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1.  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
2.  Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication 
Molecular Autism  2010;1:15.
SHANK3 is a protein in the core of the postsynaptic density (PSD) and has a critical role in recruiting many key functional elements to the PSD and to the synapse, including components of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA), metabotropic glutamate (mGlu) and N-methyl-D-aspartic acid (NMDA) glutamate receptors, as well as cytoskeletal elements. Loss of a functional copy of the SHANK3 gene leads to the neurobehavioral manifestations of 22q13 deletion syndrome and/or to autism spectrum disorders. The goal of this study was to examine the effects of haploinsufficiency of full-length Shank3 in mice, focusing on synaptic development, transmission and plasticity, as well as on social behaviors, as a model for understanding SHANK3 haploinsufficiency in humans.
We used mice with a targeted disruption of Shank3 in which exons coding for the ankyrin repeat domain were deleted and expression of full-length Shank3 was disrupted. We studied synaptic transmission and plasticity by multiple methods, including patch-clamp whole cell recording, two-photon time-lapse imaging and extracellular recordings of field excitatory postsynaptic potentials. We also studied the density of GluR1-immunoreactive puncta in the CA1 stratum radiatum and carried out assessments of social behaviors.
In Shank3 heterozygous mice, there was reduced amplitude of miniature excitatory postsynaptic currents from hippocampal CA1 pyramidal neurons and the input-output (I/O) relationship at Schaffer collateral-CA1 synapses in acute hippocampal slices was significantly depressed; both of these findings indicate a reduction in basal neurotransmission. Studies with specific inhibitors demonstrated that the decrease in basal transmission reflected reduced AMPA receptor-mediated transmission. This was further supported by the observation of reduced numbers of GluR1-immunoreactive puncta in the stratum radiatum. Long-term potentiation (LTP), induced either with θ-burst pairing (TBP) or high-frequency stimulation, was impaired in Shank3 heterozygous mice, with no significant change in long-term depression (LTD). In concordance with the LTP results, persistent expansion of spines was observed in control mice after TBP-induced LTP; however, only transient spine expansion was observed in Shank3 heterozygous mice. Male Shank3 heterozygotes displayed less social sniffing and emitted fewer ultrasonic vocalizations during interactions with estrus female mice, as compared to wild-type littermate controls.
We documented specific deficits in synaptic function and plasticity, along with reduced reciprocal social interactions in Shank3 heterozygous mice. Our results are consistent with altered synaptic development and function in Shank3 haploinsufficiency, highlighting the importance of Shank3 in synaptic function and supporting a link between deficits in synapse function and neurodevelopmental disorders. The reduced glutamatergic transmission that we observed in the Shank3 heterozygous mice represents an interesting therapeutic target in Shank3-haploinsufficiency syndromes.
PMCID: PMC3019144  PMID: 21167025
3.  Gestational nicotine exposure regulates expression of AMPA and NMDA receptors and their signaling apparatus in developing and adult rat hippocampus 
Neuroscience  2011;188:168-181.
Untimely activation of nicotinic acetylcholine receptor (nAChR) by nicotine results in short- and long-term consequences on learning and behavior. In this study, the aim was to determine how prenatal nicotine exposure affects components of glutamatergic signaling in the hippocampus during postnatal development. We investigated regulation of both nAChRs and glutamate receptors for α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA), from postnatal day (P) 1 to P63 after a temporally restricted exposure to saline or nicotine for 14 days in utero. We analyzed postsynaptic density components associated with AMPAR and NMDAR signaling: Calcium/calmodulin-dependent protein kinase II α (CaMKIIα), Calmodulin (CaM), and postsynaptic density-95 (PSD95), as well as presynaptically localized synaptosomal-associated protein 25 (SNAP25). At P1, there was significantly heightened expression of AMPAR subunit GluR1 but not GluR2, and of NMDAR subunits NR1, NR2a and NR2d but not NR2b. NR2c was not detectable. At P1, the postsynaptic proteins CaMKIIα, CaM, and PSD95 were also significantly upregulated, together with presynaptic SNAP25. This enhanced expression of glutamate receptors and signaling proteins was concomitant with elevated levels of [3H] Epibatidine (EB) binding in prenatal nicotine-exposed hippocampus, indicating that α4β2 nAChR may influence glutamatergic function in the hippocampus at P1. By P14, neither [3H]EB binding nor the expression levels of subunits GluR1, GluR2, NR1, NR2a, NR2b, NR2c, or NR2d seemed changed with prenatal nicotine. However, CaMKIIα was significantly upregulated with nicotine treatment while CaM showed downregulation at P14. The effects of nicotine persisted in young adult brains at P63. They exhibited significantly downregulated GluR2, NR1, and NR2c expression levels in hippocampal homogenates and a considerably muted overall distribution of [3H]AMPA binding in areas CA1, CA2, CA3, and the dentate gyrus. Our results suggest that prenatal nicotine exposure can regulate the glutamatergic signaling system throughout postnatal development by enhancing or inhibiting availability of AMPAR and NMDAR or their signaling components. The persistent depression, in adults, of the requisite NR1 subunit for NMDAR assembly, and of GluR2, important for assembly, trafficking, and biophysical properties of AMPAR, indicates that nicotine may alter ionotropic glutamate receptor stoichiometry and functional properties in adults after prenatally restricted exposure.
PMCID: PMC3117944  PMID: 21596105
prenatal nicotine; developing hippocampus; AMPAR; NMDAR; nAChR; postsynaptic signaling
4.  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
5.  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
6.  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
7.  Glutamate receptor composition of the post-synaptic density is altered in genetic mouse models of NMDA receptor hypo- and hyperfunction 
Brain research  2011;1392:1-7.
The N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) are ionotropic glutamate receptors responsible for excitatory neurotransmission in the brain. These excitatory synapses are found on dendritic spines, with the abundance of receptors concentrated at the postsynaptic density (PSD). We utilized two genetic mouse models, the serine racemase knockout (SR−/−) and the glycine transporter subtype 1 heterozygote mutant (GlyT1+/−), to determine how constitutive NMDAR hypo- and hyperfunction, respectively, affect the glutamate receptor composition of the PSD in the hippocampus and prefrontal cortex (PFC). Using cellular fractionation, we found that SR−/− mice had elevated protein levels of NR1 and NR2A NMDAR subunits specifically in the PSD-enriched fraction from the hippocampus, but not from the PFC. There were no changes in the amounts of AMPAR subunits (GluR1, GluR2), or PSD protein of 95kDa (PSD95) in either brain region. GlyT1+/− mice also had elevated protein expression of NR1 and NR2A subunits in the PSD, as well as an increase in total protein. Moreover, GlyT1+/− mice had elevated amounts of GluR1 and GluR2 in the PSD, and higher total amounts of GluR1. Similar to SR−/− mice, there was no protein changes observed in the PFC. These findings illustrate the complexity of synaptic adaptation to altered NMDAR function.
PMCID: PMC3225000  PMID: 21443867
serine racemase; glycine transporter; postsynaptic density; NMDA receptor; AMPA receptor; schizophrenia
8.  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
9.  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
10.  Numbers, Densities, and Colocalization of AMPA- and NMDA-Type Glutamate Receptors at Individual Synapses in the Superficial Spinal Dorsal Horn of Rats 
The Journal of Neuroscience  2008;28(39):9692-9701.
Ionotropic glutamate receptors play important roles in spinal processing of nociceptive sensory signals and induction of central sensitization in chronic pain. Here we applied highly sensitive freeze-fracture replica labeling to laminae I–II of the spinal dorsal horn of rats and investigated the numbers, densities, and colocalization of AMPA- and NMDA-type glutamate receptors at individual postsynaptic membrane specializations with a high resolution. All glutamatergic postsynaptic membranes in laminae I–II expressed AMPA receptors, and most of them (96%) were also immunoreactive for the NR1 subunit of NMDA receptors. The numbers of gold particles for AMPA and NMDA receptors at individual postsynaptic membranes showed a linear correlation with the size of postsynaptic membrane specializations and varied in the range of 8–214 and 5–232 with median values of 37 and 28, whereas their densities varied in the range of 325–3365/μm2 and 102–2263/μm2 with median values of 1115/μm2 and 777/μm2, respectively. Virtually all (99%) glutamatergic postsynaptic membranes expressed GluR2, and most of them (87%) were also immunoreactive for GluR1. The numbers of gold particles for pan-AMPA, NR1, and GluR2 subunits showed a linear correlation with the size of postsynaptic surface areas. Concerning GluR1, there may be two populations of synapses with high and low GluR1 densities. In synapses larger than 0.1 μm2, GluR1 subunits were recovered in very low numbers. Differential expression of GluR1 and GluR2 subunits suggests regulation of AMPA receptor subunit composition by presynaptic mechanism.
