MAB5416 Sigma-AldrichAnti-Glutamate Receptor 3 Antibody, clone 3B3

Protein glycosylation may contribute to the evolution of mammalian brain complexity by adapting excitatory neurotransmission in response to environmental and social cues. Balanced excitatory synaptic transmission is primarily mediated by glutamatergic neurotransmission. Previous studies have found that subunits of the AMPA subtype of glutamate receptor are N-glycosylated, which may play a critical role in AMPA receptor trafficking and function at the cell membrane. Studies have predominantly used rodent models to address altered glycosylation in human pathological conditions. Given the rate of mammalian brain evolution and the predicted rate of change in the brain-specific glycoproteome, we asked if there are species-specific changes in glycoprotein expression, focusing on the AMPA receptor. N-glycosylation of AMPA receptor subunits was investigated in rat (Rattus norvegicus), tree shrew (Tupaia glis belangeri), macaque (Macaca nemestrina), and human frontal cortex tissue using a combination of enzymatic deglycosylation and Western blot analysis, as well as lectin binding assays. We found that two AMPA receptor subunits, GluA2 and GluA4, are sensitive to deglycosylation with Endo H and PNGase F. When we enriched for glycosylated proteins using lectin binding assays, we found that all four AMPA receptor subunits are glycosylated, and were predominantly recognized by lectins that bind to glucose or mannose, N-acetylglucosamine (GlcNAc), or 1-6αfucose. We found differences in glycosylation between different subunits, as well as modest differences in glycosylation of homologous subunits between different species.

Following the exocytosis of neurotransmitter-containing synaptic vesicles, endocytosis is fundamental to re-establishing conditions for synaptic transmission. As there are distinct endocytotic pathways that each differ in their efficiency to generate releasable synaptic vesicles, we used the dye FM1-43 to track vesicle recycling, and to determine whether nerve terminals use multiple pathways of endocytosis. We identified two types of synaptic boutons in cultured cerebellar granule cells that were characterized by weak or strong FM1-43-unloading profiles. Decreasing the extent of exocytosis dramatically increased the proportion of synaptic boutons that exhibited strong FM1-43-unloading and dramatically reduced the number of endosome-like structures. Hence, we concluded that efficient recycling of synaptic vesicles is concomitant with the formation of non-releasable endosomes in both types of synaptic boutons, although to different extents. Furthermore, cell maturation in culture increased the proportion of synaptic boutons that were capable of an intense release response, whereas the chronic blockage of synaptic activity diminished the capacity of boutons to release dye.

The subunit composition of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) is an important determinant of AMPAR biophysical properties and trafficking. To date, AMPAR subunit composition has been quantitatively evaluated only for the hippocampus, where different experimental approaches have yielded different results. Here, we used quantitative co-immunoprecipitation to characterize GluA1-3 associations in the adult rat nucleus accumbens, dorsal striatum, prefrontal cortex, and hippocampus, and blue native electrophoresis (BNE) to study GluA1-3 assembly state. In all brain regions, co-immunoprecipitation experiments showed that ~90% of GluA1 was associated with GluA2 or GluA3 (most was GluA1A2). All regions contained a small number of GluA1A3 receptors. Homomeric GluA1 receptors may also exist. More than half of the GluA2 (53%-65% depending on the region) was not associated with GluA1. However, this represents an over-estimate of the percent of GluA2 present in GluA2A3 receptors, based on BNE results demonstrating that the majority of GluA2 exists as dimers, rather than functional tetrameric receptors. Relatively more GluA1 was present in tetramers. Together with other findings, our results suggest a dominant role for GluA1A2 receptors in all brain regions examined. They also help explain why different results for hippocampal AMPAR subunit composition were obtained using co-immunoprecipitation, which assesses the total cellular pool of AMPARs including partially assembled AMPARs in intracellular compartments, and electrophysiological approaches, which can selectively assess tetrameric (functional) AMPARs on the cell surface.

The physiological responses of AMPA receptors can be modulated through the differential expression of their subunits and by modifying their number at the cell surface. Here we have studied the expression of AMPA receptor subunits (GluR1-4) mRNAs in cerebellar granule cells grown in depolarizing (25mMK(+)) medium, and we have evaluated the effect of decreasing the [K(+)] in the culture medium for 24 h on both GluR1-4 expression (both mRNA and protein) and their presence at the plasma membrane. The expression of the four AMPAR subunits increases as the [K(+)] decreases, although the increase in GluR2 and GluR3 was only observed in the cell soma but not in the dendrites. Calcium entry through L-type calcium channel and CaMKIV activation are responsible for the reduction in the expression of AMPA receptor subunits in cells cultured in depolarizing conditions. Indeed, prolonged reduction of extracellular [K(+)] or blockage of L-type calcium channels enhanced both the surface insertion of the four AMPAR subunits and the AMPA response measured through intracellular calcium increase. These findings reveal a balanced increase in functional AMPA receptors at the surface of cells that can trigger strong increases in calcium in response to the persistent reduction of calcium entry.

Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+)-permeable AMPA receptors (CP-AMPARs). However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1) subunit at the serine 845 (S845) site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants), which is a substrate of cAMP-dependent kinase (PKA), show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

Oligodendrocyte precursor cells (OPCs), a major glial cell type that gives rise to myelinating oligodendrocytes in the CNS, express calcium-permeable AMPA receptors (CP-AMPARs). Although CP-AMPARs are important for OPC proliferation and neuron-glia signaling, they render OPCs susceptible to ischemic damage in early development. We identified factors controlling the dynamic regulation of AMPAR subtypes in OPCs from rat optic nerve and mouse cerebellar cortex. We found that activation of group 1 mGluRs drove an increase in the proportion of CP-AMPARs, reflected by an increase in single-channel conductance and inward rectification. This plasticity required the elevation of intracellular calcium and used PI3K, PICK-1 and the JNK pathway. In white matter, neurons and astrocytes release both ATP and glutamate. Unexpectedly, activation of purinergic receptors in OPCs decreased CP-AMPAR expression, suggesting a capacity for homeostatic regulation. Finally, we found that stargazin-related transmembrane AMPAR regulatory proteins, which are critical for AMPAR surface expression in neurons, regulate CP-AMPAR plasticity in OPCs.

Ampakines are cognitive enhancers that potentiate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents and synaptic responses by slowing receptor deactivation. Their efficacy varies greatly between classes of neurons and brain regions, but the factor responsible for this effect remains unclear. Ampakines also increase agonist affinity in binding tests in ways that are related to their physiological action. We therefore examined 1) whether ampakine effects on agonist binding vary across brain regions and 2) whether they differ across receptor subunits expressed alone and together with transmembrane AMPA receptor regulatory proteins (TARPs), which associate with AMPA receptors in the brain. We found that the maximal increase in agonist binding (E(max)) caused by the prototypical ampakine 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546) differs significantly between brain regions, with effects in hippocampus and cerebellum being nearly three times larger than that in thalamus, brainstem, and striatum, and cortex being intermediate. These differences can be explained at least in part by regional variations in receptor subunit and TARP expression because combinations prevalent in hippocampus (GluA2 with TARPs gamma3 and gamma8) exhibited E(max) values nearly twice those of combinations abundant in thalamus (GluA4 with gamma2 or gamma4). TARPs seem to be critical because GluA2 and GluA4 alone had comparable E(max) and also because hippocampal and thalamic receptors had similar E(max) after solubilization with Triton X-100, which probably removes associated proteins. Taken together, our data suggest that variations in physiological drug efficacy, such as the 3-fold difference previously seen in recordings from hippocampus versus thalamus, may be explained by region-specific expression of GluA1-4 as well as TARPs.

