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1.  Acute Inactivation of PSD-95 Destabilizes AMPA Receptors at Hippocampal Synapses 
PLoS ONE  2013;8(1):e53965.
Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.
PMCID: PMC3546964  PMID: 23342049
2.  Extinction of morphine-dependent conditioned behavior is associated with increased phosphorylation of the GluR1 subunit of AMPA receptors at hippocampal synapses 
In abstinent opiate addicts, relapse can be triggered by exposure to environmental cues associated with drug use; thus, the disruption of these learned associations may be an effective approach for reducing relapse. Interestingly, glutamatergic systems are thought to be involved in opiate-induced behavioral plasticity. In this study, changes in expression and phosphorylation levels of AMPA glutamate receptor subunits (GluR1, GluR2) in the hippocampus were investigated in rats showing a conditioned response to an opiate-paired environment as well as in animals in which this conditioned behavior was extinguished. Additionally, another set of animals went through a drug-unpaired paradigm (without conditioning) in order to examine the effects of the pharmacology of the drug itself. Subcellular fractionation techniques were used to analyze the local distribution of AMPA glutamate subunits within the synapse, especially at the postsynaptic density (PSD). Results showed that morphine-dependent conditioned responses did not alter expression or redistribution of GluR1 or GluR2; however the unpaired administration of morphine resulted in an increase in the phosphorylation of the GluR1 subunit at extrasynaptic sites. Interestingly, the extinction of the conditioned response significantly increased phosphorylation of the GluR1 subunit at the PSD. Therefore we propose that, within the synapse, the phosphorylation of the GluR1 subunit at the PSD may be a key mechanism in the extinction of opiate-associated conditioned responses.
PMCID: PMC2662612  PMID: 19077125
glutamate; CPP; extinction; PSD; rat
3.  Disruption of glutamate receptors at Shank-postsynaptic platform in Alzheimer's disease 
Brain research  2009;1292:191-198.
Synaptic loss underlies the memory deficit of Alzheimer's disease (AD). The molecular mechanism is elusive; however, excitatory synapses organized by the postsynaptic density (PSD) have been used as targets for AD treatment. To identify pathological entities at the synapse in AD, synaptic proteins were screened by quantitative proteomic profiling. The critical proteins were then selected for immunoblot analysis. The glutamate receptors N-methyl-d-aspartate (NMDA) receptor 1 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor 2 (GluR2) were substantially lost; specifically, the loss of GluR2 was up to 40% at PSD in AD. Shank proteins, the organizers of these glutamate receptors at excitatory synapses, were dramatically altered in AD. The level of Shank2 was increased, whereas the protein level of Shank3 was decreased. Further, the Shank3 protein was modified by ubiquitin, indicating that abnormal activity of the ubiquitin–proteasome system may lead to Shank3 degradation in AD. Our findings suggest that disruption of glutamate receptors at the Shank-postsynaptic platform could contribute to destruction of the PSD which underlies the synaptic dysfunction and loss in AD.
PMCID: PMC2745956  PMID: 19635471
Alzheimer's; Glutamate receptor; Synapse; Postsynaptic density; Shank; Proteomic
4.  PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs) and Synaptic Plasticity 
Current opinion in neurobiology  2011;21(2):306-312.
Activity-dependent modification of excitatory synaptic transmission is fundamental for developmental plasticity of the neural circuits and experience-dependent plasticity. Synaptic glutamatergic receptors including AMPA receptors and NMDA receptors (AMPARs and NMDARs) are embedded in the highly organized protein network in the postsynaptic density. Overwhelming data have shown that PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs), as a major family of scaffold proteins at glutamatergic synapses, regulate basal synaptic AMPAR function and trafficking. It is now clear that PSD-MAGUKs have multifaceted functions in terms of regulating synaptic transmission and plasticity. Here we discuss recent advancements in understanding the roles of PSD-95 and other family members of PSD-MAGUKs in synaptic plasticity, both as an anchoring protein for synaptic AMPARs and also as a signaling scaffold for mediating the interaction of the signaling complex and NMDARs.
