cell potassium currents. In addition, we find that ultrasound exposure activates synaptic signalling cascades that result in marked increases in excitatory synaptic transmission. Finally, we demonstrate the requirement of ionotropic glutamate receptor (AMPAR/NMDAR) activation for ultrasound-induced modulation of neuronal potassium currents. ConclusionThese results suggest specific patterns of pulsed ultrasound can induce contemporaneous enhancement of both neuronal excitability and synaptic function, with implications for the application of FUS in experimental and therapeutic settings. Further study is now required to deduce the precise molecular mechanisms through which these changes occur.

Allosteric control of NMDA receptor calcium permeability by extracellular protons

Excitatory neurotransmission in the central nervous system is primarily mediated through the activation of ionotropic glutamate receptors (iGluRs). Among iGluRs, N-methyl-D-aspartate receptors (NMDARs) are unique in their ability to pass large Ca2+ currents, which are essential to the normal physiology and development of excitatory synapses. Classically, the content of Ca2+ current passed by NMDARs has been viewed as a static property of the receptor. However, recent evidence shows that extracellular protons, which are strong negative allosteric modulators of NMDA receptors, are capable of also modulating the receptors' relative Ca2+ permeability, suggesting that NMDAR Ca2+ permeability is under dynamic regulation.

Local ionotropic glutamate receptors are required to trigger and sustain ramping of sympathetic nerve activity by hypothalamic paraventricular nucleus TNFα.

Tumor necrosis factor-α (TNFα) in the hypothalamic paraventricular nucleus (PVN) contributes to increased sympathetic nerve activity (SNA) in cardiovascular disease models, but mechanisms are incompletely understood. As previously reported, bilateral PVN TNFα (0.6 pmol, 50 nL) induced acute ramping of splanchnic SNA (SSNA) that averaged +64 ± 7% after 60 min and +109 ± 17% after 120 min (P < 0.0001, n = 10). Given that TNFα can rapidly strengthen glutamatergic transmission, we hypothesized that progressive activation of ionotropic glutamate receptors is critically involved. When compared with that of vehicle (n = 5), prior blockade of PVN AMPA or NMDA receptors in anesthetized (urethane/α-chloralose) adult male Sprague-Dawley rats dose-dependently (ED50: 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), 2.48 nmol; D-(-)-2-amino-5-phosphonopentanoic acid (APV), 12.33 nmol), but incompletely (Emax: NBQX, 64%; APV, 41%), attenuated TNFα-induced SSNA ramping (n = 5/dose). By contrast, combined receptor blockade prevented ramping (1.3 ± 2.1%, P < 0.0001, n = 5). Whereas separate blockade of PVN AMPA or NMDA receptors (n = 5/group) had little effect on continued SSNA ramping when performed 60 min after TNFα injection, combined blockade (n = 5) or PVN inhibition with the GABA-A receptor agonist muscimol (n = 5) effectively stalled, without reversing, the SSNA ramp. Notably, PVN TNFα increased local TNFα immunofluorescence after 120, but not 60 min. Findings indicate that AMPA and NMDA receptors each contribute to SSNA ramping to PVN TNFα, and that their collective availability and ongoing activity are required to initiate and sustain the ramping response. We conclude that acute sympathetic activation by PVN TNFα involves progressive local glutamatergic excitation that recruits downstream neurons capable of maintaining heightened SSNA, but incapable of sustaining SSNA ramping.NEW & NOTEWORTHY The proinflammatory cytokine TNFα contributes to heightened SNA in cardiovascular disease models, but mechanisms remain obscure. Here, we demonstrate that TNFα injection into the hypothalamic PVN triggers SNA ramping by mechanisms dependent on local ionotropic glutamate receptor availability, but largely independent of TNFα autoinduction. Continued SNA ramping depends on ionotropic glutamate receptor and neuronal activity in PVN, indicating that strengthening and/or increased efficacy of glutamatergic transmission is necessary for acute sympathoexcitation by PVN TNFα.

Mechanisms of Invasion in Glioblastoma: Extracellular Matrix, Ca2+ Signaling, and Glutamate.