A unifying principle of sensory system organization is feature extraction by modality-specific neuronal maps in which arrays of neurons show systematically varied response properties and receptive fields. Only beginning to be understood, however, are the mechanisms by which these graded systems are established. In the peripheral auditory system, we have shown previously that the intrinsic firing features of spiral ganglion neurons are influenced by brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). We now show that is but a part of a coordinated package of neurotrophin actions that also includes effects on presynaptic and postsynaptic proteins, thus encompassing the input, transmission, and output functions of the spiral ganglion neurons. Using immunocytochemical methods, we determined that proteins targeted to opposite ends of the neuron were organized and regulated in a reciprocal manner. AMPA receptor subunits GluR2 and GluR3 were enriched in base neurons compared with their apex counterparts. This distribution pattern was enhanced by exposure to BDNF but reduced by NT-3. SNAP-25 and synaptophysin were distributed and regulated in the mirror image: enriched in the apex, enhanced by NT-3 and reduced by BDNF. Moreover, we used a novel coculture to identify potential endogenous sources of neurotrophins by showing that sensory receptors from different cochlear regions were capable of altering presynaptic and postsynaptic protein levels in these neurons. From these studies, we suggest that BDNF and NT-3, which are systematically distributed in complementary gradients, are responsible for orchestrating a comprehensive set of electrophysiological specializations along the frequency contour of the cochlea.

Excitatory synapses onto dopamine (DA) neurons of the ventral tegmental area (VTA) represent a critical site of psychostimulant-induced synaptic plasticity. This plasticity involves alterations in synaptic strength through AMPA receptor (AMPAR) redistribution. Here, we report an in vitro model for studying regulation of AMPAR trafficking in DA neurons under control conditions and after elevation of DA levels, mimicking cocaine exposure. We used cocultures containing rat VTA neurons and prefrontal cortex (PFC) neurons from enhanced cyan fluorescent protein-expressing mice. In VTA-PFC cocultures, D1 receptor activation (10 min) increased synaptic and nonsynaptic glutamate receptor subunit 1 (GluR1) and GluR2 surface expression on DA neurons. NMDA or AMPA receptor antagonists blocked this effect, and it was not observed in pure VTA cultures, suggesting that DA agonists acted on D1 receptors on PFC neurons, altering their excitatory transmission onto VTA DA neurons and, thus, influencing AMPARs. To mimic the longer elevation in extracellular DA levels produced by systemic cocaine, cocultures were incubated with DA for 1 h. Synaptic GluR1 was increased 24 h later, reminiscent of the increased AMPA/NMDA ratio at excitatory synapses onto VTA DA neurons 24 h after cocaine injection (Ungless et al., 2001). In contrast, GluR2 was unchanged. Analysis of colocalization of surface GluR1-3 labeling suggested that control DA neurons express a substantial number of GluR1/2, GluR2/3, and homomeric GluR1 receptors and that the increase in surface AMPARs 24 h after DA exposure may in part reflect increased GluR1/3-containing receptors. These results help define the cellular basis for plasticity underlying the development of behavioral sensitization.

At most excitatory synapses, AMPA and NMDA receptors (AMPARs and NMDARs) occupy the postsynaptic density (PSD) and contribute to miniature excitatory postsynaptic currents (mEPSCs) elicited by single transmitter quanta. Juxtaposition of AMPARs and NMDARs may be crucial for certain types of synaptic plasticity, although extrasynaptic NMDARs may also contribute. AMPARs and NMDARs also contribute to evoked EPSCs in retinal ganglion cells (RGCs), but mEPSCs are mediated solely by AMPARs. Previous work indicates that an NMDAR component emerges in mEPSCs when glutamate uptake is reduced, suggesting that NMDARs are located near the release site but perhaps not directly beneath in the PSD. Consistent with this idea, NMDARs on RGCs encounter a lower glutamate concentration during synaptic transmission than do AMPARs. To understand better the roles of NMDARs in RGC function, we used immunohistochemical and electron microscopic techniques to determine the precise subsynaptic localization of NMDARs in RGC dendrites. RGC dendrites were labeled retrogradely with cholera toxin B subunit (CTB) injected into the superior colliculus (SC) and identified using postembedding immunogold methods. Colabeling with antibodies directed toward AMPARs and/or NMDARs, we found that nearly all AMPARs are located within the PSD, while most NMDARs are located perisynaptically, 100-300 nm from the PSD. This morphological evidence for exclusively perisynaptic NMDARs localizations suggests a distinct role for NMDARs in RGC function.