PMCID: PMC3138136  PMID: 21450454
5.  Activity patterns govern synapse-specific AMPA receptor trafficking between deliverable and synaptic pools 
Neuron  2009;62(1):84-101.
In single neurons, glutamatergic synapses receiving distinct afferent inputs may contain AMPA receptors (-Rs) with unique subunit compositions. However, the cellular mechanisms by which differential receptor transport achieves this synaptic diversity remain poorly understood. In lateral geniculate neurons, we show that retinogeniculate and corticogeniculate synapses have distinct AMPA-R subunit compositions. Under basal conditions at both synapses, GluR1-containing AMPA-Rs are transported from an anatomically defined reserve pool to a deliverable pool near the postsynaptic density (PSD), but further incorporate into the PSD or functional synaptic pool only at retinogeniculate synapses. Vision-dependent activity, stimulation mimicking retinal input, or activation of CaMKII or Ras signaling regulated forward GluR1 trafficking from the deliverable pool to the synaptic pool at both synapses, whereas Rap2 signals reverse GluR1 transport at retinogeniculate synapses. These findings suggest that synapse-specific AMPA-R delivery involves constitutive and activity-regulated transport steps between morphological pools, a mechanism that may extend to the site-specific delivery of other membrane protein complexes.
PMCID: PMC2682220  PMID: 19376069
6.  Distinct perisynaptic and synaptic localization of NMDA and AMPA receptors on ganglion cells in rat retina 
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 also may 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 have 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. Co-labeling 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.
PMCID: PMC2577313  PMID: 16927255
NMDA; AMPA; synaptic and perisynaptic distribution; postembedding immunogold; retinal ganglion cell
7.  Genetic Deletion of NR3A Accelerates Glutamatergic Synapse Maturation 
PLoS ONE  2012;7(8):e42327.
Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth.
PMCID: PMC3411625  PMID: 22870318
8.  Evidence for Low GluR2 AMPA Receptor Subunit Expression at Synapses in the Rat Basolateral Amygdala 
Journal of neurochemistry  2005;94(6):1728-1738.
Fast excitatory synaptic responses in basolateral amygdala (BLA) neurons are mainly mediated by ionotropic glutamate receptors of the AMPA subtype. AMPA receptors containing an edited GluR2 subunit are calcium impermeable, whereas those that lack this subunit are calcium permeable and also inwardly rectifying. Here we sought to determine the extent to which synapses in the rat BLA have AMPA receptors with GluR2 subunits. We assessed GluR2 protein expression in the BLA by immunocytochemistry with a GluR2 subunit-specific antiserum at the light and electron microscopic level; for comparison a parallel examination was carried out in the hippocampus. We also recorded from amygdala brain slices to examine the voltage-dependent properties of AMPA receptor-mediated evoked synaptic currents in BLA principal neurons. At the light microscopic level, GluR2 immunoreactivity was localized to the perikarya and proximal dendrites of BLA neurons; dense labeling was also present over the pyramidal cell layer of hippocampal subfields CA1 and CA3. In electron micrographs from the BLA, most of the synapses were asymmetrical with pronounced postsynaptic densities (PSD). They contained clear, spherical vesicles apposed to the PSD and were predominantly onto spines (86%), indicating that they are mainly with BLA principal neurons. Only 11% of morphological synapses in the BLA were onto postsynaptic elements that showed GluR2 immunoreactivity in contrast to hippocampal subfields CA1 and CA3 in which 76% and 71% of postsynaptic elements were labeled (p < 0.001). Synaptic staining in the BLA and hippocampus, when it occurred, was exclusively postsynaptic, and particularly heavy over the PSD. In whole-cell voltage clamp recordings, 72% of BLA principal neurons exhibited AMPA receptor-mediated synaptic currents evoked by external capsule stimulation that were inwardly rectifying. Although BLA principal neurons express perikaryal and proximal dendritic GluR2 immunoreactivity, few synapses onto these neurons express GluR2 and a preponderance of principal neurons have inwardly rectifying AMPA-mediated synaptic currents, suggesting that targeting of GluR2 to synapses is restricted. Many BLA synaptic AMPA receptors are likely to be calcium permeable and could play roles in synaptic plasticity, epileptogenesis and excitoxicity.