Glioblastoma (GBM) is the most common and malignant form of primary brain tumor with a median survival time of 14–16 months in GBM patients. Surgical treatment with chemotherapy and radiotherapy may help increase survival by removing GBM from the brain. However, complete surgical resection to eliminate GBM is almost impossible due to its high invasiveness. When GBM cells migrate to the brain, they interact with various cells, including astrocytes, neurons, endothelial cells, and the extracellular matrix (ECM). They can also make their cell body shrink to infiltrate into narrow spaces in the brain; thereby, they can invade regions of the brain and escape from surgery. Brain tumor cells create an appropriate microenvironment for migration and invasion by modifying and degrading the ECM. During those processes, the Ca2+ signaling pathway and other signaling cascades mediated by various ion channels contribute mainly to gene expression, motility, and invasion of GBM cells. Furthermore, GBM cells release glutamate, affecting migration via activation of ionotropic glutamate receptors in an autocrine manner. This review focuses on the cellular mechanisms of glioblastoma invasion and motility related to ECM, Ca2+ signaling, and glutamate. Finally, we discuss possible therapeutic interventions to inhibit invasion by GBM cells.

Neuroprotective Effects of Thymoquinone by the Modulation of ER Stress and Apoptotic Pathway in In Vitro Model of Excitotoxicity.

Experimental evidence indicates that the activation of ionotropic glutamate receptors plays an important role in neurological disorders’ models such as epilepsy, cerebral ischemia and trauma. The glutamate receptor agonist kainic acid (KA) induces seizures and excitotoxic cell death in the CA3 region of the hippocampus. Thymoquinone (TQ) is the most important component of the essential oil obtained from black cumin (Nigella sativa L.) seeds. It has many pharmacological actions including antioxidant, anti-inflammatory, and anti-apoptotic effects. TQ was used in an in vitro experimental model of primary cultures where excitotoxicity was induced. Briefly, rat organotypic hippocampal slices were exposed to 5 µM KA for 24 h. Cell death in the CA3 subregions of slices was quantified by measuring propidium iodide fluorescence. The cross-talk between TQ, ER stress and apoptotic pathways was investigated by Western blot. In untreated slices TQ (10 µM) induced a significant increase on the PSD95 levels and it decreased the excitotoxic injury induced by KA. Additionally, TQ was able to ameliorate the KA-induced increase in unfolded proteins GRP78 and GRP94 expression. Finally, TQ was able to partially rescue the reduction of the KA-induced apoptotic pathway activation. Our results suggest that TQ modulates the processes leading to post-kainate neuronal death in the CA3 hippocampal area.

Chronic and Acute Manipulation of Cortical Glutamate Transmission Induces Structural and Synaptic Changes in Co-cultured Striatal Neurons.

In contrast to the prenatal topographic development of sensory cortices, striatal circuit organization is slow and requires the functional maturation of cortical and thalamic excitatory inputs throughout the first postnatal month. While mechanisms regulating synapse development and plasticity are quite well described at excitatory synapses of glutamatergic neurons in the neocortex, comparatively little is known of how this translates to glutamate synapses onto GABAergic neurons in the striatum. Here we investigate excitatory striatal synapse plasticity in an in vitro system, where glutamate can be studied in isolation from dopamine and other neuromodulators. We examined pre-and post-synaptic structural and functional plasticity in GABAergic striatal spiny projection neurons (SPNs), co-cultured with glutamatergic cortical neurons. After synapse formation, medium-term (24 h) TTX silencing increased the density of filopodia, and modestly decreased dendritic spine density, when assayed at 21 days in vitro (DIV). Spine reductions appeared to require residual spontaneous activation of ionotropic glutamate receptors. Conversely, chronic (14 days) TTX silencing markedly reduced spine density without any observed increase in filopodia density. Time-dependent, biphasic changes to the presynaptic marker Synapsin-1 were also observed, independent of residual spontaneous activity. Acute silencing (3 h) did not affect presynaptic markers or postsynaptic structures. To induce rapid, activity-dependent plasticity in striatal neurons, a chemical NMDA receptor-dependent “long-term potentiation (LTP)” paradigm was employed. Within 30 min, this increased spine and GluA1 cluster densities, and the percentage of spines containing GluA1 clusters, without altering the presynaptic signal. The results demonstrate that the growth and pruning of dendritic protrusions is an active process, requiring glutamate receptor activity in striatal projection neurons. Furthermore, NMDA receptor activation is sufficient to drive glutamatergic structural plasticity in SPNs, in the absence of dopamine or other neuromodulators.

