Glutamate Spillover Research Articles

Gap junctions

In vertebrates and invertebrates, signaling among neurons is most commonly mediated by chemical synapses. At these synapses neurotransmitter released by presynaptic neurons is detected by receptors on the postsynaptic neurons, leading to an influx of ions through the receptors themselves or through channels activated by intracellular signaling downstream of the receptors. But neurons can communicate with each other in a more direct way, by passing signals composed of small molecules and ions through pores called gap junctions. Gap junctions that transmit electrical signals are called electrical synapses. Unlike most chemical synapses, electrical synapses interact through axon-to-axon or dendrite-to-dendrite contacts. Found throughout the nervous system, they are probably best known for linking the relatively few inhibitory, GABAergic, neurons into large, effective networks within vertebrate brains. They are particularly important early in development before the formation of most chemical synapses, but recent work shows gap junctions play important roles in the adult nervous system, too. Gap junctions are sometimes thought to be mere passageways between cells. But, as recent work shows, their properties can be complex and surprising. Gap junctions help generate, propagate, and regulate neural oscillations, can filter electrical signals, and can be modulated in a variety of ways. Here we discuss recent work highlighting the diversity and importance of gap junctions throughout the nervous system.

Dysregulation of synaptic and extrasynaptic N-methyl-D-aspartate receptors induced by amyloid-β

The toxicity of amyloid-beta (Aβ) is strongly associated with Alzheimer's disease (AD), which has a high incidence in the elderly worldwide. Recent evidence showed that alteration in the activity of N-methyl-D-aspartate receptors (NMDARs) plays a key role in Aβ-induced neurotoxicity. However, the activation of synaptic and extrasynaptic NMDARs has distinct consequences for plasticity, gene regulation, neuronal death, and Aβ production. This review focuses on the dysregulation of synaptic and extrasynaptic NMDARs induced by Aβ. On one hand, Aβ downregulates the synaptic NMDAR response by promoting NMDAR endocytosis, leading to either neurotoxicity or neuroprotection. On the other hand, Aβ enhances the activation of extrasynaptic NMDARs by decreasing neuronal glutamate uptake and inducing glutamate spillover, subsequently causing neurotoxicity. In addition, selective enhancement of synaptic activity by low doses of NMDA, or reduction of extrasynaptic activity by memantine, a non-competitive NMDAR antagonist, halts Aβ-induced neurotoxicity. Therefore, future neuroprotective drugs for AD should aim at both the enhancement of synaptic activity and the disruption of extrasynaptic NMDAR-dependent death signaling.

Activation of Extrasynaptic NMDARs at Individual Parallel Fiber–Molecular Layer Interneuron Synapses in Cerebellum

NMDA receptors (NMDARs) expressed by cerebellar molecular layer interneurons (MLIs) are not activated by single exocytotic events but can respond to glutamate spillover following coactivation of adjacent parallel fibers (PFs), indicating that NMDARs are perisynaptic. Several types of synaptic plasticity rely on these receptors but whether they are activated at isolated synapses is not known. Using a combination of electrophysiological and optical recording techniques in acute slices of rat cerebellum, along with modeling, we find that repetitive activation of single PF-MLI synapses can activate NMDARs in MLIs. High-frequency stimulation, multivesicular release (MVR), or asynchronous release can each activate NMDARs. Frequency facilitation was found at all PF-MLI synapses but, while some showed robust MVR with increased release probability, most were limited to univesicular release. Together, these results reveal a functional diversity of PF synapses, which use different mechanisms to activate NMDARs.

Spillover-Mediated Feedforward Inhibition Functionally Segregates Interneuron Activity

Neurotransmitter spillover represents a form of neural transmission not restricted to morphologically defined synaptic connections. Communication between climbing fibers (CFs) and molecular layer interneurons (MLIs) in the cerebellum is mediated exclusively by glutamate spillover. Here, we show how CF stimulation functionally segregates MLIs based on their location relative to glutamate release. Excitation of MLIs that reside within the domain of spillover diffusion coordinates inhibition of MLIs outside the diffusion limit. CF excitation of MLIs is dependent on extrasynaptic NMDA receptors that enhance the spatial and temporal spread of CF signaling. Activity mediated by functionally segregated MLIs converges onto neighboring Purkinje cells (PCs) to generate a long-lasting biphasic change in inhibition. These data demonstrate how glutamate release from single CFs modulates excitability of neighboring PCs, thus expanding the influence of CFs on cerebellar cortical activity in a manner not predicted by anatomical connectivity.

