Synaptic AMPA-type Glutamate Receptors Research Articles

Mechanisms underlying contextual fear learning

The hippocampus plays a central role in learning and memory. Although synaptic delivery of AMPA-type glutamate receptors (AMPARs) contributes to experience-dependent synaptic strengthening, its role in hippocampus-dependent learning remains elusive. In a recent study,we found that the inhibitory avoidance (IA) task, a hippocampus-dependent contextual fear learning paradigm, drives GluR1-containing AMPARs into CA3-CA1 synapses of the dorsal hippocampus.We expressed mutated membrane-proxymal region (14 amino acids) of the GluR1-cytoplasmic tail (serines mutated to phospho-mimicking aspartates:MPR-DD) in the dorsal hippocampus to block the synaptic delivery of endogenous AMPARs. Learning-driven synaptic AMPARs delivery in CA1 neurons was prevented by the expression of MPR-DD. Bilateral expression of MPR-DD in CA1 region of the dorsal hippocampusattenuated IA learning, indicating that synaptic GluR1 trafficking in the hippocampus is required for encoding contextual fear memories. Furthermore, fraction of CA1 neurons with synaptic strengthening positively correlated with the performance in the IA fear memory task. Thus, the robustness of a contextual fear memory may depend on thenumber of hipppocampal neurons that are involved in the encoding of a memory trace.

MGluR and NMDAR activation internalize distinct populations of AMPARs

Activation of metabotropic- (mGluRs) or NMDA-type glutamate receptors (NMDARs) each can induce long-term depression (LTD) of synaptic transmission in CA1 hippocampal neurons. These two forms of LTD are triggered by diverse signaling pathways yet both are expressed by the internalization of AMPA-type glutamate receptors (AMPARs). An unanswered question remains as to whether the convergence of the mGluR and NMDAR signaling pathways on AMPAR endocytosis renders these two forms of plasticity functionally equivalent, with both pathways inducing endocytosis of the same population of synaptic AMPARs. We now report evidence that these pathways couple to the endocytosis of distinct populations of AMPARs defined by their mobility in the membrane surface. NMDAR activation enhances removal of surface AMPARs that rapidly cycle into and out of the membrane surface, while activation of mGluRs with DHPG results in the internalization of a non-mobile population of AMPARs. Glutamate Receptor Interacting Proteins 1 and 2 (GRIP1/2) play a key role in defining the non-cycling receptor population. GRIP1/2 knockdown with siRNA increases the proportion of rapidly cycling surface AMPARs and inhibits mGluR- but not NMDAR-mediated AMPAR internalization. Additionally, we find that mGluR activation dissociates surface AMPARs from GRIP1/2 while stimulation of NMDARs elicits the loss of membrane receptors not bound to GRIP1/2. We propose that these two receptor pathways can drive the endocytosis of distinct populations of AMPARs: NMDARs activation induces the endocytosis of rapidly cycling surface AMPARs not directly associated with GRIP1/2 while mGluR activation induces the endocytosis of non-cycling GRIP-bound surface AMPARs.

Contextual learning requires synaptic AMPA receptor delivery in the hippocampus

The hippocampus plays a central role in learning and memory. Although synaptic delivery of AMPA-type glutamate receptors (AMPARs) contributes to experience-dependent synaptic strengthening, its role in hippocampus-dependent learning remains elusive. By combining viral-mediated in vivo gene delivery with in vitro patch-clamp recordings, we found that the inhibitory avoidance task, a hippocampus-dependent contextual fear-learning paradigm, delivered GluR1-containing AMPARs into CA3-CA1 synapses of the dorsal hippocampus. To block the synaptic delivery of endogenous AMPARs, we expressed a fragment of the GluR1-cytoplasmic tail (the 14-aa GluR1 membrane-proximal region with two serines mutated to phospho-mimicking aspartates: MPR-DD). MPR-DD prevented learning-driven synaptic AMPAR delivery in CA1 neurons. Bilateral expression of MPR-DD in the CA1 region of the rat impaired inhibitory avoidance learning, indicating that synaptic GluR1 trafficking in the CA1 region of the hippocampus is required for encoding contextual fear memories. The fraction of CA1 neurons that underwent synaptic strengthening positively correlated with the performance in the inhibitory avoidance fear memory task. These data suggest that the robustness of a contextual memory depends on the number of hippocampal neurons that participate in the encoding of a memory trace.