NMDA and AMPA Receptor Autoantibodies in Brain Disorders: From Molecular Mechanisms to Clinical Features.

The role of autoimmunity in central nervous system (CNS) disorders is rapidly expanding. In the last twenty years, different types of autoantibodies targeting subunits of ionotropic glutamate receptors have been found in a variety of patients affected by brain disorders. Several of these antibodies are directed against NMDA receptors (NMDAR), mostly in autoimmune encephalitis, whereas a growing field of research has identified antibodies against AMPA receptor (AMPAR) subunits in patients with different types of epilepsy or frontotemporal dementia. Several in vitro and in vivo studies performed in the last decade have dramatically improved our understanding of the molecular and functional effects induced by both NMDAR and AMPAR autoantibodies at the excitatory glutamatergic synapse and, consequently, their possible role in the onset of clinical symptoms. In particular, the method by which autoantibodies can modulate the localization at synapses of specific target subunits leading to functional impairments and behavioral alterations has been well addressed in animal studies. Overall, these preclinical studies have opened new avenues for the development of novel pharmacological treatments specifically targeting the synaptic activation of ionotropic glutamate receptors.

Changes in Behavior and the Expression of Ionotropic Glutamate Receptor Genes in the Brains of Adult Rats after Neonatal Administration of Bacterial Lipopolysaccharide

Many studies have shown that early experience, particularly of neonatal infections, has a role in forming high anxiety levels in later life. One of the mechanisms of these changes may consist of impairments to the functional activity of ionotropic glutamate receptors associated with rearrangements in their subunit composition. The aim of the present work was to study measures of anxiety and levels of expression of genes for NMDA receptor (Grin1, Grin2a, Grin2b) and AMPA receptor (Gria1, Gria2) subunits in the medial prefrontal cortex, ventral and dorsal parts of the hippocampus in adult rats which at an early age had received bacterial lipopolysaccharide (LPS) at doses inducing the development of neuroinflammatory processes. Gene expression was studied by real-time reverse transcription PCR (qRT-PCR). Administration of LPS at doses of 25 and 50 μg/kg to male Wistar rats on days 15, 18, and 21 of life induced increases in the expression of genes for proinflammatory cytokines IL-1β and tumor necrosis factor in parts of the hippocampus. Increases in the expression of the Grin2b, Gria1, and Gria2 genes in the ventral parts of the hippocampus were seen three months after LPS administration (after injection of 50 μg/kg LPS), as were increases in expression of the Gria2 gene in the dorsal part of the hippocampus (after injection of 25 μg/kg LPS). These changes were accompanied by impairment to exploratory behavior in the open field test and decreased anxiety levels in the elevated plus maze. These studies showed that administration of bacterial LPS in early postnatal ontogeny leads to time-delayed changes in the expression of genes for NMDA and AMPA receptor subunits in the hippocampus and the associated forms of behavior.

Plastic smoke aerosol: Nano-sized particle distribution, absorption/fluorescent properties, dysregulation of oxidative processes and synaptic transmission in rat brain nerve terminals

