Glutamate receptor dynamics in dendritic microdomains

Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity.     

Glutamate receptor desensitization and its role in synaptic transmission

Responses of excitatory amino acid receptors to rapidly applied glutamate were measured in outside-out membrane patches from chick spinal neurons. The peak current varied with glutamate concentration, with a half-maximal response at 510 microM and a Hill coefficient near 2. Currents activated by 1 mM glutamate desensitized and recovered in two phases. The faster time constant was identical to the time constant of decay of synaptic currents, suggesting that glutamatergic synaptic currents are terminated, in part, by receptor desensitization. Steady-state desensitization was evident following application of only 2-3 microM glutamate, concentrations comparable to levels in the extracellular space in the intact brain. Thus, glutamate receptor desensitization can affect synaptic efficacy in two ways: at high concentrations, rapid desensitization of receptors may curtail synaptic currents; at low concentrations, there is a significant reduction in the number of activatable receptors.     

Bidirectional, activity-dependent regulation of glutamate receptors in the adult hippocampus in vivo

Experience-dependent regulation of synaptic strength has been suggested as a physiological mechanism by which memory storage occurs in the brain. Although modifications in postsynaptic glutamate receptor levels have long been hypothesized to be a molecular basis for long-lasting regulation of synaptic strength, direct evidence obtained in the intact brain has been lacking. Here we show that in the adult brain in vivo, synaptic glutamate receptor trafficking is bidirectionally, and reversibly, modified by NMDA receptor-dependent synaptic plasticity and that changes in glutamate receptor protein levels accurately predict changes in synaptic strength. These findings support the idea that memories can be encoded by the precise experience-dependent assignment of glutamate receptors to synapses in the brain.     

AMPA receptor trafficking at excitatory synapses

Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate the postsynaptic depolarization that initiates neuronal firing, whereas NMDA receptors initiate synaptic plasticity. Recent studies have emphasized that distinct mechanisms control synaptic expression of these two receptor classes. Whereas NMDA receptor proteins are relatively fixed, AMPA receptors cycle synaptic membranes on and off. A large family of interacting proteins regulates AMPA receptor turnover at synapses and thereby influences synaptic strength. Furthermore, neuronal activity controls synaptic AMPA receptor trafficking, and this dynamic process plays a key role in the synaptic plasticity that is thought to underlie aspects of learning and memory.     

The regulation of glutamate receptor trafficking and function by TARPs and other transmembrane auxiliary subunits

At excitatory synapses in the brain, glutamate released from nerve terminals binds to glutamate receptors to mediate signaling between neurons. Glutamate receptors expressed in heterologous cells show ion channel activity. Recently, native glutamate receptors were shown to contain auxiliary subunits that modulate the trafficking and/or channel properties. The AMPA receptor (AMPAR) can contain TARP and CNIHs as the auxiliary subunits, whereas kainate receptor (KAR) can contain the Neto auxiliary subunit. Each of these auxiliary subunits uniquely modulates the glutamate receptors, and determines properties of native glutamate receptors. A thorough elucidation of the properties of native glutamate receptor complexes is indispensable for the understanding of the molecular machinery that regulates glutamate receptors and excitatory synaptic transmission in the brain.     

Synaptic strength regulated by palmitate cycling on PSD-95

Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. Here, we identify palmitate cycling on PSD-95 at the synapse and find that palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. We also find that rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity.     

Effect of ambient extracellular glutamate on <Emphasis Type="Italic">Drosophila</Emphasis> glutamate receptor trafficking and function