Serotonin as a mediator of cross-modal cortical reorganization.

When one type of sensory system is disrupted, other intact remaining sensory function can be improved. Although this form of plasticity, cross-modal plasticity, is widely known, the molecular and cellular mechanisms underlying it are poorly understood. In a recent study, we demonstrated that visual deprivation increases extracellular serotonin in the juvenile rat barrel cortex and resulted in facilitation of synaptic delivery of AMPA-type glutamate receptors (AMPARs) at layer4-2/3 synapses in the barrel cortex via the activation of serotonin 5HT2A/2C receptors and ERK. This caused sharpening of functional whisker-barrel map at layer2/3 of the barrel cortex. Thus, sensory dysfunction of one modality leads to improvement of remaining modalities by the refinement of cortical organization through serotonin signaling-mediated facilitation of synaptic AMPARs delivery.

Serotonin as a mediator of cross-modal cortical reorganization

When one type of sensory system is disrupted, other intact remaining sensory function can be improved. Although this form of plasticity, cross-modal plasticity, is widely known, the molecular and cellular mechanisms underlying it are poorly understood. In a recent study, we demonstrated that visual deprivation increases extracellular serotonin in the juvenile rat barrel cortex and resulted in facilitation of synaptic delivery of AMPA-type glutamate receptors (AMPARs) at layer4-2/3 synapses in the barrel cortex via the activation of serotonin 5HT2A/2C receptors and ERK. This caused sharpening of functional whisker-barrel map at layer2/3 of the barrel cortex. Thus, sensory dysfunction of one modality leads to improvement of remaining modalities by the refinement of cortical organization through serotonin signaling-mediated facilitation of synaptic AMPARs delivery.

Acute knockdown of AMPA receptors reveals a trans-synaptic signal for presynaptic maturation

Newly formed glutamatergic synapses often lack postsynaptic AMPA-type glutamate receptors (AMPARs). Aside from 'unsilencing' the postsynaptic site, however, the significance of postsynaptic AMPAR insertion during synapse maturation remains unclear. To investigate the role of AMPAR in synapse maturation, we used RNA interference (RNAi) to knockdown AMPARs in cultured hippocampal neurons. Surprisingly, loss of postsynaptic AMPARs increased the occurrence of presynaptically inactive synapses without changing the release probability of the remaining active synapses. Additionally, heterologous synapses formed between axons and AMPAR-expressing HEK cells develop significantly fewer inactive presynaptic terminals. The extracellular domain of the AMPAR subunit GluA2 was sufficient to reproduce this effect at heterologous synapses. Indeed, the retrograde signalling by AMPARs is independent of their channel function as RNAi-resistant AMPARs restore synaptic transmission in neurons lacking AMPARs despite chronic receptor antagonist treatment. Our findings suggest that postsynaptic AMPARs perform an organizational function at synapses that exceeds their standard role as ionotropic receptors by conveying a retrograde trans-synaptic signal that increases the transmission efficacy at a synapse.

CaMKII Autonomy Is Substrate-dependent and Further Stimulated by Ca2+/Calmodulin

A hallmark feature of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca(2+)-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active ( approximately 70-100%), implicating that it is no longer regulated and that its dramatically increased Ca(2+)/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15-25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca(2+)/CaM, both in vitro and within cells. Altered K(m) and V(max) made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca(2+) signals, but prevents complete uncoupling from subsequent cellular stimulation.

Two populations of neurokinin 1 receptor-expressing projection neurons in lamina I of the rat spinal cord that differ in AMPA receptor subunit composition and density of excitatory synaptic input