PMCID: PMC1352164  PMID: 16045445
AMPA receptor; GluR2 subunit; basolateral amygdala; hippocampus; electron microscopy; patch clamp recording; BLA, basolateral amygdala
9.  Ontogeny of Postsynaptic Density Proteins at Glutamatergic Synapses 
In glutamatergic synapses, glutamate receptors (GluRs) associate with many other proteins involved in scaffolding and signal transduction. The ontogeny of these postsynaptic density (PSD) proteins involves changes in their composition during development, paralleling changes in GluR type and function. In the CA1 region of the hippocampus, at postnatal day 2 (P2), many synapses already have a distinct PSD. We used immunoblot analysis, subcellular fractionation, and quantitative immunogold electron microscopy to examine the distribution of PSD proteins during development of the hippocampus. Synapses at P2 contained substantial levels of NR1 and NR2B and most GluR-associated proteins, including SAP102, SynGAP, the chain of proteins from GluRs/SAP102 through GKAP/Shank/Homer and metabotropic glutamate receptors, and the adhesion factors, cadherin, catenin, neuroligin, and Nr-CAM. Development was marked by substantial decreases in NR2B and SAP102 and increases in NR2A, PSD-95, AMPA receptors and CaMKII. Other components showed more moderate changes.
PMCID: PMC1414063  PMID: 15894489
10.  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 exo- 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 label for GluR2 varies, but is consistent throughout cell body and dendrites in each individual neuron, except at postsynaptic densities (PSD), where it is typically higher. GluR2 label at PSDs significantly increases after synaptic activation by glycine treatment and increases 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 their 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, exo- 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.
PMCID: PMC3138201  PMID: 21451021
postsynaptic density; endocytosis; exocytosis; clathrin; transferrin receptor; electron microscopy
11.  Electrophysiological Characterization of AMPA and NMDA Receptors in Rat Dorsal Striatum 
The striatum receives glutamatergic afferents from the cortex and thalamus, and these synaptic transmissions are mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to characterize glutamate receptors by analyzing NMDA/AMPA ratio and rectification of AMPA and NMDA excitatory postsynaptic currents (EPSCs) using a whole-cell voltage-clamp method in the dorsal striatum. Receptor antagonists were used to isolate receptor or subunit specific EPSC, such as (DL)-2-amino-5-phosphonovaleric acid (APV), an NMDA receptor antagonist, ifenprodil, an NR2B antagonist, CNQX, an AMPA receptor antagonist and IEM-1460, a GluR2-lacking AMPA receptor blocker. AMPA and NMDA EPSCs were recorded at -70 and +40 mV, respectively. Rectification index was calculated by current ratio of EPSCs between +50 and -50 mV. NMDA/AMPA ratio was 0.20±0.05, AMPA receptor ratio of GluR2-lacking/GluR2-containing subunit was 0.26±0.05 and NMDA receptor ratio of NR2B/NR2A subunit was 0.32±0.03. The rectification index (control 2.39±0.27) was decreased in the presence of both APV and combination of APV and IEM-1460 (1.02±0.11 and 0.93±0.09, respectively). These results suggest that the major components of the striatal glutamate receptors are GluR2-containing AMPA receptors and NR2A-containing NMDA receptors. Our results may provide useful information for corticostriatal synaptic transmission and plasticity studies.
PMCID: PMC2766737  PMID: 19885039
Striatum; AMPA; Glutamate receptor; NMDA; Patch clamp
12.  β1-Integrins Are Required for Hippocampal AMPA Receptor-Dependent Synaptic Transmission, Synaptic Plasticity, and Working Memory 
Integrins comprise a large family of cell adhesion receptors that mediate diverse biological events through cell–cell and cell–extracellular matrix interactions. Recent studies have shown that several integrins are localized to synapses with suggested roles in synaptic plasticity and memory formation. We generated a postnatal forebrain and excitatory neuron-specific knock-out of β1-integrin in the mouse. Electrophysiological studies demonstrated that these mutants have impaired synaptic transmission through AMPA receptors and diminished NMDA receptor-dependent long-term potentiation. Despite the impairment in hippocampal synaptic transmission, the mutants displayed normal hippocampal-dependent spatial and contextual memory but were impaired in a hippocampal-dependent, nonmatching-to-place working memory task. These phenotypes parallel those observed in animals carrying knock-outs of the GluR1 (glutamate receptor subunit 1) subunit of the AMPA receptor. These observations suggest a new function of β1-integrins as regulators of synaptic glutamate receptor function and working memory.
PMCID: PMC2408376  PMID: 16399691
integrins; AMPA receptors; basal synaptic transmission; LTP; working memory; synaptic plasticity
13.  PSD-95 is required to sustain the molecular organization of the postsynaptic density 
PSD-95, a membrane-associated guanylate kinase (MAGUK), is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNAi knockdown of PSD-95 leads to loss of entire patches of PSD material, and EM tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA, but not NMDA receptor type structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.
PMCID: PMC3099547  PMID: 21525273
PSD-95; molecular conformation; postsynaptic density; hippocampal spines; RNAi; EM tomography; Immuno-EM
14.  Ultrastructural localisation and differential agonist-induced regulation of AMPA and kainate receptors present at the presynaptic active zone and postsynaptic density 
Journal of neurochemistry  2006;99(2):549-560.
Activity-dependent changes in ionotropic glutamate receptors at the postsynaptic membrane are well established and this regulation plays a central role in the expression of synaptic plasticity. However, very little is known about the distributions and regulation of ionotropic receptors at presynaptic sites. To determine if presynaptic receptors are subject to similar regulatory processes we investigated the localisation and modulation of AMPA (GluR1, GluR2, GluR3) and kainate (GluR6/7, KA2) receptor subunits by ultrasynaptic separation and immunoblot analysis of rat brain synaptosomes. All of the subunits were enriched in the postsynaptic fraction but were also present in the presynaptic and non-synaptic synaptosome fractions. AMPA stimulation resulted in a marked decrease in postsynaptic GluR2 and GluR3 subunits, but an increase in GluR6/7. Conversely, GluR2 and GluR3 increased in the presynaptic fraction whereas GluR6/7 decreased. There were no significant changes in any of the compartments for GluR1. NMDA treatment decreased postsynaptic GluR1, GluR2 and GluR6/7 but increased presynaptic levels of these subunits. NMDA treatment did not evoke changes in GluR3 localisation. Our results demonstrate that presynaptic and postsynaptic subunits are regulated in opposite directions by AMPA and NMDA stimulation.
PMCID: PMC3314506  PMID: 16903873
AMPA; kainate; synaptosomes; post-synaptic; pre-synaptic; regulation
15.  The distribution of NMDA and AMPA receptor subunits at thalamo-amygdala dendritic spines 
Brain research  2007;1134(1):87-94.
Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for ∝-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdala afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdala spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdala spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1 and NR2B subunits. Our data also show that in adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdala spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdala spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.
PMCID: PMC2359729  PMID: 17207780
GluR2; GluR3; excitatory amino acids; immunogold; NR1; NR2B; postembedding; immunohistochemistry; tracing; electron microscopy
16.  Expression of AMPA receptor subunits at synapses in laminae I–III of the rodent spinal dorsal horn 
Molecular Pain  2008;4:5.
Glutamate receptors of the AMPA type (AMPArs) mediate fast excitatory transmission in the dorsal horn and are thought to underlie perception of both acute and chronic pain. They are tetrameric structures made up from 4 subunits (GluR1-4), and subunit composition determines properties of the receptor. Antigen retrieval with pepsin can be used to reveal the receptors with immunocytochemistry, and in this study we have investigated the subunit composition at synapses within laminae I–III of the dorsal horn. In addition, we have compared staining of AMPArs with that for PSD-95, a major constituent of glutamatergic synapses. We also examined tissue from knock-out mice to confirm the validity of the immunostaining.
As we have shown previously, virtually all AMPAr-immunoreactive puncta were immunostained for GluR2. In laminae I–II, ~65% were GluR1-positive and ~60% were GluR3-positive, while in lamina III the corresponding values were 34% (GluR1) and 80% (GluR3). Puncta stained with antibody against the C-terminus of GluR4 (which only detects the long form of this subunit) made up 23% of the AMPAr-containing puncta in lamina I, ~8% of those in lamina II and 46% of those in lamina III. Some overlap between GluR1 and GluR3 was seen in each region, but in lamina I GluR1 and GluR4 were present in largely non-overlapping populations. The GluR4 puncta often appeared to outline dendrites of individual neurons in the superficial laminae. Virtually all of the AMPAr-positive puncta were immunostained for PSD-95, and 98% of PSD-95 puncta contained AMPAr-immunoreactivity. Staining for GluR1, GluR2 and GluR3 was absent in sections from mice in which these subunits had been knocked out, while the punctate staining for PSD-95 was absent in mice with a mutation that prevents accumulation of PSD-95 at synapses.
Our results suggest that virtually all glutamatergic synapses in laminae I–III of adult rat spinal cord contain AMPArs. They show that synapses in laminae I–II contain GluR2 together with GluR1 and/or GluR3, while the long form of GluR4 is restricted to specific neuronal populations, which may include some lamina I projection cells. They also provide further evidence that immunostaining for AMPAr subunits following antigen retrieval is a reliable method for detecting these receptors at glutamatergic synapses.
PMCID: PMC2248168  PMID: 18215271
17.  Synaptic and Extrasynaptic Localization of Brain-Derived Neurotrophic Factor and the Tyrosine Kinase B Receptor in Cultured Hippocampal Neurons 
Brain-derived neurotrophic factor (BDNF) regulates synapses, but the distribution of BDNF and its receptor TrkB relative to the location of glutamatergic and γ-aminobutyric acidergic (GABAergic) synapses is presently unknown. Immunocytochemistry was performed in primary hippocampal neuron cultures to determine whether BDNF and TrkB are preferentially localized to excitatory or inhibitory markers at 7, 14, and 21 days in vitro (DIV). Glutamatergic sites were localized with vesicular glutamate transporter type 1 (VGLUT1) as presynaptic marker and the NR1 subunit of the NMDA receptor and the GluR1 subunit of the AMPA receptor as receptor markers. GABAergic sites were labeled with the 65-kDa isoform of glutamic acid decarboxylase (GAD-65) as presynaptic marker and the γ2 subunit of the GABAA receptor as receptor marker. During development, <30% of BDNF punctae and TrkB clusters were localized to glutamatergic and GABAergic markers. Because their rates of colocalization did not change from 7 to 21 DIV, this study details the distribution of BDNF and TrkB at 14 DIV. BDNF was preferentially colocalized with glutamatergic markers VGLUT1 and NR1 (~30% each). TrkB was also relatively highly colocalized with VGLUT1 and NR1 (~20% each) but was additionally highly colocalized with GABAergic markers GAD-65 (~20%) and γ2 (~30%). NR1 clusters colocalized with BDNF puncta and TrkB clusters were mostly extrasynaptic, as were γ2 clusters colocalized with TrkB clusters. These results show that, whereas most BDNF and TrkB protein is extrasynaptic, BDNF is preferentially associated with excitatory markers and that TrkB is associated equally with excitatory and inhibitory markers.
PMCID: PMC2892721  PMID: 15384067
neurotrophins; glutamate; GABA; synaptogenesis
18.  Diffusional Trapping of GluR1 AMPA Receptors by Input-Specific Synaptic Activity 
Neuron  2007;54(3):447-460.
Synaptic activity regulates the postsynaptic accumulation of AMPA receptors over timescales ranging from minutes to days. Indeed, the regulated trafficking and mobility of GluR1 AMPA receptors underlies many forms of synaptic potentiation at glutamatergic synapses throughout the brain. However, the basis for synapse-specific accumulation of GluR1 is unknown. Here we report that synaptic activity locally immobilizes GluR1 AMPA receptors at individual synapses. Using single-molecule tracking together with the silencing of individual presynaptic boutons, we demonstrate that local synaptic activity reduces diffusional exchange of GluR1 between synaptic and extraynaptic domains, resulting in postsynaptic accumulation of GluR1. At neighboring inactive synapses, GluR1 is highly mobile with individual receptors frequently escaping the synapse. Within the synapse, spontaneous activity confines the diffusional movement of GluR1 to restricted subregions of the postsynaptic membrane. Thus, local activity restricts GluR1 mobility on a submicron scale, defining a novel input-specific mechanism for regulating AMPA receptor composition and abundance.
PMCID: PMC1993808  PMID: 17481397
19.  Immunogold electron microscopic evidence of differential regulation of GluN1, GluN2A and GluN2B, NMDA-type glutamate receptor subunits in rat hippocampal CA1 synapses during benzodiazepine withdrawal 
The Journal of comparative neurology  2010;518(21):4311-4328.
Benzodiazepine withdrawal-anxiety is associated with enhanced AMPA receptor (AMPAR)-mediated glutamatergic transmission in rat hippocampal CA1 synapses due to enhanced synaptic insertion and phosphorylation of GluA1 homomers. Interestingly, attenuation of withdrawal-anxiety is associated with a reduction in NMDA receptor (NMDAR)-mediated currents and subunit expression, secondary to AMPA receptor potentiation. Therefore, in this study ultrastructural evidence for possible reductions in NMDAR GluN1, GluN2A and GluN2B subunits was sought at CA1 stratum radiatum synapses in proximal dendrites using postembedding immunogold labeling of tissues from rats withdrawn for 2-days from 1-week daily oral administration of the benzodiazepine, flurazepam (FZP). GluN1-immunogold density and the percentage of immunopositive synapses were significantly decreased in tissues from FZP-withdrawn rats. Similar decreases were observed for GluN2B subunits, however the relative lateral distribution of GluN2B-immunolabeling within the postsynaptic density did not change after BZ withdrawal. In contrast to the GluN2B subunit, the percentage of synapses labeled with the GluN2A subunit antibody and the density of immunogold labeling for this subunit was unchanged. The spatial localization of immunogold particles asssociated with each NMDAR subunit was consistent with a predominantly postsynaptic localization. The data therefore provide direct evidence for reduced synaptic GluN1/GluN2B receptors and preservation of GluN1/GluN2A receptors in the CA1 stratum radiatum region during BZ withdrawal. Based on collective findings in this benzodiazepine withdrawal-anxiety model, we propose a functional model illustrating the changes in glutamate receptor populations at excitatory synapses during benzodiazepine withdrawal.
PMCID: PMC2943829  PMID: 20853509
Electron microscopy; Plasticity; Dependence; Glutamate; LTP; Anxiety
20.  Purification and crystallization of a non-GluR2 AMPA-receptor ligand-binding domain: a case of cryo-incompatibility addressed by room-temperature data collection 
High-resolution diffraction was obtained from GluR4 AMPA-receptor ligand-binding domain crystals using a combination of cryogenic and room-temperature data-collection strategies.