Measurements suggest that the hemolymph glutamate concentrations in Drosophila are relatively high. This raises the possibility that extracellular glutamate could be an important regulator of glutamatergic transmission in vivo. Using voltage clamp electrophysiology, we found that synaptic currents in D. melanogaster larval neuromuscular junctions are reduced by extracellular glutamate (EC50: ~0.4 mM), such that only 10–30% of receptors were functionally available in 1 mM extracellular glutamate. The kinetics of synaptic currents were also slowed in a dose-dependent fashion (EC50: ~1 mM), consistent with the idea that extracellular glutamate preferentially removes the fastest-desensitizing receptors from the functional pool. Prolonged exposure (several hours) to extracellular glutamate also triggers loss of glutamate receptor immunoreactivity from neuromuscular junctions. To determine whether this receptor loss requires that glutamate bind directly to the lost receptors, we examined glutamate-dependent loss of receptor immunoreactivity in larvae with glutamate receptor ligand binding mutations. Our results suggest that glutamate-dependent receptor loss requires binding of glutamate directly to the lost receptors. To determine whether lost receptor protein is degraded or merely redistributed, we used immunoblots. Results suggest that glutamate receptor protein is redistributed, but not degraded, after prolonged exposure to high extracellular glutamate. K. Chen and H. Augustin contributed equally to this work.     

Interaction between ephrins/Eph receptors and excitatory amino acid receptors: possible relevance in the regulation of synaptic plasticity and in the pathophysiology of neuronal degeneration

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.     

Activation of presynaptic ionotropic glutamate receptors stimulates GABA release from hippocampal and cortical nerve terminals in rats

One of the pathways implicated in a fine-tuning control of neurosecretory process is the activation of presynaptic receptors. The present study was focused on the role of presynaptic glutamate receptor activation in the regulation of inhibitory synaptic transmission in the rat hippocampus and cortex. We aimed to clarify what types of ionotropic glutamate receptors are involved in the modulation of GABA secretion, and what mechanism underlies this modulation. We have revealed that specific agonists of kainate and NMDA receptors, kainate and NMDA, like glutamate, induced the release of [3H]GABA from hippocampal and cortical nerve terminals suggesting the involvement of both types in the regulation of GABAergic transmission. Our results indicate preferential involvement of vesicular, but not cytosolic, pool in response to glutamate receptor activation. This is based on the finding that NO-711 (a specific inhibitor of plasma membrane GABA transporters), fails to attenuate [3H]GABA release. We have concluded that presynaptic glutamate receptor-induced modulation of the strength of synaptic response is due to increasing the release probability of synaptic vesicles.     

Posttranslational regulation of AMPA receptor trafficking and function

In the mammalian central nervous system, the majority of fast excitatory synaptic transmission is mediated by glutamate acting on AMPA-type ionotropic glutamate receptors. The abundance of AMPA receptors at the synapse can be modulated through receptor trafficking, which dynamically regulates many fundamental brain functions, including learning and memory. Reversible posttranslational modifications, including phosphorylation, palmitoylation and ubiquitination of AMPA receptor subunits are important regulatory mechanisms for controlling synaptic AMPA receptor expression and function. In this review, we highlight recent advances in the study of AMPA receptor posttranslational modifications and discuss how these modifications regulate AMPA receptor trafficking and function at synapses.     

Excitatory synaptic currents in Purkinje cells

The N-methyl-D-aspartate (NMDA) and non-NMDA classes of glutamate receptor combine in many regions of the central nervous system to form a dual-component excitatory postsynaptic current. Non-NMDA receptors mediate synaptic transmission at the resting potential, whereas NMDA receptors contribute during periods of postsynaptic depolarization and play a role in the generation of long-term synaptic potentiation. To investigate the receptor types underlying excitatory synaptic transmission in the cerebellum, we have recorded excitatory postsynaptic currents (EPSCS), by using whole-cell techniques, from Purkinje cells in adult rat cerebellar slices. Stimulation in the white matter or granule-cell layer resulted in an all-or-none synaptic current as a result of climbing-fibre activation. Stimulation in the molecular layer caused a graded synaptic current, as expected for activation of parallel fibres. When the parallel fibres were stimulated twice at an interval of 40 ms, the second EPSC was facilitated; similar paired-pulse stimulation of the climbing fibre resulted in a depression of the second EPSC. Both parallel-fibre and climbing-fibre responses exhibited linear current-voltage relations. At a holding potential of -40 mV or in the nominal absence of Mg2+ these synaptic responses were unaffected by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), but were blocked by the non-NMDA receptor antagonist 6-cyano-2,3-dihydro-7-nitroquinoxalinedione (CNQX). NMDA applied to the bath failed to evoke an inward current, whereas aspartate or glutamate induced a substantial current; this current was, however, largely reduced by CNQX, indicating that non-NMDA receptors mediate this response. These results indicate that both types of excitatory input to adult Purkinje cells are mediated exclusively by glutamate receptors of the non-NMDA type, and that these cells entirely lack NMDA receptors.     