Lamina I of the spinal cord contains many projection neurons that express the neurokinin 1 receptor (NK1r). It has been reported that these cells can undergo long-term potentiation (LTP), which may result from insertion of AMPA-type glutamate receptors (AMPArs) containing GluA1 or GluA4 subunits. We therefore investigated synaptic AMPAr expression on these cells with immunocytochemistry following antigen-retrieval. We also examined their density of glutamatergic input (by analysing AMPAr synaptic puncta and contacts from glutamatergic boutons), and phosphorylation of extracellular signal-regulated kinases (pERKs) following noxious stimulation. Our results indicate that there are two populations of NK1r-expressing projection neurons: large GluA4+/GluA1− cells with a high density of glutamatergic input and small GluA1+/GluA4− cells with a much lower input density. Results from pERK experiments suggested that the two groups may not differ in the types of noxious stimulus that activate them. Glutamatergic synapses on distal dendrites of the large cells were significantly longer than those on proximal dendrites, which presumably compensates for the greater attenuation of distally-generated excitatory postsynaptic currents (EPSCs). Both types of cell received contacts from peptidergic primary afferents, however, on the large cells these appeared to constitute over half of the glutamatergic synapses, and were often associated with elongated AMPAr puncta. This suggests that these afferents, which probably contain substance P, provide a powerful, secure synaptic input to large NK1r-expressing projection neurons. These results demonstrate the importance of GluA4-containing AMPArs in nociceptive transmission and raise the possibility that different forms of LTP in lamina I projection neurons may be related to differential expression of GluA1/GluA4.

Angelman Syndrome: Finding the Lost Arc

Angelman syndrome is a neurodevelopmental disorder caused by mutations in the maternally inherited UBE3A gene, which encodes a ubiquitin ligase. Greer et al. (2010) now identify a UBE3A substrate called Arc that promotes endocytosis of neuronal AMPA receptors, providing insight into synaptic defects that may underlie the cognitive deficits in Angelman syndrome.

Synaptic SAP97 Isoforms Regulate AMPA Receptor Dynamics and Access to Presynaptic Glutamate

The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasma membrane. Whether SAP97 also plays a role in scaffolding GluR1 at the postsynaptic membrane is controversial, attributable to its expression as a collection of alternatively spliced isoforms with ill-defined spatial and temporal distributions. In the present study, we have used live imaging and electrophysiology to demonstrate that two postsynaptic, N-terminal isoforms of SAP97 directly modulate the levels, dynamics, and function of synaptic GluR1-containing AMPARs. Specifically, the unique N-terminal domains confer distinct subsynaptic localizations onto SAP97, targeting the palmitoylated alpha-isoform to the postsynaptic density (PSD) and the L27 domain-containing beta-isoform primarily to non-PSD, perisynaptic regions. Consequently, alpha- and betaSAP97 differentially influence the subsynaptic localization and dynamics of AMPARs by creating binding sites for GluR1-containing receptors within their respective subdomains. These results indicate that N-terminal splicing of SAP97 can control synaptic strength by regulating the distribution of AMPARs and, hence, their responsiveness to presynaptically released glutamate.

TNFalpha-induced AMPA-receptor trafficking in CNS neurons; relevance to excitotoxicity?

Injury and disease in the CNS increases the amount of tumor necrosis factor alpha (TNFalpha) that neurons are exposed to. This cytokine is central to the inflammatory response that occurs after injury and during prolonged CNS disease, and contributes to the process of neuronal cell death. Previous studies have addressed how long-term apoptotic-signaling pathways that are initiated by TNFalpha might influence these processes, but the effects of inflammation on neurons and synaptic function in the timescale of minutes after exposure are largely unexplored. Our published studies examining the effect of TNFalpha on trafficking of AMPA-type glutamate receptors (AMPARs) in hippocampal neurons demonstrate that glial-derived TNFalpha causes a rapid (<15 minute) increase in the number of neuronal, surface-localized, synaptic AMPARs leading to an increase in synaptic strength. This indicates that TNFalpha-signal transduction acts to facilitate increased surface localization of AMPARs from internal postsynaptic stores. Importantly, an excess of surface localized AMPARs might predispose the neuron to glutamate-mediated excitotoxicity and excessive intracellular calcium concentrations, leading to cell death. This suggests a new mechanism for excitotoxic TNFalpha-induced neuronal death that is initiated minutes after neurons are exposed to the products of the inflammatory response. Here we review the importance of AMPAR trafficking in normal neuronal function and how abnormalities that are mediated by glial-derived cytokines such as TNFalpha can be central in causing neuronal disorders. We have further investigated the effects of TNFalpha on different neuronal cell types and present new data from cortical and hippocampal neurons in culture. Finally, we have expanded our investigation of the temporal profile of the action of this cytokine relevant to neuronal damage. We conclude that TNFalpha-mediated effects on AMPAR trafficking are common in diverse neuronal cell types and very rapid in their onset. The abnormal AMPAR trafficking elicited by TNFalpha might present a novel target to aid the development of new neuroprotective drugs.