Activation of extrasynaptic kainate receptors drives hilar mossy cell activity

Mossy cells (MCs) of the dentate gyrus (DG) are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place field encoding, pattern separation and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished in GluK2 KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs, and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-15 containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus.

Dysregulation of Ambient Glutamate and Glutamate Receptors in Epilepsy: An Astrocytic Perspective

Given the important functions that glutamate serves in excitatory neurotransmission, understanding the regulation of glutamate in physiological and pathological states is critical to devising novel therapies to treat epilepsy. Exclusive expression of pyruvate carboxylase and glutamine synthetase in astrocytes positions astrocytes as essential regulators of glutamate in the central nervous system (CNS). Additionally, astrocytes can significantly alter the volume of the extracellular space (ECS) in the CNS due to their expression of the bi-directional water channel, aquaporin-4, which are enriched at perivascular endfeet. Rapid ECS shrinkage has been observed following epileptiform activity and can inherently concentrate ions and neurotransmitters including glutamate. This review highlights our emerging knowledge on the various potential contributions of astrocytes to epilepsy, particularly supporting the notion that astrocytes may be involved in seizure initiation via failure of homeostatic responses that lead to increased ambient glutamate. We also review the mechanisms whereby ambient glutamate can influence neuronal excitability, including via generation of the glutamate receptor subunit GluN2B-mediated slow inward currents, as well as indirectly affect neuronal excitability via actions on metabotropic glutamate receptors that can potentiate GluN2B currents and influence neuronal glutamate release probabilities. Additionally, we discuss evidence for upregulation of System xc-, a cystine/glutamate antiporter expressed on astrocytes, in epileptic tissue and changes in expression patterns of glutamate receptors.

Incomplete removal of extracellular glutamate controls synaptic transmission and integration at a cerebellar synapse

Synapses of glutamatergic mossy fibers onto cerebellar unipolar brush cells (UBCs) generate slow excitatory (ON) or inhibitory (OFF) postsynaptic responses dependent on the complement of glutamate receptors expressed on the UBC's large dendritic brush. Using mouse brain slice recording and computational modeling of synaptic transmission, we found that substantial glutamate is maintained in the UBC synaptic cleft, sufficient to modify spontaneous firing in OFF UBCs and tonically desensitize AMPARs of ON UBCs. The source of this ambient glutamate was spontaneous, spike-independent exocytosis from the mossy fiber terminal, and its level was dependent on activity of glutamate transporters EAAT1-2. Increasing levels of ambient glutamate shifted the polarity of evoked synaptic responses in ON UBCs and altered the phase of responses to in vivo-like synaptic activity. Unlike classical fast synapses, receptors at the UBC synapse are virtually always exposed to a significant level of glutamate, which varies in a graded manner during transmission.

Incomplete removal of extracellular glutamate controls synaptic transmission and integration at a cerebellar synapse

Synapses of glutamatergic mossy fiber onto cerebellar unipolar brush cells (UBCs) generate slow excitatory (ON) or inhibitory (OFF) postsynaptic responses dependent on the complement of glutamate receptors expressed on the UBC’s large dendritic brush. Using brain slice recording and computational modeling of synaptic transmission, we found that substantial glutamate is maintained in the UBC synaptic cleft, sufficient to modify spontaneous firing in OFF UBCs and tonically desensitize AMPARs of ON UBC. The source of ambient glutamate was spontaneous, spike-independent exocytosis from the mossy fiber terminal, and its level was dependent on activity of glutamate transporters EAAT1-2. Changing levels of ambient glutamate shifted the polarity of evoked synaptic responses in ON UBCs and altered the phase of responses to in vivo-like synaptic activity. Unlike classical fast synapses, receptors at the UBC synapse are virtually always exposed to a significant level of glutamate, which varies in a graded manner during transmission.

Permanent dynamic transporter-mediated turnover of glutamate across the plasma membrane of presynaptic nerve terminals: arguments in favor and against

Mechanisms for maintenance of the extracellular level of glutamate in brain tissue and its regulation still remain almost unclear, and criticism of the current paradigm of glutamate transport and homeostasis has recently appeared. The main premise for this study is the existence of a definite and non-negligible concentration of ambient glutamate between the episodes of exocytotic release in our experiments with rat brain nerve terminals (synaptosomes), despite the existence of a very potent Na+-dependent glutamate uptake. Glutamate transporter reversal is considered as the main mechanisms of glutamate release under special conditions of energy deprivation, hypoxia, hypoglycemia, brain trauma, and stroke, underlying an increase in the ambient glutamate concentration and development of excitotoxicity. In the present study, a new vision on transporter-mediated release of glutamate as one of the main mechanisms involved in the maintenance of definite concentration of ambient glutamate under normal energetical status of nerve terminals is forwarded. It has been suggested that glutamate transporters act effectively in outward direction in a non-pathological manner, and this process is thermodynamically synchronized with uptake and provides effective outward glutamate current, thereby establishing and maintaining permanent and dynamic glutamatein/glutamateout gradient and turnover across the plasma membrane. In this context, non-transporter tonic glutamate release by diffusion, spontaneous exocytosis, cystine-glutamate exchanger, and leakage through anion channels can be considered as a permanently added ‘new’ exogenous substrate using two-substrate kinetic model calculations. Permanent glutamate turnover is of value for tonic activation of post/presynaptic glutamate receptors, long-term potentiation, memory formation, etc. Counterarguments against this mechanism are also considered.

Different pools of glutamate receptors mediate sensitivity to ambient glutamate in the cochlear nucleus

Ambient glutamate plays an important role in pathological conditions, such as stroke, but its role during normal activity is not clear. In addition, it is not clear how ambient glutamate acts on glutamate receptors with varying affinities or subcellular localizations. To address this, we studied “endbulb of Held” synapses, which are formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. When ambient glutamate was increased by applying the glutamate reuptake inhibitor TFB-TBOA, BCs depolarized as a result of activation of N-methyl-d-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs). Application of antagonists against NMDARs (in 0 Mg2+) or mGluRs caused hyperpolarization, indicating that these receptors were bound by a tonic source of glutamate. AMPA receptors did not show these effects, consistent with their lower glutamate affinity. We also evaluated the subcellular localization of the receptors activated by ambient glutamate. The mGluRs were not activated by synaptic stimulation and thus appear to be exclusively extrasynaptic. By contrast, NMDARs in both synaptic and extrasynaptic compartments were activated by ambient glutamate, as shown using the use-dependent antagonist MK-801. Levels of ambient glutamate appeared to be regulated in a spike-independent manner, and glia likely play a major role. These low levels of ambient glutamate likely have functional consequences, as even low concentrations of TBOA caused significant increases in BC spiking following synaptic stimulation. These results indicate that normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate.