Number Of AMPA Receptors Research Articles

Assembly of AMPA receptors: mechanisms and regulation.

AMPA receptors (AMPARs) play a critical role in excitatory glutamatergic neurotransmission. The number and subunit composition of AMPARs at synapses determines the dynamics of fast glutamatergic signalling. Functional AMPARs on the cell surface are tetramers. Thus tetrameric assembly of AMPARs represents a promising target for modulating AMPAR-mediated signalling in health and disease. Multiple structural domains within the receptor influence AMPAR assembly. In a proposed model for AMPAR assembly, the amino-terminal domain underlies the formation of a dimer pool. The transmembrane domain facilitates the formation and enhances the stability of the tetramer. The ligand-binding domain influences assembly through a process referred to as 'domain swapping'. We propose that this core AMPAR assembly process could be regulated by neuronal signals and speculate on possible mechanisms for such regulation.

LTD expression is independent of glutamate receptor subtype.

Long-term depression (LTD) is a form of synaptic plasticity that plays a major role in the activity-dependent reshaping of synaptic transmission. LTD is expressed as a decrease in synaptic AMPA receptor number, though the exact mechanism remains controversial. Several lines of evidence have suggested necessary roles for both the GluA1 and GluA2 subunits, and specifically certain interactions with their cytoplasmic tails. However, it is unclear if either GluA1 or GluA2 are absolutely required for LTD. We tested this hypothesis using constitutive knock-outs and single-cell molecular replacement of AMPA receptor subunits in mouse hippocampus. We found that neither GluA1 or GluA2 are required for normal expression of LTD, and indeed a normal decrease in synaptic transmission was observed in cells in which all endogenous AMPA receptors have been replaced by kainate receptors. Thus, LTD does not require removal of specific AMPA receptor subunits, but likely involves a more general modification of the synapse and its ability to anchor a broad range of receptor proteins.

Postsynaptic GABABReceptor Activity Regulates Excitatory Neuronal Architecture and Spatial Memory

Cognitive dysfunction is a common symptom in many neuropsychiatric disorders and directly correlates with poor patient outcomes. The majority of prolonged inhibitory signaling in the brain is mediated via GABAB receptors (GABABRs), but the molecular function of these receptors in cognition is ill defined. To explore the significance of GABABRs in neuronal activity and cognition, we created mice with enhanced postsynaptic GABABR signaling by mutating the serine 783 in receptor R2 subunit (S783A), which decreased GABABR degradation. Enhanced GABABR activity reduced the expression of immediate-early gene-encoded protein Arc/Arg3.1, effectors that are critical for long-lasting memory. Intriguingly, S783A mice exhibited increased numbers of excitatory synapses and surface AMPA receptors, effects that are consistent with decreased Arc/Arg3.1 expression. These deficits in Arc/Arg3.1 and neuronal morphology lead to a deficit in spatial memory consolidation. Collectively our results suggest a novel and unappreciated role for GABABR activity in determining excitatory neuronal architecture and spatial memory via their ability to regulate Arc/Arg3.1.

Moderate AMPA receptor clustering on the nanoscale can efficiently potentiate synaptic current

The prevailing view at present is that postsynaptic expression of the classical NMDA receptor-dependent long-term potentiation relies on an increase in the numbers of local AMPA receptors (AMPARs). This is thought to parallel an expansion of postsynaptic cell specializations, for instance dendritic spine heads, which accommodate synaptic receptor proteins. However, glutamate released into the synaptic cleft can normally activate only a hotspot of low-affinity AMPARs that occur in the vicinity of the release site. How the enlargement of the AMPAR pool is causally related to the potentiated AMPAR current remains therefore poorly understood. To understand possible scenarios of postsynaptic potentiation, here we explore a detailed Monte Carlo model of the typical small excitatory synapse. Simulations suggest that approximately 50% increase in the synaptic AMPAR current could be provided by expanding the existing AMPAR pool at the expense of 100–200% new AMPARs added at the same packing density. Alternatively, reducing the inter-receptor distances by only 30–35% could achieve a similar level of current potentiation without any changes in the receptor numbers. The NMDA receptor current also appears sensitive to the NMDA receptor crowding. Our observations provide a quantitative framework for understanding the ‘resource-efficient’ ways to enact use-dependent changes in the architecture of central synapses.

Balanced Synaptic Impact via Distance-Dependent Synapse Distribution and Complementary Expression of AMPARs and NMDARs in Hippocampal Dendrites

Neuronal computation involves the integration of synaptic inputs that are often distributed over expansive dendritic trees, suggesting the need for compensatory mechanisms that enable spatially disparate synapses to influence neuronal output. In hippocampal CA1 pyramidal neurons, such mechanisms have indeed been reported, which normalize either the ability of distributed synapses to drive action potential initiation in the axon or their ability to drive dendritic spiking locally. Here we report that these mechanisms can coexist, through an elegant combination of distance-dependent regulation of synapse number and synaptic expression of AMPA and NMDA receptors. Together, these complementary gradients allow individual dendrites in both the apical and basal dendritic trees of hippocampal neurons to operate as facile computational subunits capable of supporting both global integration in the soma/axon and local integration in the dendrite.

Distance-Dependent Scaling of AMPARs Is Cell-Autonomous and GluA2 Dependent

The extensive dendritic arbor of a pyramidal cell introduces considerable complexity to the integration of synaptic potentials. Propagation of dendritic potentials is largely passive, in contrast to regenerative axonal potentials that are maintained by voltage-gated sodium channels, leading to a declination in amplitude as dendritic potentials travel toward the soma in a manner that disproportionally affects distal synaptic inputs. To counteract this amplitude filtering, Schaffer collateral synapses onto CA1 pyramidal cells contain a varying number of AMPA receptors (AMPARs) per synapse that increases with distance from the soma, a phenomenon known as distance-dependent scaling. Here, we undertake an investigation into the molecular mechanisms of distance-dependent scaling. Using dendritic recordings from rat pyramidal neurons, we confirm the basic scaling phenomenon and find that it is expressed and can be manipulated cell autonomously. Finally, we show that it depends on the presence of both a reserve pool of AMPARs and the AMPAR subunit GluA2.

Initial state of single spines affects probability of induction of cerebellar long term depression in a stochastic model

Cerebellar long-term depression (LTD) is a type of synaptic plasticity which shows a robust and persistent decrease in the synaptic transmission between parallel fibers (PF) and Purkinje cells (PC), expressed as a reduction in the number of synaptic AMPA receptors. Our lab recently developed a stochastic model of the LTD signaling network, including a PKC-ERK-cPLA2 positive feedback loop and mechanisms of AMPAR trafficking, and tuned the model to replicate calcium uncaging experiments [1]. Probabilistically, signaling activity switches between two discrete stable states (LTD or non-LTD). In single spines, the probability of LTD occurrence is only modulated by the concentration and duration of the signal used to trigger it, and inputs with the same magnitude give rise to one of two responses. The detailed kinetic model of the signaling and trafficking network involved in the induction of cerebellar LTD was developed based on extensive experimental data. The model was solved stochastically and deterministically using STEPS (http://steps.sourceforge.net/), a well validated simulator that implements the Stochastic Simulation Algorithm (SSA)[2]. The reasons for the bistable behavior in single spines is not fully understood. Molecular memory of the compartment, i.e. initial state of molecules before calcium injection, may play a role in altering the probability of a spine to undergo LTD induction or not. We address the possible effects of this initial state on the observed bistable behavior quantitatively. Preliminary analysis of these initial conditions for several simulations lead to their categorization on the basis of LTD probability observed for a particular calcium concentration and pulse duration (for simplicity we named them as High LTD likelihood, Normal LTD likelihood and Low LTD likelihood). We then confirmed these categories for different calcium conditions. The probability of LTD induction has a dependence on calcium pulse duration well-described by the Hill equation. The High and Normal LTD likelihood cases showed similar characteristics, though the Low LTD likelihood category showed different Hill parameters and also didn't saturate at 100 percent. Observations when varying the time before calcium input showed that the High and Normal LTD likelihood states exist only on the order of minutes and dissipate afterwards. In depth understanding of these different initial states may give better insights into the underlying molecular mechanisms causing bistable behavior. This is an important phenomena, as it is also observed in other forms of long-term synaptic plasticity and in many other biological processes.

MiR-132/212 Knockout Mice Reveal Roles for These miRNAs in Regulating Cortical Synaptic Transmission and Plasticity

miR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNβ induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP) induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity.

Biophysical mechanisms regulating AMPA receptor accumulation at synapses

Controlling the number of AMPA receptors at synapses is fundamental for fast synaptic transmission as well as for long term adaptations in synaptic strength. In this review, we examine the biophysical mechanisms implicated in regulating AMPAR levels at the cell surface and at synapses. We first describe the structure and function of AMPARs, as well as their interactions with various proteins regulating their traffic and function. Second we review the vesicular trafficking mechanism involving exocytosis and endocytosis, by which AMPARs reach the cell surface and are internalized, respectively. Third, we examine the properties of lateral diffusion of AMPARs and their trapping at post-synaptic densities. Finally, we discuss how these two parallel mechanisms are integrated in time and space to control changes in synaptic AMPAR levels in response to plasticity protocols. This review highlights the important role of the extra-synaptic AMPAR pool, which makes an obligatory link between vesicular trafficking and trapping or release at synapses.

SUMOylation, Arc and the regulation homeostatic synaptic scaling

Neurons compensate for changes in network activity by altering the sensitivity of transmission across collections of synapses by up- or downregulating the number of synaptic AMPA receptors. We recently reported that, in parallel to increasing AMPA receptor surface expression, suppression of network activity with TTX increases protein SUMOylation by decreasing levels of the deSUMOylating enzyme SENP1. SUMOylation of the immediate early gene product Arc is required for synaptic scaling. These results reveal a previously unsuspected role for protein SUMOylation in activity-dependent AMPA receptor trafficking and the regulation of neuronal network activity, processes which play important roles in neurodegenerative disease.

The Balance between Receptor Recycling and Trafficking toward Lysosomes Determines Synaptic Strength during Long-Term Depression

The strength of excitatory synaptic transmission depends partly on the number of AMPA receptors (AMPARs) at the postsynaptic surface and, thus, can be modulated by membrane trafficking events. These processes are critical for some forms of synaptic plasticity, such as long-term potentiation and long-term depression (LTD). In the case of LTD, AMPARs are internalized and dephosphorylated in response to NMDA receptor activation. However, the fate of the internalized receptors upon LTD induction and its relevance for synaptic function is still a matter of debate. Here we examined the functional contribution of receptor recycling versus degradation for LTD in rat hippocampal slices, and their correlation with receptor dephosphorylation. We observed that GluA1 undergoes sequential dephosphorylation and degradation in lysosomes after LTD induction. However, this degradation does not have functional consequences for the regulation of synaptic strength, and therefore, for the expression of LTD. In contrast, the partition of internalized AMPARs between Rab7-dependent trafficking (toward lysosomes) or Rab11-dependent endosomes (recycling back toward synapses) is the key factor determining the extent of synaptic depression upon LTD induction. This sorting decision is related to the phosphorylation status of GluA1 Ser845, the dephosphorylated receptors being those preferentially targeted for lysosomal degradation. Altogether, these new data contribute to clarify the fate of AMPARs during LTD and emphasize the importance of membrane sorting decisions to determine the outcome of synaptic plasticity.

PICK1 Mediates Transient Synaptic Expression of GluA2-Lacking AMPA Receptors during Glycine-Induced AMPA Receptor Trafficking

The number and subunit composition of postsynaptic AMPA receptors (AMPARs) is a key determinant of synaptic transmission. The vast majority of AMPARs contain GluA2 subunit, which renders the channel impermeable to calcium. However, a small proportion are GluA2 lacking and therefore calcium permeable (CP-AMPARs). It has been proposed recently that long-term potentiation (LTP) involves not only an increase in the total number of AMPARs at the synapse but also a transient switch to CP-AMPARs in the first few minutes after LTP induction. The molecular mechanisms that underlie this switch to CP-AMPARs and the subsequent switch back to calcium-impermeable AMPARs are unknown. Here, we show that endogenous GluA1 is rapidly inserted at the synaptic plasma membrane of rat hippocampal neurons immediately after stimulation with elevated glycine, a treatment known to induce LTP. In contrast, GluA2 is restricted from trafficking to the cell surface by a glycine-induced increase in PICK1-GluA2 binding on endosomal compartments. Between 5 and 20 min after stimulus, activation of CP-AMPARs triggers a release of GluA2 from PICK1, allowing GluA2-containing AMPARs to traffic to the synaptic plasma membrane. These results define a PICK1-dependent mechanism that underlies transient alterations in the subunit composition and calcium permeability of synaptic AMPARs that is important during the early phase after stimulation with glycine and therefore is likely to be important during the expression of LTP.

Light-Induced Plasticity of Synaptic AMPA Receptor Composition in Retinal Ganglion Cells

Light-evoked responses of all three major classes of retinal ganglion cells (RGCs) are mediated by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Although synaptic activity at RGC synapses is highly dynamic, synaptic plasticity has not been observed in adult RGCs. Here, using patch-clamp recordings in dark-adapted mouse retina, we report a retina-specific form of AMPAR plasticity. Both chemical and light activation of NMDARs caused the selective endocytosis of GluA2-containing, Ca(2+)-impermeable AMPARs on RGCs and replacement with GluA2-lacking, Ca(2+)-permeable AMPARs. The plasticity was expressed in ON but not OFF RGCs and was restricted solely to the ON responses in ON-OFF RGCs. Finally, the plasticity resulted in a shift in the light responsiveness of ON RGCs. Thus, physiologically relevant light stimuli can induce a change in synaptic receptor composition of ON RGCs, providing a mechanism by which the sensitivity of RGC responses may be modified under scotopic conditions.

A Stochastic Signaling Network Mediates the Probabilistic Induction of Cerebellar Long-Term Depression

Many cellular processes involve a small number of molecules and undergo stochastic fluctuations in their levels of activity. Cerebellar long-term depression (LTD) is a form of synaptic plasticity expressed as a reduction in the number of synaptic AMPA receptors (AMPARs) in Purkinje cells. We developed a stochastic model of the LTD signaling network, including a PKC-ERK-cPLA(2) positive feedback loop and mechanisms of AMPAR trafficking, and tuned the model to replicate calcium uncaging experiments. The signaling network activity in single synapses switches between two discrete stable states (LTD and non-LTD) in a probabilistic manner. The stochasticity of the signaling network causes threshold dithering and allows at the macroscopic level for many different and stable mean magnitudes of depression. The probability of LTD occurrence in a single spine is only modulated by the concentration and duration of the signal used to trigger it, and inputs with the same magnitude can give rise to two different responses; there is no threshold for the input signal. The stochasticity is intrinsic to the signaling network and not mostly dependent on noise in the calcium input signal, as has been suggested previously. The activities of the ultrasensitive ERK and of cPLA(2) undergo strong stochastic fluctuations. Conversely, PKC, which acts as a noise filter, is more constantly activated. Systematic variation of the biochemical population size demonstrates that threshold dithering and the absence of spontaneous LTD depend critically on the number of molecules in a spine, indicating constraints on spine size in Purkinje cells.

Cortical development of AMPA receptor trafficking proteins

AMPA-receptor trafficking plays a central role in excitatory plasticity, especially during development. Changes in the number of AMPA receptors and time spent at the synaptic surface are important factors of plasticity that directly affect long-term potentiation (LTP), long-term depression (LTD), synaptic scaling, and the excitatory-inhibitory (E/I) balance in the developing cortex. Experience-dependent changes in synaptic strength in visual cortex (V1) use a molecularly distinct AMPA trafficking pathway that includes the GluA2 subunit. We studied developmental changes in AMPA receptor trafficking proteins by quantifying expression of GluA2, pGluA2 (GluA2serine880), GRIP1, and PICK1 in rat visual and frontal cortex. We used Western Blot analysis of synaptoneurosome preparations of rat visual and frontal cortex from animals ranging in age from P0 to P105. GluA2 and pGluA2 followed different developmental trajectories in visual and frontal cortex, with a brief period of over expression in frontal cortex. The over expression of GluA2 and pGluA2 in immature frontal cortex raises the possibility that there may be a period of GluA2-dependent vulnerability in frontal cortex that is not found in V1. In contrast, GRIP1 and PICK1 had the same developmental trajectories and were expressed very early in development of both cortical areas. This suggests that the AMPA-interacting proteins are available to begin trafficking receptors as soon as GluA2-containing receptors are expressed. Finally, we used all four proteins to analyze the surface-to-internalization balance and found that this balance was roughly equal across both cortical regions, and throughout development. Our finding of an exquisite surface-to-internalization balance highlights that these AMPA receptor trafficking proteins function as a tightly controlled system in the developing cortex.

Memory maintenance by PKMζ — an evolutionary perspective

Long-term memory is believed to be maintained by persistent modifications of synaptic transmission within the neural circuits that mediate behavior. Thus, long-term potentiation (LTP) is widely studied as a potential physiological basis for the persistent enhancement of synaptic strength that might sustain memory. Whereas the molecular mechanisms that initially induce LTP have been extensively characterized, the mechanisms that persistently maintain the potentiation have not. Recently, however, a candidate molecular mechanism linking the maintenance of LTP and the storage of long-term memory has been identified. The persistent activity of the autonomously active, atypical protein kinase C (aPKC) isoform, PKMζ, is both necessary and sufficient for maintaining LTP. Furthermore, blocking PKMζ activity by pharmacological or dominant negative inhibitors disrupts previously stored long-term memories in a variety of neural circuits, including spatial and trace memories in the hippocampus, aversive memories in the basolateral amygdala, appetitive memories in the nucleus accumbens, habit memory in the dorsal lateral striatum, and elementary associations, extinction, and skilled sensorimotor memories in the neocortex. During LTP and memory formation, PKMζ is synthesized de novo as a constitutively active kinase. This molecular mechanism for memory storage is evolutionarily conserved. PKMζ formation through new protein synthesis likely originated in early vertebrates ~500 million years ago during the Cambrian period. Other mechanisms for forming persistently active PKM from aPKC are found in invertebrates, and inhibiting this atypical PKM disrupts long-term memory in the invertebrate model systems Drosophila melanogaster and Aplysia californica. Conversely, overexpressing PKMζ enhances memory in flies and rodents. PKMζ persistently enhances synaptic strength by maintaining increased numbers of AMPA receptors at postsynaptic sites, a mechanism that might have evolved from the general function of aPKC in trafficking membrane proteins to the apical compartment of polarized cells. This mechanism of memory may have had adaptive advantages because it is both stable and reversible, as demonstrated by the downregulation of experience-dependent, long-term increases in PKMζ after extinction and reconsolidation blockade that attenuate learned behavior. Thus, PKMζ, the “working end” of LTP, is a component of an evolutionarily conserved molecular mechanism for the persistent, yet flexible storage of long-term memory.

Trafficking of AMPA Receptors at Plasma Membranes of Hippocampal Neurons

The number of AMPA receptors at synapses depends on receptor cycling. Because receptors diffuse rapidly in plasma membranes, their exocytosis and endocytosis need not occur near synapses. Here, pre-embedding immunogold electron microscopy is applied to dissociated rat hippocampal cultures to provide sensitive, high-resolution snapshots of the distribution of surface AMPA receptors in spines, dendrites, and cell bodies that will be informative about trafficking of AMPA receptors. The density of the label for GluR2 varies, but is consistent throughout cell body and dendrites in each individual neuron, except at postsynaptic densities (PSDs), where it is typically higher. Glutamate receptor 2 (GluR2) labels at PSDs significantly increase after synaptic activation by glycine treatment and increase further upon depolarization by high K(+). Islands of densely packed labels have consistent size and density but vary in frequency under different experimental conditions. These patches of label, which occur on plasma membranes of cell bodies and dendrites but not near PSDs, are taken to be the aftermath of exocytosis of AMPA receptors. A subpopulation of clathrin-coated pits in cell bodies and dendrites label for GluR2, and the number and amount of label in individual pits increase after NMDA treatment. Coated pits near synapses typically lack GluR2 label under basal conditions, but ∼40% of peri-PSD pits label for GluR2 after NMDA treatment. Thus, exocytosis and endocytosis of AMPA receptors occur mainly at extrasynaptic locations on cell bodies and dendrites. Receptors are not preferentially exocytosed near PSDs, but may be removed via endocytosis at peri-PSD locations after activation of NMDA receptors.

Glutamate Receptor δ2 Is Essential for Input Pathway-Dependent Regulation of Synaptic AMPAR Contents in Cerebellar Purkinje Cells

The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor δ2 (GluRδ2 or GluD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRδ2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluRδ2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluRδ2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluRδ2. These results suggest that GluRδ2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.

Alleviating Neuropathic Pain Hypersensitivity by Inhibiting PKMζ in the Anterior Cingulate Cortex

Synaptic plasticity is a key mechanism for chronic pain. It occurs at different levels of the central nervous system, including spinal cord and cortex. Studies have mainly focused on signaling proteins that trigger these plastic changes, whereas few have addressed the maintenance of plastic changes related to chronic pain. We found that protein kinase M zeta (PKMζ) maintains pain-induced persistent changes in the mouse anterior cingulate cortex (ACC). Peripheral nerve injury caused activation of PKMζ in the ACC, and inhibiting PKMζ by a selective inhibitor, ζ-pseudosubstrate inhibitory peptide (ZIP), erased synaptic potentiation. Microinjection of ZIP into the ACC blocked behavioral sensitization. These results suggest that PKMζ in the ACC acts to maintain neuropathic pain. PKMζ could thus be a new therapeutic target for treating chronic pain.

Potentiation of Synaptic AMPA Receptors Induced by the Deletion of NMDA Receptors Requires the GluA2 Subunit

Deletion of N-methyl-D-aspartate receptors (NMDARs) early in development results in an increase in the number of synaptic AMPA receptors (AMPARs), suggesting a role for NMDARs in negatively regulating AMPAR trafficking at developing synapses. Substantial evidence has shown that AMPAR subunits function differentially in AMPAR trafficking. However, the role of AMPAR subunits in the enhancement of AMPARs following NMDAR ablation remains unknown. We have now performed single-cell genetic deletions in double-floxed mice in which the deletion of GluN1 is combined with the deletion of GluA1 or GluA2. We find that the AMPAR enhancement following NMDAR deletion requires the GluA2 subunit, but not the GluA1 subunit, indicating a key role for GluA2 in the regulation of AMPAR trafficking in developing synapses.