Presynaptic NMDA receptors act as local high‐gain glutamate detector in developing cerebellar molecular layer interneurons

In the classical view, NMDA receptors (NMDARs) are located postsynaptically and play a pivotal role in excitatory transmission and synaptic plasticity. In developing cerebellar molecular layer interneurons (MLIs) however, NMDARs are known to be solely extra- or presynaptic and somewhat poorly expressed. Somatodendritic NMDARs are exclusively activated by glutamate spillover from adjacent synapses, but the mode of activation of axonal NMDARs remains unclear. Our data suggest that a volume transmission is likely to stimulate presynaptic NMDARs (preNMDARs) since NMDA puffs directed to the axon led to inward currents and Ca²⁺ transients restricted to axonal varicosities. Using local glutamate photoliberation, we show that pre- and post-synaptic NMDARs share the same voltage dependence indicating their containing NR2A/B subunits. Ca²⁺ transients elicited by NMDA puffs are eventually followed by delayed events reminding of the spontaneous Ca²⁺ transients (ScaTs) described at the basket cell/Purkinje cell terminals. Moreover, the presence of Ca²⁺ transients at varicosities located more than 5 μm away from the uncaging site indicates that the activation of preNMDARs sensitizes the Ca²⁺ stores in adjacent varicosities, a process that is abolished in the presence of a high concentration of ryanodine. Altogether, the data demonstrate that preNMDARs act as high-gain glutamate detectors.

Extrasynaptic glutamate and inhibitory neurotransmission modulate ganglion cell participation during glutamatergic retinal waves

During the first 2 wk of mouse postnatal development, transient retinal circuits give rise to the spontaneous initiation and lateral propagation of depolarizations across the ganglion cell layer (GCL). Glutamatergic retinal waves occur during the second postnatal week, when GCL depolarizations are mediated by ionotropic glutamate receptors. Bipolar cells are the primary source of glutamate in the inner retina, indicating that the propagation of waves depends on their activation. Using the fluorescence resonance energy transfer-based optical sensor of glutamate FLII81E-1μ, we found that retinal waves are accompanied by a large transient increase in extrasynaptic glutamate throughout the inner plexiform layer. Using two-photon Ca(2+) imaging to record spontaneous Ca(2+) transients in large populations of cells, we found that despite this spatially diffuse source of depolarization, only a subset of neurons in the GCL and inner nuclear layer (INL) are robustly depolarized during retinal waves. Application of the glutamate transporter blocker dl-threo-β-benzyloxyaspartate (25 μM) led to a significant increase in cell participation in both layers, indicating that the concentration of extrasynaptic glutamate affects cell participation in both the INL and GCL. In contrast, blocking inhibitory transmission with the GABAA receptor antagonist gabazine and the glycine receptor antagonist strychnine increased cell participation in the GCL without significantly affecting the INL. These data indicate that during development, glutamate spillover provides a spatially diffuse source of depolarization, but that inhibitory circuits dictate which neurons within the GCL participate in retinal waves.

Glutamate-Dependent Neuroglial Calcium Signaling Differs Between Young and Adult Brain

An extensive literature shows that astrocytes exhibit metabotropic glutamate receptor 5 (mGluR5)-dependent increases in cytosolic calcium ions (Ca(2+)) in response to glutamatergic transmission and, in turn, modulate neuronal activity by their Ca(2+)-dependent release of gliotransmitters. These findings, based on studies of young rodents, have led to the concept of the tripartite synapse, in which astrocytes actively participate in neurotransmission. Using genomic analysis, immunoelectron microscopy, and two-photon microscopy of astrocytic Ca(2+) signaling in vivo, we found that astrocytic expression of mGluR5 is developmentally regulated and is undetectable after postnatal week 3. In contrast, mGluR3, whose activation inhibits adenylate cyclase but not calcium signaling, was expressed in astrocytes at all developmental stages. Neuroglial signaling in the adult brain may therefore occur in a manner fundamentally distinct from that exhibited during development.

『グルタミン酸伝達を“観る”技術』 グルタミン酸スピルオーバーの可視化解析

『グルタミン酸伝達を“観る”技術』 グルタミン酸スピルオーバーの可視化解析

Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held

Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle (N(Glu)) and its diffusion coefficient (D(Glu)) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of N(Glu) and D(Glu) with an optimum of 7000 and 0.3 μm(2) ms(-1) reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals.