Glutamate is the major excitatory neurotransmitter in the brain. Among the cognate ionotropic glutamate receptors, the subfamily selective for AMPA (α-­amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) is responsible for most fast excitatory synaptic signaling and plays key roles in synaptic plasticity. AMPA receptors (AMPA-Rs) have also been implicated in a number of neurological disorders. To investigate subunit-specific differences in the ligand binding and activation of AMPA-Rs, the GluR4 AMPA-R ligand-binding domain (LBD) was crystallized in complex with full and partial agonists. This is the first non-GluR2 AMPA-R LBD available for structural analysis. Standard cryo­protection protocols yielded high-resolution diffraction from flash-cooled crystals of the complex with the full agonist glutamate. However, for cocrystals with the partial agonist kainate, systematic screening and optimization of cryoprotection conditions yielded at best mosaic, weak diffraction at 100 K. In contrast, room-temperature data collection from capillary-mounted kainate cocrystals exhibited reproducible diffraction to better than 3 Å resolution. Together, these crystals lay the foundation for a structural comparison of LBD–agonist interactions in distinct AMPA-R subunits.
PMCID: PMC2531279  PMID: 18765917
ionotropic glutamate receptor; AMPA-receptor ion channel; GluR4 ligand-binding domain; recombinant expression; metal-affinity chromatography; capillary mounting
21.  AKAP150-Anchored Calcineurin Regulates Synaptic Plasticity by Limiting Synaptic Incorporation of Ca2+-Permeable AMPA Receptors 
AMPA receptors (AMPARs) are tetrameric ion channels assembled from GluA1-GluA4 subunits that mediate the majority of fast excitatory synaptic transmission in the brain. In the hippocampus, most synaptic AMPARs are composed of GluA1/2 or GluA2/3 with the GluA2 subunit preventing Ca2+ influx. However, a small number of Ca2+-permeable GluA1 homomeric receptors reside in extrasynaptic locations where they can be rapidly recruited to synapses during synaptic plasticity. Phosphorylation of GluA1 S845 by the cAMP-dependent protein kinase (PKA) primes extrasynaptic receptors for synaptic insertion in response to NMDA receptor (NMDAR) Ca2+ signaling during long-term potentiation (LTP), while phosphatases dephosphorylate S845 and remove synaptic and extrasynaptic GluA1 during long-term depression (LTD). PKA and the Ca2+-activated phosphatase calcineurin (CaN) are targeted to GluA1 through binding to A-kinase anchoring protein (AKAP) 150 in a complex with PSD-95, but we do not understand how the opposing activities of these enzymes are balanced to control plasticity. Here, we generated AKAP150ΔPIX knock-in mice to selectively disrupt CaN anchoring in vivo. We found that AKAP150ΔPIX mice lack LTD but express enhanced LTP at CA1 synapses. Accordingly, basal GluA1 S845 phosphorylation is elevated in AKAP150ΔPIX hippocampus, and LTD-induced dephosphorylation and removal of GluA1, AKAP150, and PSD-95 from synapses is impaired. In addition, basal synaptic activity of GluA2-lacking AMPARs is increased in AKAP150ΔPIX mice and pharmacologic antagonism of these receptors restores normal LTD and inhibits the enhanced LTP. Thus, AKAP150-anchored CaN opposes PKA phosphorylation of GluA1 to restrict synaptic incorporation of Ca2+-permeable AMPARs both basally and during LTP and LTD.
PMCID: PMC3504485  PMID: 23100425
22.  Ubiquitination Regulates PSD-95 Degradation and AMPA Receptor Surface Expression 
Neuron  2003;40(3):595-607.
PSD-95 is a major scaffolding protein of the postsynaptic density, tethering NMDA- and AMPA-type glutamate receptors to signaling proteins and the neuronal cytoskeleton. Here we show that PSD-95 is regulated by the ubiquitin-proteasome pathway. PSD-95 interacts with and is ubiquitinated by the E3 ligase Mdm2. In response to NMDA receptor activation, PSD-95 is ubiquitinated and rapidly removed from synaptic sites by proteasome-dependent degradation. Mutations that block PSD-95 ubiquitination prevent NMDA-induced AMPA receptor endocytosis. Likewise, proteasome inhibitors prevent NMDA-induced AMPA receptor internalization and synaptically induced long-term depression. This is consistent with the notion that PSD-95 levels are an important determinant of AMPA receptor number at the synapse. These data suggest that ubiquitination of PSD-95 through an Mdm2-mediated pathway is critical in regulating AMPA receptor surface expression during synaptic plasticity.
PMCID: PMC3963808  PMID: 14642282
23.  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, due 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 α-isoform to the postsynaptic density (PSD) and the L27 domain-containing β-isoform primarily to non-PSD, perisynaptic regions. Consequently, α- and βSAP97 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.
PMCID: PMC3230533  PMID: 19357261
AMPAR; FRAP; GluR1; palmitoylation; SAP97; PSD-95
24.  Excitatory Mechanisms in the Suprachiasmatic Nucleus: The Role of AMPA/KA Glutamate Receptors 
Journal of neurophysiology  2002;88(2):817-828.
A variety of evidence suggests that the effects of light on the mammalian circadian system are mediated by direct retinal ganglion cell projection to the suprachiasmatic nucleus (SCN). This synaptic connection is glutamatergic and the release of glutamate is detected by both N-methyl-d-asparate (NMDA) and amino-methyl proprionic acid/kainate (AMPA/KA) iontotropic glutamate receptors (GluRs). It is well established that NMDA GluRs play a critical role in mediating the effects of light on the circadian system; however, the role of AMPA/KA GluRs has received less attention. In the present study, we sought to better understand the contribution of AMPA/KA-mediated currents in the circadian system based in the SCN. First, whole cell patch-clamp electrophysiological techniques were utilized to measure spontaneous excitatory postsynaptic currents (sEPSCs) from SCN neurons. These currents were widespread in the SCN and not just restricted to the retino-recipient region. The sEPSC frequency and amplitude did not vary with the daily cycle. Similarly, currents evoked by the exogenous application of AMPA onto SCN neurons were widespread within the SCN and did not exhibit a diurnal rhythm in their magnitude. Fluorometric techniques were utilized to estimate AMPA-induced calcium (Ca2+) concentration changes in SCN neurons. The resulting data indicate that AMPA-evoked Ca2+ transients were widespread in the SCN and that there was a daily rhythm in the magnitude of AMPA-induced Ca2+ transients that peaked during the night. By itself, blocking AMPA/KA GluRs with a receptor blocker decreased the spontaneous firing of some SCN neurons as well as reduced resting Ca2+ levels, suggesting tonic glutamatergic excitation. Finally, immunohistochemical techniques were used to describe expression of the AMPA-preferring GluR subunits GluR1 and GluR2/3s within the SCN. Overall, our data suggest that glutamatergic synaptic transmission mediated by AMPA/KA GluRs play an important role throughout the SCN synaptic circuitry.
PMCID: PMC2579743  PMID: 12163533
25.  AMPA Receptor Activation Causes Silencing of AMPA Receptor-Mediated Synaptic Transmission in the Developing Hippocampus 
PLoS ONE  2012;7(4):e34474.
Agonist-induced internalization of transmembrane receptors is a widespread biological phenomenon that also may serve as a mechanism for synaptic plasticity. Here we show that the agonist AMPA causes a depression of AMPA receptor (AMPAR) signaling at glutamate synapses in the CA1 region of the hippocampus in slices from developing, but not from mature, rats. This developmentally restricted agonist-induced synaptic depression is expressed as a total loss of AMPAR signaling, without affecting NMDA receptor (NMDAR) signaling, in a large proportion of the developing synapses, thus creating AMPAR silent synapses. The AMPA-induced AMPAR silencing is induced independently of activation of mGluRs and NMDARs, and it mimics and occludes stimulus-induced depression, suggesting that this latter form of synaptic plasticity is expressed as agonist-induced removal of AMPARs. Induction of long-term potentiation (LTP) rendered the developing synapses resistant to the AMPA-induced depression, indicating that LTP contributes to the maturation-related increased stability of these synapses. Our study shows that agonist binding to AMPARs is a sufficient triggering stimulus for the creation of AMPAR silent synapses at developing glutamate synapses.
PMCID: PMC3317613  PMID: 22485173

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