Smoke from plastic waste incineration in an open air travels worldwide and is a major source of air pollution particulate matter (PM) that is very withstand to degradation and hazard to human health. Suspension of smoke aerosol components in water occurs during rains and fire extinguishing. Here, water-suspended plastic smoke aerosol (WPS) preparations suitable for biotesting were synthesized. It has been revealed using dynamic light scattering that WPS contained major nano-sized (∼30 nm) PM fraction, and this result was confirmed by electron microscopy. Optical absorption of WPS was in the UV region and an increase in λex led to a red-shift in fluorescence emission with a corresponding decrease in fluorescence intensity. WPS was analyzed in neurotoxicity studies in vitro using presynaptic rat cortex nerve terminals (synaptosomes). Generation of spontaneous reactive oxygen species (ROS) detected using fluorescent dye 2′,7-dichlorofluorescein in nerve terminals was decreased by WPS (10–50 μg/ml) in a dose-dependent manner. WPS also reduced the H2O2-evoked ROS production in synaptosomes, thereby influencing cellular oxidative processes and this effect was similar to that for carbon nanodots. WPS (0.1 mg/ml) decreased the synaptosomal membrane potential and synaptic vesicle acidification in fluorimetric experiments. WPS (1.0 mg/ml) attenuated the synaptosomal transporter-mediated uptake of excitatory and inhibitory neurotransmitters, L-[14C]glutamate and [3H]GABA, respectively. This can lead to an excessive increase in the glutamate concentration in the synaptic cleft and neurotoxicity via over activation of ionotropic glutamate receptors. Therefore, WPS was neurotoxic and provoked presynaptic malfunction through changes of oxidative activity, reduction of the membrane potential, synaptic vesicle acidification, and transporter-mediated uptake of excitatory and inhibitory neurotransmitters in nerve terminals. In summary, synthesis and emission to the environment of ultrafine PM occur during combustion of plastics, thereby polluting air and water resources, and possibly triggering development of neuropathologies.

Specialized cytonemes induce self-organization of stem cells

Spatial cellular organization is fundamental for embryogenesis. Remarkably, coculturing embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) recapitulates this process, forming embryo-like structures. However, mechanisms driving ESC-TSC interaction remain elusive. We describe specialized ESC-generated cytonemes that react to TSC-secreted Wnts. Cytoneme formation and length are controlled by actin, intracellular calcium stores, and components of the Wnt pathway. ESC cytonemes select self-renewal-promoting Wnts via crosstalk between Wnt receptors, activation of ionotropic glutamate receptors (iGluRs), and localized calcium transients. This crosstalk orchestrates Wnt signaling, ESC polarization, ESC-TSC pairing, and consequently synthetic embryogenesis. Our results uncover ESC-TSC contact-mediated signaling, reminiscent of the glutamatergic neuronal synapse, inducing spatial self-organization and embryonic cell specification.

The involvement of TRP channels in memory formation and task retrieval in a passive avoidance task in one-day old chicks.

An increase in the intracellular Ca2+ level in neurons is one of the main steps in the memory formation cascade. The increase results from extracellular Ca2+ influx by activation of ionotropic glutamate receptors and release from intracellular stores by the stimulation of IP3 receptors (IP3Rs) via group I metabotropic glutamate receptors (mGluR1/5). Recent data indicate an additional mechanism resulting in Ca2+ influx into neurons, triggered by intracellular signals that are directly connected to the activation of group I mGluRs. This influx occurs through transient receptor potential (TRP) channels, which are permeable to Na+, K+ and, mainly, Ca2+. These channels are activated by increases in intracellular Ca2+, diacylglycerol (DAC) and inositol 1,4,5-triphosphate (IP3) level resulting from a group I mGluR activation. The aim of the present study was to investigate the participation of TRP channels, especially from TRPC and TRPV groups, in memory consolidation and reconsolidation and memory retrieval processes in a passive avoidance task in one-day old chicks. TRP channels were blocked by the injection of the unspecific channel modulators SKF 96365 (2.5µl 30µM/hemisphere) and 2-APB (2.5µl 10µM/hemisphere) directly into the intermediate medial mesopallium (IMM) region of the chick brain immediately after initial training or after a reminder. The inhibition of specific TRP channels (TRPV1, TRPV3 or TRPC3) was achieved by the application of selective antibodies. Our results demonstrate that the inhibition of TRP channels by the application of both modulators disrupted memory consolidation, resulting in permanent task amnesia. The inhibition of the TRPV1, TRPC3 and TRPV3 channels by specific antibodies resulted in similar amnesia. Moreover, the inhibition of TRP channels by SKF 96365 and 2-APB at different time points after initial training or after the reminder also resulted in amnesia, indicating the role of TRP channels in memory retrieval. The inhibition of calcium influx through these channels resulted in permanent memory disruption, which suggests that the calcium signal generated by TRP channels is crucial for memory formation and retrieval processes. For the first time, the important role of TRPV3 channels in memory formation was demonstrated.