Metabotropic glutamate receptors and glutamate transporters shape transmission at the developing retinogeniculate synapse

Over the first few postnatal weeks, extensive remodeling occurs at the developing murine retinogeniculate synapse, the connection between retinal ganglion cells (RGCs) and the visual thalamus. Although numerous studies have described the role of activity in the refinement of this connection, little is known about the mechanisms that regulate glutamate concentration at and around the synapse over development. Here we show that interactions between glutamate transporters and metabotropic glutamate receptors (mGluRs) dynamically control the peak and time course of the excitatory postsynaptic current (EPSC) at the immature synapse. Inhibiting glutamate transporters by bath application of TBOA (DL-threo-β-benzyloxyaspartic acid) prolonged the decay kinetics of both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) currents at all ages. Moreover, at the immature synapse, TBOA-induced increases in glutamate concentration led to the activation of group II/III mGluRs and a subsequent reduction in neurotransmitter release at RGC terminals. Inhibition of this negative-feedback mechanism resulted in a small but significant increase in peak NMDAR EPSCs during basal stimulation and a substantial increase in the peak with coapplication of TBOA. Activation of mGluRs also shaped the synaptic response during high-frequency trains of stimulation that mimic spontaneous RGC activity. At the mature synapse, however, the group II mGluRs and the group III mGluR7-mediated response are downregulated. Our results suggest that transporters reduce spillover of glutamate, shielding NMDARs and mGluRs from the neurotransmitter. Furthermore, mechanisms of glutamate clearance and release interact dynamically to control the glutamate transient at the developing retinogeniculate synapse.

Paired‐pulse facilitation of multivesicular release and intersynaptic spillover of glutamate at rat cerebellar granule cell–interneurone synapses

A simple form of presynaptic plasticity, paired-pulse facilitation (PPF), has been explained as a transient increase in the probability of vesicular release. Using the whole-cell patch-clamp technique to record synaptic activity in rat cerebellar slices, we found different forms of presynaptically originated short-term plasticity during glutamatergic excitatory neurotransmission from granule cells (GCs) to molecular-layer interneurones (INs). Paired-pulse activation of GC axons at short intervals (30-100 ms) elicited not only a facilitation in the peak amplitude (PPF(amp)), but also a prolongation in the decay-time constant (PPP(decay)) of the EPSCs recorded from INs. The results of pharmacological tests and kinetics analyses suggest that the mechanisms underlying the respective types of short-term plasticity were different. PPF(amp) was elicited by a transient increase in the number of released vesicles. On the other hand, PPP(decay) was caused not only by delayed release as has been reported but also by extrasynaptic spillover of the GC transmitter and the subsequent intersynaptic pooling. Both PPF(amp) and PPP(decay) closely rely on repetitive-activation-induced multivesicular release. Using a dynamic clamp technique, we further examined the physiological significance of different presynaptic plasticity, and found that PPF(amp) and PPP(decay) can differentially encode and process neuronal information by influencing the total synaptic charge transferred to postsynaptic INs to reflect activation frequency of the presynaptic GCs.

Activity-Dependent Regulation of Retinogeniculate Signaling by Metabotropic Glutamate Receptors

Thalamocortical neurons in dorsal lateral geniculate nucleus (dLGN) dynamically convey visual information from retina to the neocortex. Activation of metabotropic glutamate receptors (mGluRs) exerts multiple effects on neural integration in dLGN; however, their direct influence on the primary sensory input, namely retinogeniculate afferents, is unknown. In the present study, we found that pharmacological or synaptic activation of type 1 mGluRs (mGluR(1)s) significantly depresses glutamatergic retinogeniculate excitation in rat thalamocortical neurons. Pharmacological activation of mGluR(1)s attenuates excitatory synaptic responses in thalamocortical neurons at a magnitude sufficient to decrease suprathreshold output of these neurons. The reduction in both NMDA and AMPA receptor-dependent synaptic responses results from a presynaptic reduction in glutamate release from retinogeniculate terminals. The suppression of retinogeniculate synaptic transmission and dampening of thalamocortical output was mimicked by tetanic activation of retinogeniculate afferent in a frequency-dependent manner that activated mGluR(1)s. Retinogeniculate excitatory synaptic transmission was also suppressed by the glutamate transport blocker TBOA (dl-threo-β-benzyloxyaspartic acid), suggesting that mGluR(1)s were activated by glutamate spillover. The data indicate that presynaptic mGluR(1) contributes to an activity-dependent mechanism that regulates retinogeniculate excitation and therefore plays a significant role in the thalamic gating of visual information.