PMCID: PMC3844880  PMID: 18815255
ionotropic glutamate receptors; NR1; GluR1; GluR2; molecular anatomy; postsynaptic active zones; SDS-FRL
11.  Kalirin-7 Is an Essential Component of both Shaft and Spine Excitatory Synapses in Hippocampal Interneurons 
Kalirin, a multifunctional Rho GDP/GTP exchange factor, plays a vital role in cytoskeletal organization, affecting process initiation and outgrowth in neurons. Through alternative splicing, the Kalirin gene generates multiple functionally distinct proteins. Kalirin-7 (Kal7) is the most prevalent isoform in the adult rat hippocampus; it terminates with a PDZ binding motif, is localized to the post-synaptic density, interacts with PSD95 and causes the formation of dendritic spines when over-expressed in pyramidal neurons. Levels of Kal7 are low in the dendrites of hippocampal aspiny interneurons. In these interneurons, Kal7 is localized to the postsynaptic side of excitatory synapses onto dendritic shafts, overlapping clusters of PSD95 and NMDA receptor subunit NR1. Selectively decreasing levels of Kal7 decreases the density of PSD95 positive, bassoon positive clusters along the dendritic shaft of hippocampal interneurons. Over-expression of Kal7 increases dendritic branching, inducing formation of spine-like structures along the dendrites and on the soma of normally aspiny hippocampal interneurons. Essentially all of the spine-like structures formed in response to Kal7 are apposed to VGLUT1 positive, bassoon positive presynaptic endings; GAD positive, VGAT positive inhibitory endings are unaffected. Almost every Kal7 positive dendritic cluster contains PSD95 along with NMDA (NR1) and AMPA (GluR1 and GluR2) receptor subunits. Kal7-induced formation of spine-like structures requires its PDZ binding motif, and interruption of interactions between the PDZ binding motif and its interactors decreases Kal7-induced formation of spine-like structures. Kal7 thus joins Shank3 and GluR2 as molecules whose level of expression at excitatory synapses titrates the number of dendritic spines.
PMCID: PMC2570025  PMID: 18199770
Rho GEF; PSD95; GABA; dendritic growth; synaptogenesis
12.  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
13.  Genetic Deletion of NP1 Prevents Hypoxic‐Ischemic Neuronal Death Via Reducing AMPA Receptor Synaptic Localization in Hippocampal Neurons 
Trafficking of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptors (AMPARs) to excitatory synapses is critical to their synaptic functions. Previously, we have shown induction of neuronal pentraxin 1 (NP1) and its colocalization with AMPAR subunit GluR1 in hypoxic‐ischemic (HI) brain injury. However, the role of NP1 in mediating GluR1 surface expression, trafficking, and clustering at synapses in HI neuronal death is unclear.
Methods and Results
Primary hippocampal neurons, isolated from wild‐type (WT) and NP1‐knockout (C57BL/6 background) mice at DIV 12 to 14 were exposed to 2 to 8 hours of oxygen glucose deprivation (OGD)—in vitro conditions that mimic human stroke. OGD exposure resulted in time‐dependent induction of NP1 (∼4‐fold), enhanced redistribution of AMAP GluR1 receptors at excitatory synapses, and increased neuronal death. We observed a significant increase in surface and synaptic GluR1 clusters that colocalized with PSD‐95 on dendrites with a simultaneous decrease in internalized GluR1. Surface cross‐linking with BS3 showed enhanced membrane insertions of GluR1, and increased phosphorylation at Ser‐845 further supported enhanced surface availability of GluR1 after OGD. NP1 protein colocalized with GluR1 and PSD‐95, and OGD significantly increased their synaptic coclustering. Most strikingly, the genetic deletion of NP1 resulted in decreases in surface GluR1 cluster density, synaptic localization, phospho‐GluR1 (Ser‐845) levels, and neuronal death after OGD compared with WT neurons. AMPA (50 μmol/L) induced NP1 and significant cell death in WT but not in NP1−/− neurons.
Our results indicate that NP1 plays a key role in synaptic clustering of GluR1, suggesting that targeting NP1 might be a practical approach to preventing ischemic brain damage.
PMCID: PMC3603251  PMID: 23525449
AMPA receptors; neuronal pentraxin 1; oxygen glucose deprivation; receptor trafficking; surface expression; synaptic clustering
14.  Long-Term Potentiation in Isolated Dendritic Spines 
PLoS ONE  2009;4(6):e6021.
In brain, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation can induce long-lasting changes in synaptic α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor (AMPAR) levels. These changes are believed to underlie the expression of several forms of synaptic plasticity, including long-term potentiation (LTP). Such plasticity is generally believed to reflect the regulated trafficking of AMPARs within dendritic spines. However, recent work suggests that the movement of molecules and organelles between the spine and the adjacent dendritic shaft can critically influence synaptic plasticity. To determine whether such movement is strictly required for plasticity, we have developed a novel system to examine AMPAR trafficking in brain synaptosomes, consisting of isolated and apposed pre- and postsynaptic elements.
Methodology/Principal Findings
We report here that synaptosomes can undergo LTP-like plasticity in response to stimuli that mimic synaptic NMDAR activation. Indeed, KCl-evoked release of endogenous glutamate from presynaptic terminals, in the presence of the NMDAR co-agonist glycine, leads to a long-lasting increase in surface AMPAR levels, as measured by [3H]-AMPA binding; the increase is prevented by an NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). Importantly, we observe an increase in the levels of GluR1 and GluR2 AMPAR subunits in the postsynaptic density (PSD) fraction, without changes in total AMPAR levels, consistent with the trafficking of AMPARs from internal synaptosomal compartments into synaptic sites. This plasticity is reversible, as the application of AMPA after LTP depotentiates synaptosomes. Moreover, depotentiation requires proteasome-dependent protein degradation.
Together, the results indicate that the minimal machinery required for LTP is present and functions locally within isolated dendritic spines.
PMCID: PMC2695539  PMID: 19547754
15.  Impaired synaptic clustering of postsynaptic density proteins and altered signal transmission in hippocampal neurons, and disrupted learning behavior in PDZ1 and PDZ2 ligand binding-deficient PSD-95 knockin mice 
Molecular Brain  2012;5:43.
Postsynaptic density (PSD)-95-like membrane-associated guanylate kinases (PSD-MAGUKs) are scaffold proteins in PSDs that cluster signaling molecules near NMDA receptors. PSD-MAGUKs share a common domain structure, including three PDZ (PDZ1/2/3) domains in their N-terminus. While multiple domains enable the PSD-MAGUKs to bind various ligands, the contribution of each PDZ domain to synaptic organization and function is not fully understood. Here, we focused on the PDZ1/2 domains of PSD-95 that bind NMDA-type receptors, and studied the specific roles of the ligand binding of these domains in the assembly of PSD proteins, synaptic properties of hippocampal neurons, and behavior, using ligand binding-deficient PSD-95 cDNA knockin (KI) mice.
The KI mice showed decreased accumulation of mutant PSD-95, PSD-93 and AMPA receptor subunits in the PSD fraction of the hippocampus. In the hippocampal CA1 region of young KI mice, basal synaptic efficacy was reduced and long-term potentiation (LTP) was enhanced with intact long-term depression. In adult KI mice, there was no significant change in the magnitude of LTP in CA1, but robustly enhanced LTP was induced at the medial perforant path-dentate gyrus synapses, suggesting that PSD-95 has an age- and subregion-dependent role. In a battery of behavioral tests, KI mice showed markedly abnormal anxiety-like behavior, impaired spatial reference and working memory, and impaired remote memory and pattern separation in fear conditioning test.
These findings reveal that PSD-95 including its ligand binding of the PDZ1/2 domains controls the synaptic clustering of PSD-MAGUKs and AMPA receptors, which may have an essential role in regulating hippocampal synaptic transmission, plasticity, and hippocampus-dependent behavior.
PMCID: PMC3575367  PMID: 23268962
PSD-MAGUK; Synaptic clustering; PDZ domain; PSD-95; Synaptic transmission; Dentate gyrus; Behavioral test battery
16.  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
17.  α7 Nicotinic Acetylcholine Receptors Occur at Postsynaptic Densities of AMPA Receptor-Positive and -Negative Excitatory Synapses in Rat Sensory Cortex 
NMDA receptor (NMDAR) activation requires concurrent membrane depolarization, and glutamatergic synapses lacking AMPA receptors (AMPARs) are often considered “silent” in the absence of another source of membrane depolarization. During the second postnatal week, NMDA currents can be enhanced in rat auditory cortex through activation of the α7 nicotinic acetylcholine receptor (α7nAChR). Electrophysiological results support a mainly presynaptic role for α7nAChR at these synapses. However, immunocytochemical evidence that α7nAChR is prevalent at postsynaptic sites of glutamatergic synapses in hippocampus and neocortex, along with emerging electrophysiological evidence for postsynaptic nicotinic currents in neocortex and hippocampus, has prompted speculation that α7nAChR allows for activation of NMDAR postsynaptically at synapses lacking AMPAR. Here we used dual immunolabeling and electron microscopy to examine the distribution of α7nAChR relative to AMPAR (GluR1, GluR2, and GluR3 subunits combined) at excitatory synapses in somatosensory cortex of adult and 1-week-old rats. α7nAChR occurred discretely over most of the thick postsynaptic densities in all cortical layers of both age groups. AMPAR immunoreactivity was also detectable at most synapses; its distribution was independent of that of α7nAChR. In both age groups, approximately one-quarter of asymmetrical synapses were α7nAChR positive and AMPAR negative. The variability of postsynaptic α7nAChR labeling density was greater at postnatal day (PD) 7 than in adulthood, and PD 7 neuropil contained a subset of small AMPA receptor-negative synapses with a high density of α7nAChR immunoreactivity. These observations support the idea that acetylcholine receptors can aid in activating glutamatergic synapses and work together with AMPA receptors to mediate postsynaptic excitation throughout life.
PMCID: PMC2839916  PMID: 12077196
sensory cortex; receptive field properties; α7 nicotinic acetylcholine receptor; glutamate; AMPA receptor; NMDA receptor; synaptic plasticity; early postnatal development; postsynaptic density; immunocytochemistry; electron microscopy
18.  Subunit- and pathway-specific localization of NMDA receptors and scaffolding proteins at ganglion cell synapses in rat retina 
Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from ON and OFF bipolar cells in distinct sublaminae of the inner plexiform layer (IPL). AMPA and NMDA receptors (AMPARs and NMDARs) mediate excitatory inputs in both synaptic layers, but specific roles for NMDARs at RGC synapses remain unclear. NMDARs comprise NR1 and NR2 subunits and are anchored by membrane associated guanylate kinases (MAGUKs), but it is unknown whether particular NR2 subunits associate preferentially with particular NR1 splice variants and MAGUKs. Here, we used postembedding immunogold electron microscopy (EM) techniques to examine the subsynaptic localization of NMDAR subunits and MAGUKs at ON and OFF synapses onto rat RGCs. We found that the NR2A subunit, the NR1C2‘ splice variant and MAGUKs PSD-95 and PSD-93 are localized to the postsynaptic density (PSD), preferentially at OFF synapses, whereas the NR2B subunit, the NR1C2 splice variant and the MAGUK SAP102 are localized perisynaptically, with NR2B exhibiting a preference for ON synapses. Consistent with these anatomical data, spontaneous EPSCs (sEPSCs) recorded from OFF cells exhibited an NMDAR component that was insensitive to the NR2B antagonist Ro 25-6981. In ON cells, sEPSCs expressed an NMDAR component, partially sensitive to Ro 25-6981, only when glutamate transport was inhibited, indicating perisynaptic expression of NR2B NMDARs. These results provide the first evidence for preferential association of particular NR1 splice variants, NR2 subunits and MAGUKs at central synapses and suggest that different NMDAR subtypes may play specific roles at functionally distinct synapses in the retinal circuitry.
PMCID: PMC4283557  PMID: 19339621
19.  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
20.  Astrocytes increase the activity of synaptic GluN2B NMDA receptors 
Astrocytes regulate excitatory synapse formation and surface expression of glutamate AMPA receptors (AMPARs) during development. Less is known about glial modulation of glutamate NMDA receptors (NMDARs), which mediate synaptic plasticity and regulate neuronal survival in a subunit- and subcellular localization-dependent manner. Using primary hippocampal cultures with mature synapses, we found that the density of NMDA-evoked whole-cell currents was approximately twice as large in neurons cultured in the presence of glia compared to neurons cultured alone. The glial effect was mediated by (an) astrocyte-secreted soluble factor(s), was Mg2+ and voltage independent, and could not be explained by a significant change in the synaptic density. Instead, we found that the peak amplitudes of total and NMDAR miniature excitatory postsynaptic currents (mEPSCs), but not AMPAR mEPSCs, were significantly larger in mixed than neuronal cultures, resulting in a decreased synaptic AMPAR/NMDAR ratio. Astrocytic modulation was restricted to synaptic NMDARs that contain the GluN2B subunit, did not involve an increase in the cell surface expression of NMDAR subunits, and was mediated by protein kinase C (PKC). Taken together, our findings indicate that astrocyte-secreted soluble factor(s) can fine-tune synaptic NMDAR activity through the PKC-mediated regulation of GluN2B NMDAR channels already localized at postsynaptic sites, presumably on a rapid time scale. Given that physiologic activation of synaptic NMDARs is neuroprotective and that an increase in the synaptic GluN2B current is associated with improved learning and memory, the astrocyte-induced potentiation of synaptic GluN2B receptor activity is likely to enhance cognitive function while simultaneously strengthening neuroprotective signaling pathways.
PMCID: PMC4400914  PMID: 25941471
NMDA receptor; GluN2B subunit; astrocytes; synapses; protein kinase C
21.  Estrogen receptor alpha and beta specific agonists regulate expression of synaptic proteins in rat hippocampus 
Brain research  2009;1290:1-11.
Changes in hippocampal CA1 dendritic spine density and synaptic number across the estrous cycle in female rats correlate with increased hippocampal-dependent cognitive performance in a manner that is dependent on estrogen receptors (ERs). Two isoforms of the estrogen receptor, α and β are present in the rat hippocampus and distinct effects on cognitive behavior have been described for each receptor. The present study generated a profile of synaptic proteins altered by administration of estradiol benzoate, the ERα selective agonist PPT (1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole) and the ERβ selective agonist DPN (2,3-bis (4-hydroxyphenyl) propionitrile) alone and in combination in comparison to vehicle in the CA1 region of the dorsal hippocampus. In the stratum radiatum, estradiol, DPN, and PPT increased PSD-95 and AMPA-type glutamate receptor subunit GluR1. Only DPN administration regulated expression of AMPA receptor subunits GluR2 and GluR3, increasing and decreasingly levels respectively. DPN also increased GluR2 expression in the other lamina of the CA1. These results support previous reports that estradiol and isoform specific agonists differentially activate ERα and ERβ to regulate protein expression. The distinct effects of DPN and PPT administration on synaptic proteins, suggest that the desired therapeutic outcome of estrogen may be accomplished by using specific estrogen receptor agonists. Moreover, the effects of estradiol treatment on PSD-95 expression are consistent with a growing body of evidence that this postsynaptic protein is a key marker of estrogen action related to spine synapse formation.
PMCID: PMC2778245  PMID: 19596275
Hippocampus; Synaptic proteins; Estradiol benzoate; PPT (1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole); DPN (2,3-bis(4-hydroxyphenyl) propionitrile); Estrogen receptor agonists
22.  Soluble epoxide hydrolase inhibitor enhances synaptic neurotransmission and plasticity in mouse prefrontal cortex 
The soluble epoxide hydrolase (sEH) is an important enzyme chiefly involved in the metabolism of fatty acid signaling molecules termed epoxyeicosatrienoic acids (EETs). sEH inhibition (sEHI) has proven to be protective against experimental cerebral ischemia, and it is emerging as a therapeutic target for prevention and treatment of ischemic stroke. However, the role of sEH on synaptic function in the central nervous system is still largely unknown. This study aimed to test whether sEH C-terminal epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) affects basal synaptic transmission and synaptic plasticity in the prefrontal cortex area (PFC). Whole cell and extracellular recording examined the miniature excitatory postsynaptic currents (mEPSCs) and field excitatory postsynaptic potentials (fEPSPs); Western Blotting determined the protein levels of glutamate receptors and ERK phosphorylation in acute medial PFC slices.
Application of the sEH C-terminal epoxide hydrolase inhibitor, AUDA significantly increased the amplitude of mEPSCs and fEPSPs in prefrontal cortex neurons, while additionally enhancing long term potentiation (LTP). Western Blotting demonstrated that AUDA treatment increased the expression of the N-methyl-D-aspartate receptor (NMDA) subunits NR1, NR2A, NR2B; the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR1, GluR2, and ERK phosphorylation.
Inhibition of sEH induced an enhancement of PFC neuronal synaptic neurotransmission. This enhancement of synaptic neurotransmission is associated with an enhanced postsynaptic glutamatergic receptor and postsynaptic glutamatergic receptor mediated synaptic LTP. LTP is enhanced via ERK phosphorylation resulting from the delivery of glutamate receptors into the PFC by post-synapse by treatment with AUDA. These findings provide a possible link between synaptic function and memory processes.
PMCID: PMC4618874  PMID: 26494028
Soluble epoxide hydrolase; Prefrontal cortex; Excitatory synaptic neurotransmission
Neuroscience  2008;153(1):44-53.
Development of the mammalian CNS requires formation and stabilization of neuronal circuits and synaptic connections. Sensory stimulation provided by the environment orchestrates neuronal circuit formation in the waking state. Endogenous sources of activation are also implicated in these processes. Accordingly we hypothesized that sleep, especially rapid eye movement sleep (REMS), the stage characterized by high neuronal activity that is more prominent in development than adulthood, provides endogenous stimulation, which, like sensory input, helps to stabilize and refine neuronal circuits during CNS development. Young (Y: postnatal day (PN) 16) and adolescent (A: PN44) rats were rapid eye movement sleep-deprived (REMSD) by gentle cage-shaking for only 4 h on 3 consecutive days (total 12 h). The effect of REMS deprivation in Y and A rats was tested 3-7 days after the last deprivation session (Y, PN21-25; A, PN49-53) and was compared with younger (immature, I, PN9-12) untreated, age-matched, treated and normal control groups.
REMS deprivation negatively affected the stability of long-term potentiation (LTP) in Y but not A animals. LTP instability in Y-REMSD animals was similar to the instability in even the more immature, untreated animals. Utilizing immunoblots, we identified changes in molecular components of glutamatergic synapses known to participate in mechanisms of synaptic refinement and plasticity. Overall, N-methyl-d-aspartate receptor subunit 2B (NR2B), N-methyl-d-aspartate receptor subunit 2A, AMPA receptor subunit 1 (GluR1), postsynaptic density protein 95 (PSD-95), and calcium/calmodulin kinase II tended to be lower in Y REMSD animals (NR2B, GluR1 and PSD-95 were significantly lower) compared with controls, an effect not present in the A animals. Taken together, these data indicate that early-life REMS deprivation reduces stability of hippocampal neuronal circuits, possibly by hindering expression of mature glutamatergic synaptic components. The findings support a role for REMS in the maturation of hippocampal neuronal circuits.
PMCID: PMC2389877  PMID: 18359575
synaptic plasticity; rat; CaMKII; NMDA; AMPA; PSD-95
24.  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
25.  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

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