The physiological role of kainate receptors in the amygdala

The kainate subtype of glutamate receptors has received considerable attention in recent years, and a wealth of knowledge has been obtained regarding the function of these receptors. Kainate receptors have been shown to mediate synaptic transmission in some brain regions, modulate presynaptic release of glutamate and gamma-aminobutyric acid (GABA), and mediate synaptic plasticity or the development of seizure activity. This article focuses on the function of kainate receptors in the amygdala, a brain region that plays a central role in emotional behavior and certain psychiatric illnesses. Evidence is reviewed indicating that postsynaptic kainate receptors containing the glutamate receptor 5 kainate receptor (GLUk5) subunit are present on interneurons and pyramidal cells in the basolateral amygdala and mediate a component of the synaptic responses of these neurons to glutamatergic input. In addition, GLUk5-containing kainate receptors are present on presynaptic terminals of GABAergic neurons, where they modulate the release of GABA in an agonist concentration-dependent, bidirectional manner. GLUk5-containing kainate receptors also mediate a longlasting synaptic facilitation induced by low-frequency stimulation in the external capsule to the basolateral nucleus pathway, and they appear to be partly responsible for the susceptibility of the amygdala to epileptogenesis. Taken together, these findings have suggested a prominent role of GLUk5-containing kainate receptors in the regulation of neuronal excitability in the amygdala.     

Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD-95

Both tyrosine phosphorylation and calpain-mediated truncation of ionotropic glutamate receptors are important mechanisms for synaptic plasticity. Previous work from our laboratory has shown that calpain activation results in truncation of the C-terminal domains of several glutamate receptor subunits. To test whether and how tyrosine phosphorylation of glutamate ionotropic receptor subunits modulates calpain susceptibility, synaptic membranes were phosphorylated by Fyn or Src, two members of the Src family tyrosine kinases. Tyrosine phosphorylation of synaptic membranes by Src significantly reduced calpain-mediated truncation of both NR2A and NR2B subunits of NMDA receptors, but not of GluR1 subunits of AMPA receptors. In contrast, phosphorylation with Fyn significantly protected calpain-mediated truncation of GluR1 subunits of AMPA receptors, but enhanced calpain-mediated truncation of NR2A subunits of NMDA receptors. Similar results were observed with NR2A and NR2B C-terminal domain fusion proteins phosphorylated by Fyn or Src before incubation with calpain and calcium. In addition, phosphorylation of NR2A and NR2B C-terminal fusion proteins by Fyn or Src enhanced their binding to spectrin and PSD-95. Thus, tyrosine phosphorylation impairs or facilitates calpain-mediated truncation of glutamate receptor subunits, depending on which tyrosine kinase is activated. Such mechanisms could serve to regulate receptor integrity and location, in addition to modulating channel properties.     

Glial glutamate receptors: likely actors in brain signaling.

It has become clear that the neurotransmitter glutamate does not confine its excitatory effects to central nervous system neurons but interacts also with glial cells. Neurons and glia share the same types of ionotropic and metabotropic glutamate receptors except for the N-methyl-D-aspartate receptor, which is not found on glia. Applied on cultured glial cells, glutamate regulates the opening of receptor channels, activates second messengers, and causes the release of neuroactive compounds. Although glutamate and glutamate receptors confer on cultured glia the ability to receive and emit signals, it remains to be established whether glial signaling takes place in vivo. The chick Bergmann glial cells provide a unique experimental system with which to test the contribution of glial glutamate receptors to neuronal electrical activity. These cells are the exclusive carriers in the cerebellum of functional kainate receptors. The synaptic location of these receptors, their ion channel properties, and their regulation by phosphorylation reactions suggest that glial kainate receptors play a role in regulating synaptic efficacy and plasticity. If proved, this concept may require a modification of the anatomical and functional definition of a synapse to include a glial component as well.     