Identifying neurotransmitter spill-over in hippocampal field recordings

A model of synaptic and extra-synaptic excitatory signaling in the hippocampus is presented. The model is used to analytically evaluate the potential contributions of homosynaptic and heterosynaptic glutamate spill-over to receptor signaling during an electrophysiological experiment in which glutamate transporters are pharmacologically blocked. Inhibition of glutamate uptake selectively prolongs the decay kinetics of the second field excitatory postsynaptic potential evoked by paired pulse stimulation of Schaffer collateral axons in area CA1. The model includes AMPA and NMDA glutamate receptors, and the removal of glutamate by transporters and diffusion. We establish analytically that the prolongation cannot be caused by local effects, i.e., the transporters acting within or near the synapse. In contrast, a time profile of glutamate consistent with spill-over from adjacent synapses can explain the effect. The different reaction kinetics of AMPA and NMDA receptors have a significant role in reproducing the experimental results, as explained by analysis of the ODEs governing the reactions.

CPP alters theta/gamma oscillations in rat hippocampus: simulation and experiment

Several different NMDA receptor (NMDAR) antagonists are used to produce pharmacological models for schizophrenia. These antagonists have different molecular mechanisms of action: non-competitive (e.g. ketamine, which binds at a site different from glutamate) and competitive (e.g. CPP, which displaces glutamate). Because competitive antagonism results in more glutamate availability, this may result in glutamate spill-over either onto other receptors of the same synapse or to neighboring synapses. In this study, we modeled the effects of competitive blockade by simultaneously blocking NMDAR and increasing activity of AMPAR (spillover). We looked at alterations of network oscillations and synchronization using our biophysical model of the CA1 region of hippocampus using the NEURON simulator. We then compared the results with data obtained from the CA1 region of hippocampus in rats injected with CPP. Our network consisted of 800 five-compartment pyramidal cells (PYR), 200 one-compartment basket cell interneurons (BAS), and 200 one-compartment oriens lacunosum-moleculare interneurons (OLM) [1]. All cells contained leak current, transient sodium current and delayed rectifier current. Additionally, pyramidal cells contained potassium type A current and pyramidal and OLM cells had Ih current. Cell classes were interconnected probabilistically with AMPA/NMDA synapses, and two classes of GABAa synapses. The OLM cells formed synapses on the distal dendrites of pyramidal cells, while the basket cells synapsed proximally on pyramidal and other basket cells. Pyramidal cells synapsed on both types of interneurons with AMPA/NMDA synapses. All synapses were bombarded with external Poisson inputs to generate network activity. Competitive inhibition was modeled by setting the conductance across NMDAR to zero, and increasing current conductance across AMPAR on the same set of synapses. We modeled this on synaptic sites on all cell populations. Also, given the possible variation of the affinity of competitive inhibitors to NMDAR on different cells, we studied this effect when it took place at the receptors of the 3 different cell populations separately. Experimental recordings were made from tetrode arrays implanted in the CA1 region of Long Evans rats chasing sugar pellets in a box. CPP at a dose of 5 mg/kg was injected intraperitoneally in the rats. Recordings were for 16 min sessions, separated by 30 min breaks during which the rats were returned to their cages. Recordings from CA1 of rat hippocampus under the effect of CPP, showed that there is a reduction in theta and an increase in gamma. Modeling competitive inhibition to glutamate at all synapse locations (on OLM, BAS, and PYR) resulted in a reduction in the theta power and an increase in gamma power, differing from the noncompetitive effect with ketamine, which reduced both theta and gamma when given across all synapses. However, this result was also seen when the effect of competitive inhibition was restricted to only OLM cell NMDARs. Therefore, our model demonstrates both of these interpretations of CPP action. We will examine further correlates between simulation and experiment to reduce this uncertainty.