Mitochondrial division inhibitor 1 disrupts oligodendrocyte Ca2+ homeostasis and mitochondrial function.

Mitochondrial fission mediated by cytosolic dynamin related protein 1 (Drp1) is essential for mitochondrial quality control but may contribute to apoptosis as well. Blockade of Drp1 with mitochondrial division inhibitor 1 (mdivi-1) provides neuroprotection in several models of neurodegeneration and cerebral ischemia and has emerged as a promising therapeutic drug. In oligodendrocytes, overactivation of AMPA-type ionotropic glutamate receptors (AMPARs) induces intracellular Ca2+ overload and excitotoxic death that contributes to demyelinating diseases. Mitochondria are key to Ca2+ homeostasis, however it is unclear how it is disrupted during oligodendroglial excitotoxicity. In the current study, we have analyzed mitochondrial dynamics during AMPAR activation and the effects of mdivi-1 on excitotoxicity in optic nerve-derived oligodendrocytes. Sublethal AMPAR activation triggered Drp1-dependent mitochondrial fission, whereas toxic AMPAR activation produced Drp1-independent mitochondrial swelling. Accordingly, mdivi-1 efficiently inhibited Drp1-mediated mitochondrial fission and did not prevent oligodendrocyte excitotoxicity. Unexpectedly, mdivi-1 also induced mitochondrial depolarization, ER Ca2+ depletion and modulation of AMPA-induced Ca2+ signaling. These off-target effects of mdivi-1 sensitized oligodendrocytes to excitotoxicity and ER stress and eventually produced oxidative stress and apoptosis. Interestingly, in cultured astrocytes mdivi-1 induced nondetrimental mitochondrial depolarization and oxidative stress that did not cause toxicity or sensitization to apoptotic stimuli. In summary, our results provide evidence of Drp1-mediated mitochondrial fission during activation of ionotropic glutamate receptors in oligodendrocytes, and uncover a deleterious and Drp1-independent effect of mdivi-1 on mitochondrial and ER function in these cells. These off-target effects of mdivi-1 limit its therapeutic potential and should be taken into account in clinical studies.

Astrocytic glutamate transporters reduce the neuronal and physiological influence of metabotropic glutamate receptors in nucleus tractus solitarii.

Astrocytic excitatory amino acid transporters (EAATs) are critical to restraining synaptic and neuronal activity in the nucleus tractus solitarii (nTS). Relief of nTS EAAT restraint generates two opposing effects, an increase in neuronal excitability that reduces blood pressure and breathing and an attenuation in afferent [tractus solitarius (TS)]-driven excitatory postsynaptic current (EPSC) amplitude. Although the former is due, in part, to activation of ionotropic glutamate receptors, there remains a substantial contribution from another unidentified glutamate receptor. In addition, the mechanism(s) by which EAAT inhibition reduced TS-EPSC amplitude is unknown. Metabotropic glutamate receptors (mGluRs) differentially modulate nTS excitability. Activation of group I mGluRs on nTS neuron somas leads to depolarization, whereas group II/III mGluRs on sensory afferents decrease TS-EPSC amplitude. Thus we hypothesize that EAATs control postsynaptic excitability and TS-EPSC amplitude via restraint of mGluR activation. To test this hypothesis, we used in vivo recording, brain slice electrophysiology, and imaging of glutamate release and TS-afferent Ca2+. Results show that EAAT blockade in the nTS with (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA) induced group I mGluR-mediated depressor, bradycardic, and apneic responses that were accompanied by neuronal depolarization, elevated discharge, and increased spontaneous synaptic activity. Conversely, upon TS stimulation TFB-TBOA elevated extracellular glutamate to decrease presynaptic Ca2+ and TS-EPSC amplitude via activation of group II/III mGluRs. Together, these data suggest an important role of EAATs in restraining mGluR activation and overall cardiorespiratory function.