Dynamic Regulation of N-Methyl-d-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors by Posttranslational Modifications

Many molecular mechanisms underlie the changes in synaptic glutamate receptor content that are required by neuronal networks to generate cellular correlates of learning and memory. During the last decade, posttranslational modifications have emerged as critical regulators of synaptic transmission and plasticity. Notably, phosphorylation, ubiquitination, and palmitoylation control the stability, trafficking, and synaptic expression of glutamate receptors in the central nervous system. In the current review, we will summarize some of the progress made by the neuroscience community regarding our understanding of phosphorylation, ubiquitination, and palmitoylation of the NMDA and AMPA subtypes of glutamate receptors.     

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

Conditioned reflex-related plasticity in the neocortex

The spike activity of neurons of the sensorimotor cortex was analyzed in cats before, during, and after iontophoretic application of substances influencing synaptic transmission. It was shown that, in addition to glutamate ionotropic receptors, glutamate metabotropic receptors, as well as adrenergic and dopaminergic receptor systems, are involved in plastic rearrangements of synaptic connections among neocortical neurons. The pattern of aftereffects allows us to suppose that potentiation of synaptic events during conditioned reflex-related learning can be maintained for 10 to 20 min, an interval sufficient for consolidation of a memory track.     

Mykhailo F. Shuba

The spike activity of neurons of the sensorimotor cortex was analyzed in cats before, during, and after iontophoretic application of substances influencing synaptic transmission. It was shown that, in addition to glutamate ionotropic receptors, glutamate metabotropic receptors, as well as adrenergic and dopaminergic receptor systems, are involved in plastic rearrangements of synaptic connections among neocortical neurons. The pattern of aftereffects allows us to suppose that potentiation of synaptic events during conditioned reflex-related learning can be maintained for 10 to 20 min, an interval sufficient for consolidation of a memory track.     

Spillover of glutamate onto synaptic AMPA receptors enhances fast transmission at a cerebellar synapse

Diffusion of glutamate from the synaptic cleft can activate high-affinity receptors, but is not thought to contribute to fast AMPA receptor-mediated transmission. Here, we show that single AMPA receptor EPSCs at the cerebellar mossy fiber-granule cell connection are mediated by both direct release of glutamate and rapid diffusion of glutamate from neighboring synapses. Immunogold localization revealed that AMPA receptors are located exclusively in postsynaptic densities, indicating that spillover of glutamate occurs between synaptic contacts. Spillover currents contributed half the synaptic charge and exhibited little trial-to-trial variability. We propose that spillover of glutamate improves transmission efficacy by both increasing the amplitude and duration of the EPSP and reducing fluctuations arising from the probabilistic nature of transmitter release.     

Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane

Intracellular membrane trafficking of glutamate receptors at excitatory synapses is critical for synaptic function. However, little is known about the specialized trafficking events occurring at the postsynaptic membrane. We have found that two components of the exocyst complex, Sec8 and Exo70, separately control synaptic targeting and insertion of AMPA-type glutamate receptors. Sec8 controls the directional movement of receptors towards synapses through PDZ-dependent interactions. In contrast, Exo70 mediates receptor insertion at the postsynaptic membrane, but it does not participate in receptor targeting. Thus, interference with Exo70 function accumulates AMPA receptors inside the spine, forming a complex physically associated, but not yet fused with the postsynaptic membrane. Electron microscopic analysis of these complexes indicates that Exo70 mediates AMPA receptor insertion directly within the postsynaptic density, rather than at extrasynaptic membranes. Therefore, we propose a molecular and anatomical model that dissects AMPA receptor sorting and synaptic delivery within the spine, and uncovers new functions of the exocyst at the postsynaptic membrane.