Disruptions in the Regulation of Extracellular Glutamate by Neurons and Glia in the Rat Striatum Two Days after Diffuse Brain Injury

Disrupted regulation of extracellular glutamate in the central nervous system contributes to and can exacerbate the acute pathophysiology of traumatic brain injury (TBI). Previously, we reported increased extracellular glutamate in the striatum of anesthetized rats 2 days after diffuse brain injury. To determine the mechanism(s) responsible for increased extracellular glutamate, we used enzyme-based microelectrode arrays (MEAs) coupled with specific pharmacological agents targeted at in vivo neuronal and glial regulation of extracellular glutamate. After TBI, extracellular glutamate was significantly increased in the striatum by (∼90%) averaging 4.1±0.6 μM compared with sham 2.2±0.4 μM. Calcium-dependent neuronal glutamate release, investigated by local application of an N-type calcium channel blocker, was no longer a significant source of extracellular glutamate after TBI, compared with sham. In brain-injured animals, inhibition of glutamate uptake with local application of an excitatory amino acid transporter inhibitor produced significantly greater increase in glutamate spillover (∼ 65%) from the synapses compared with sham. Furthermore, glutamate clearance measured by locally applying glutamate into the extracellular space revealed significant reductions in glutamate clearance parameters in brain-injured animals compared with sham. Taken together, these data indicate that disruptions in calcium-mediated glutamate release and glial regulation of extracellular glutamate contribute to increased extracellular glutamate in the striatum 2 days after diffuse brain injury. Overall, these data suggest that therapeutic strategies used to regulate glutamate release and uptake may improve excitatory circuit function and, possibly, outcomes following TBI.

Positive allosteric modulation reveals a specific role for mGlu2 receptors in sensory processing in the thalamus.

Group II metabotropic glutamate receptor (mGlu) modulation of sensory processing in the rat ventrobasal thalamic nucleus (VB) has been extensively studied in vivo. However, it is not yet known what the relative contributions are of the Group II mGlu receptor subtypes (mGlu2 and mGlu3) to this modulation, nor to what extent these receptors may be activated under physiological conditions during this process. Using single-neurone recording in the rat VB in vivo with local application of the selective Group II agonist LY354740 and the subtype selective mGlu2 positive allosteric modulator (PAM) LY487379, our findings were twofold. Firstly, we found that there is an mGlu2 component to the effects of LY354740 on sensory responses in the VB. Secondly, we have demonstrated that application of the PAM alone can modulate sensory responses of single neurones in vivo. This indicates that mGlu2 receptors can be activated by endogenous agonist following physiological sensory stimulation. We speculate that the mGlu2 subtype could be activated under physiological stimulus-evoked conditions by 'glutamate spillover' from synapses between excitatory sensory afferents and VB neurones that can lead to a reduction in sensory-evoked inhibition arising from the thalamic reticular nucleus (TRN). We propose that this potential mGlu2 receptor modulation of inhibition could play an important role in discerning relevant information from background activity upon physiological sensory stimulation. Furthermore, this could be a site of action for mGlu2 PAMs to modulate cognitive processes.

Parasynaptic NMDA Receptor Signaling Couples Neuronal Glutamate Transporter Function to AMPA Receptor Synaptic Distribution and Stability

At synapses, two major processes occur concomitantly after the release of glutamate: activation of AMPA receptors (AMPARs) to conduct synaptic transmission and activation of excitatory amino acid transporters (EAATs) for transmitter removal. Although crosstalk between the receptors and EAATs is conceivable, whether and how the transporter activity affects AMPAR synaptic localization remain unknown. Using cultured hippocampal and cortical rat neurons, we show that inhibition of glutamate transporters leads to rapid reduction in AMPAR synaptic accumulation and total AMPAR abundance. EAAT inactivity also results in elevated internalization and reduced surface expression of AMPARs. The reduction in AMPAR amount is accompanied by receptor ubiquitination and can be blocked by suppression of proteasome activity, indicating the involvement of proteasome-mediated receptor degradation. Consistent with glutamate spillover, effect of EAAT inhibition on AMPAR distribution and stability is dependent on the activation of parasynaptically localized NR2B-containing NMDA receptors (NMDARs). Moreover, we show that neuronal glutamate transporters, especially those localized at the postsynaptic sites, are responsible for the observed effect during EAAT suppression. These results indicate a role for neuron-specific glutamate transporters in AMPAR synaptic localization and stability.

Creatine Protects against Excitoxicity in an In Vitro Model of Neurodegeneration

Creatine has been shown to be neuroprotective in aging, neurodegenerative conditions and brain injury. As a common molecular background, oxidative stress and disturbed cellular energy homeostasis are key aspects in these conditions. Moreover, in a recent report we could demonstrate a life-enhancing and health-promoting potential of creatine in rodents, mainly due to its neuroprotective action. In order to investigate the underlying pharmacology mediating these mainly neuroprotective properties of creatine, cultured primary embryonal hippocampal and cortical cells were challenged with glutamate or H2O2. In good agreement with our in vivo data, creatine mediated a direct effect on the bioenergetic balance, leading to an enhanced cellular energy charge, thereby acting as a neuroprotectant. Moreover, creatine effectively antagonized the H2O2-induced ATP depletion and the excitotoxic response towards glutamate, while not directly acting as an antioxidant. Additionally, creatine mediated a direct inhibitory action on the NMDA receptor-mediated calcium response, which initiates the excitotoxic cascade. Even excessive concentrations of creatine had no neurotoxic effects, so that high-dose creatine supplementation as a health-promoting agent in specific pathological situations or as a primary prophylactic compound in risk populations seems feasible. In conclusion, we were able to demonstrate that the protective potential of creatine was primarily mediated by its impact on cellular energy metabolism and NMDA receptor function, along with reduced glutamate spillover, oxidative stress and subsequent excitotoxicity.

Glutamate transporters control metabotropic glutamate receptors activation to prevent the genesis of paroxysmal burst in the developing hippocampus

Metabotropic glutamate receptors (mGluR) can control neuronal excitability by modulating several ionic channels. In hippocampal pyramidal cells, groups I/II mGluR are located extrasynaptically, suggesting that their endogenous activation is dependent on the glutamate clearance rate and therefore on excitatory amino-acid transporters (EAAT) efficiency. Deficiency of glutamate uptake can generate seizures in rodents and has been suggested as a mechanism of seizure generation in some human epileptic syndromes. However, the cellular mechanisms linking EAAT dysfunction and pathological cortical activities remain elusive. Here, we investigate the possible role of mGluR on paroxysmal burst of multiple unit activities (MUA) generated in the CA1 region of developing hippocampal slices using an EAAT inhibitor, TBOA. These bursts are generated by a synaptic release of glutamate and involve extrasynaptic NMDA receptors (NMDAR) activated by transmitter spillover. Here, we show that postsynaptic mGluR (groups I/II) are tonically activated by the rise in ambient glutamate concentration after EAAT inhibition and strongly contribute to paroxysmal burst genesis. The inhibition of mGluR with broad spectrum antagonists or addition of a glutamate scavenger strongly reduced the occurrence of paroxysmal burst and the frequency/number of MUA during the burst. Moreover, this endogenous activation of groups I/II mGluR leads to (i) the reduction of the slow afterhyperpolarization current (IsAHP), increasing the firing pattern of pyramidal cells, and (ii) the potentiation of extrasynaptic NMDAR-mediated responses, enabling glutamate spillover to generate a suprathreshold depolarization for several seconds. Our data show that an insufficient buffering of extracellular glutamate enables a cross talk between groups I/II mGluR and NMDAR, which, combined with a decrease of IsAHP, leads to the hyperexcitability of the hippocampal network, facilitating the genesis of epileptical-like activity in response to glutamate release. These findings highlight the importance of the control exerted by EAAT on mGluR.

Activation of group III metabotropic glutamate receptors by endogenous glutamate protects against glutamate‐mediated excitotoxicity in the hippocampus in vivo

Perfusion of 4-aminopyridine (4-AP) by microdialysis in the hippocampus produces intense epileptiform behavioral and electrical activity and neurodegeneration, resulting from a stimulated release of glutamate from nerve endings. In contrast, accumulation of extracellular glutamate by blockade of its transport in vivo in anesthetized rats is innocuous, and studies in vitro in brain slices suggest that under these conditions glutamate may activate presynaptic group III metabotropic glutamate receptors (mGluRs) and inhibit its own release. Therefore, using microdialysis, EEG recording, and histological evaluation, we studied the effect of increased endogenous extracellular glutamate by blockade of its transport with pyrrolidine dicarboxylic acid (PDC) on the excitotoxic action of 4-AP in the hippocampus of awake rats. We found that up to a 20-fold increase in extracellular glutamate during >90 min with PDC does not induce any sign of excitotoxicity. On the contrary, this glutamate increase notably protected against the 4-AP-induced seizures and neurodegeneration, and, remarkably, this protection was dependent on the time of perfusion with PDC and thus on the duration of extracellular glutamate accumulation. To test whether this protective action was mediated by the activation of group III mGluRs, we used specific antagonists of these receptors and found that they clearly prevented the protective effect of PDC, without affecting the accumulation of extracellular glutamate. We conclude that the spillover of the excess extracellular glutamate activates presynaptic group III mGluRs and inhibits the stimulatory effect of 4-AP on its release, thus preventing the activation of postsynaptic N-methyl-D-aspartate receptors and its deleterious consequences.