Two distinct electrophysiological mechanisms underlie extensive depolarization elicited by 2,4 diaminobutyric acid in leech Retzius neurons

Recent studies suggest that 2,4-DABA, a neurotoxic excitatory amino acid present in virtually all environments, but predominantly in aquatic ecosystems may be a risk factor for development of neurodegenerative diseases in animals and humans. Despite its neurotoxicity and potential environmental importance, mechanisms underlying the excitatory and putative excitotoxic action of 2,4-DABA in neurons are still unexplored. We previously reported on extensive two-stage membrane depolarization and functional disturbances in leech Retzius neurons induced by 2,4-DABA. Current study presents the first detailed look into the electrophysiological processes leading to this depolarization. Intracellular recordings were performed on Retzius neurons of the leech Haemopis sanguisuga using glass microelectrodes and input membrane resistance (IMR) was measured by injecting hyperpolarizing current pulses through these electrodes. Results show that the excitatory effect 2,4-DABA elicits on neurons’ membrane potential is dependent on sodium ions. Depolarizing effect of 5·10−3 mol/L 2,4-DABA in sodium-free solution was significantly diminished by 91% reducing it to 3.26 ± 0.62 mV and its two-stage nature was abrogated. In addition to being sodium-dependent, the depolarization of membrane potential induced by this amino acid is coupled with an increase of membrane permeability, as 2,4-DABA decreases IMR by 8.27 ± 1.47 MΩ (67.60%). Since present results highlight the role of sodium ions, we investigated the role of two putative sodium-dependent mechanisms in 2,4-DABA-induced excitatory effect – activation of ionotropic glutamate receptors and the electrogenic transporter for neutral amino acids. Excitatory effect of 5·10−3 mol/L 2,4-DABA was partially blocked by 10-5 mol/L 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) a non-NMDA receptor antagonist as the first stage of membrane depolarization was significantly reduced by 2.59 ± 0.98 mV (40%), whilst second stage remained unaltered. Moreover, involvement of the sodium-dependent transport system for neutral amino acids was investigated by equimolar co-application of 5·10−3 mol/L 2,4-DABA and L-alanine, a competitive inhibitor of this transporter. Although L-alanine exhibited no effect on the first stage of membrane depolarization elicited by 2,4-DABA, it substantially reduced the second stage (the overall membrane depolarization) from 39.63 ± 2.22 mV to 16.28 ± 2.58 mV, by 58.92%. We therefore propose that the electrophysiological effect of 2,4-DABA on Retzius neurons is mediated by two distinct mechanisms, i.e. by activation of ionotropic glutamate receptor that initiates the first stage of membrane depolarization followed by the stimulation of an electrogenic sodium-dependent neutral amino acid transporter, leading to additional influx of positive charge into the cell and the second stage of depolarization.

Involvement of metabotropic glutamate receptor 5 in ethanol regulation of NMDA receptor activity in rat substantia gelatinosa neurons

AimsGlutamatergic receptors are important targets of ethanol. Intake of ethanol may produce analgesic effects. The present study examined the effects of ethanol on the activity of ionotropic glutamate receptors in spinal cord substantia gelatinosa (SG) neurons, critical neurons involved in nociceptive transmission. Main methodsWhole-cell recordings were made from SG neurons of the lumbar spinal cord slices from 15 to 20-day-old rats. Ethanol and glutamate receptor agonists or antagonists were applied by superfusion. Key findingEthanol (50 and 100 mM) applied by superfusion for 5 min dose-dependently decreased the amplitude of evoked excitatory postsynaptic potential in SG neurons. Superfusion of ethanol (100 mM) for 15 min consistently inhibited NMDA- or AMPA-induced depolarizations in SG neurons. Ethanol (100 mM) also inhibited the depolarizations induced by glutamate. However, ethanol inhibition of glutamate-induced responses significantly decreased at 10–15 min following continuous superfusion, suggesting the development of acute tolerance to the inhibition during prolonged exposure. Application of MPEP hydrochloride (an antagonist of metabotropic glutamate receptor [mGluR] 5) or GF109203X (a protein kinase C [PKC] inhibitor), together with ethanol significantly blocked the tolerance. The inhibition by ethanol of the NMDA-induced, but not AMPA-induced, depolarizations significantly decreased at 15 min during continuous superfusion while ACPD (a mGluR agonist) was co-applied with ethanol. SignificanceThe results suggest that (1) ethanol exposure may inhibit ionotropic glutamate receptor-mediated neurotransmission; (2) regulation of NMDA receptor function by mGluR5/PKC pathways may be involved in the development of the tolerance to ethanol inhibition of glutamate-induced responses during prolonged exposure in SG neurons.

Local ionotropic glutamate receptor activity is required for the ramp‐like increase of sympathetic activity induced by PVN TNFα

Central nervous system actions of pro‐inflammatory cytokines (PIC) play a pivotal role in driving exaggerated sympathetic nerve activity (SNA), a major aggravating factor in cardiovascular and metabolic diseases. We previously reported that delivery of tumor necrosis factor alpha (TNFα), a prototypical early onset PIC, into the hypothalamic paraventricular nucleus (PVN) elicits a progressive, ramp‐like, increase of splanchnic SNA (SSNA) that resembles the response to sympathoexcitatory stimuli such as plasma insulin and leptin. Here we investigated the contribution of local ionotropic glutamate receptors both in triggering and sustaining this SNA response. In anesthetized (α‐chloralose/urethane) male Sprague‐Dawley rats, unilateral PVN injection of vehicle (aCSF) was without affect, but subsequent injection of TNFα (0.6 pmol/50 nl) elicited a significant (P&lt;0.0001) ramp‐like increase of SSNA that averaged +55 ± 5.5% over a period of 60 min. Mean arterial pressure and heart rate were unaffected. Separate PVN pretreatment with the AMPA receptor antagonist NBQX (5.2 nmol/50 nl) and the NMDA antagonist AP5 (24 nmol/50 nl) (n = 5/group) each significantly attenuated (P&lt;0.05) the TNFα elicited SSNA response (55 ± 5.5 % vs. NBQX: 24 ± 5% vs. AP5: 32 ± 6.5%) at the 60 min post‐TNFα time point. PVN pretreatment with a cocktail of NBQX and AP5 abolished the TNFα‐induced SSNA ramp (55 ± 5.5 % vs. 1.3 ± 2.1%; P&lt;0.0001). When delivered separately into the PVN 60 min after TNFα, neither NBQX nor AP5 significantly altered the rate of SSNA ramping over an additional 60 min period. These results indicate that ionotropic glutamate receptor activity in the PVN is required to initiate the progressive increase of SSNA triggered by TNFα, and hint that sympathoexcitatory stimuli such as TNFα that elicit a PVN‐driven ramp‐like SNA response might do so by inducing a long‐term potentiation (LTP)‐like form of glutamatergic synaptic plasticity.Support or Funding InformationCapes (AAM, GRP), NIH HL088052 &amp; AHA 25710176 (GMT)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting metabotropic glutamate receptors in the treatment of epilepsy: rationale and current status

ABSTRACTIntroduction: Several drugs targeting the GABAergic system are used in the treatment of epilepsy, but only one drug targeting glutamate receptors is on the market. This is surprising because an imbalance between excitatory and inhibitory neurotransmission lies at the core of the pathophysiology of epilepsy. One possible explanation is that drug development has been directed towards the synthesis of molecules that inhibit the activity of ionotropic glutamate receptors. These receptors mediate fast excitatory synaptic transmission in the central nervous system (CNS) and their blockade may cause severe adverse effects such as sedation, cognitive impairment, and psychotomimetic effects. Metabotropic glutamate (mGlu) receptors are more promising drug targets because these receptors modulate synaptic transmission rather than mediate it.Areas covered: We review the current evidence that links mGlu receptor subtypes to the pathophysiology and experimental treatment of convulsive and absence seizures.Expert opinion: While mGlu5 receptor negative allosteric modulators have the potential to be protective against convulsive seizures and hyperactivity-induced neurodegeneration, drugs that enhance mGlu5 and mGlu7 receptor function may have beneficial effects in the treatment of absence epilepsy. Evidence related to the other mGlu receptor subtypes is more fragmentary; further investigations are required for an improved understanding of their role in the generation and propagation of seizures.