Conversion of silent synapses into the active pool by selective GluR1-3 and GluR4 AMPAR trafficking during in vitro classical conditioning

The conversion of silent synapses into active sites is hypothesized to be a primary mechanism underlying learning and memory processes. Here we used an in vitro model of classical conditioning from turtles that demonstrates a neural correlate of eyeblink conditioning to examine whether the conversion of silent synapses has a role in this form of associative learning. This was accomplished by direct visualization of AMPA receptor (AMPAR) and N-methyl-d-aspartate receptor (NMDAR) subunits colocalized with synaptophysin (Syn) using immunofluorescence and confocal microscopy. In naive preparations, there was a relatively high level of synapses immunopositive for NR1-Syn alone interpreted to be silent synapses. After early stages of conditioning during acquisition of conditioned responses (CRs), there was a significant increase in the colocalization of GluR1-3 AMPAR subunits at NR1-immunopositive synaptic sites. Later in conditioning, levels of GluR1-3 declined and enhanced colocalization of GluR4-containing AMPAR subunits at synapses was observed. The trafficking of these subunits during conditioning was NMDAR mediated and was accompanied by protein synthesis of GluR4 subunits. Examination of the postsynaptic density fraction confirmed the early and late synaptic insertion of GluR1-3 and GluR4, respectively, during conditioning. These findings suggest that there is differential trafficking of synaptic AMPARs during classical conditioning. Existing GluR1-3 AMPAR subunits are initially delivered to silent synapses early in conditioning to unsilence them followed by synthesis and insertion of GluR4 AMPAR subunits that are required for acquisition and expression of CRs.     

Subunit interaction with PICK and GRIP controls Ca2+ permeability of AMPARs at cerebellar synapses

At many excitatory central synapses, activity produces a lasting change in the synaptic response by modifying postsynaptic AMPA receptors (AMPARs). Although much is known about proteins involved in the trafficking of Ca2+-impermeable (GluR2-containing) AMPARs, little is known about protein partners that regulate subunit trafficking and plasticity of Ca2+-permeable (GluR2-lacking) AMPARs. At cerebellar parallel fiber-stellate cell synapses, activity triggers a novel type of plasticity: Ca2+ influx through GluR2-lacking synaptic AMPARs drives incorporation of GluR2-containing AMPARs, generating rapid, lasting changes in excitatory postsynaptic current properties. Here we examine how glutamate receptor interacting protein (GRIP, also known as AMPAR binding protein or ABP) and protein interacting with C-kinase-1 (PICK) regulate subunit trafficking and plasticity. We find that repetitive synaptic activity triggers loss of synaptic GluR2-lacking AMPARs by selectively disrupting their interaction with GRIP and that PICK drives activity-dependent delivery of GluR2-containing receptors. This dynamic regulation of AMPARs provides a feedback mechanism for controlling Ca2+ permeability of synaptic receptors.     

GluN2B subunits of the NMDA receptor contribute to the AMPA receptor internalization during long-term depression in the lateral amygdala of juvenile rats

The lateral nucleus of the amygdala (LA) is a critical structure involved in fear conditioning. We recently showed that regulated exocytosis and endocytosis of postsynaptic A-amino-3-hydroxy-5-methylisoxazole-4-propionate subtype of glutamate receptors (AMPARs) are involved in the expression of N-methyl-D-aspartate subtype glutamate receptors (NMDARs) dependent long-term potentiation (LTP) and long-term depression (LTD) in coronal slices of the LA. However, the molecular mechanisms of this effect remain unclear. In the present study, we investigated the role of distinct NMDAR subtypes in the endocytosis of AMPARs during LTD expression at the synapses of the thalamic inputs to the LA neurons. Here we show that the NMDARs antagonist DL-2-amino-5-phosphonovalerate (D-APV) blocked the induction of LTD and thus prevented endocytosis of surface AMPARs, indicating that NMDAR activation enhanced the internalization of AMPARs in LTD expression. Furthermore, the selective blocking of GluN2B-containing NMDARs completely abolished the NMDAR-induced AMPAR endocytosis, whereas preferential inhibition of GluN2A-containing NMDARs did not block the NMDAR-induced AMPAR endocytosis during LTD expression. These results suggest that there exist a preferred NMDAR subtype for AMPAR internalization and activation of GluN2B-containing NMDARs represent the predominate pathway triggered during the early stages of this NMDAR-induced endocytosis of AMPARs during LTD in the thalamic inputs to the LA of juvenile rats.     

Activation of metabotropic glutamate receptors does not alter the phosphorylation state of GluR1 AMPA receptor subunit at serine 845 in perirhinal cortical neurons

Activation of group I and group II metabotropic glutamate receptors (mGluRs) is thought to be required for long-term depression (LTD) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor-mediated synaptic transmission in the perirhinal cortex. However, little is known about how activation of mGluRs leads to this form of synaptic plasticity. AMPA receptor phosphorylation has been implicated in several forms of modulation of synaptic transmission. In the CA1 area of the hippocampus, N-methyl-d-aspartate (NMDA) receptor-dependent LTD is associated with the reduced phosphorylation of the GluR1 AMPA receptor subunit at serine 845 (GluR1-S845). Immunoblot analysis of perirhinal cortical neurons using GluR1 and GluR1-S845 phosphorylation state specific antibodies showed that stimulation of adenylyl cyclase (AC) with forskolin (FSK) dramatically increased PKA-mediated phosphorylation of GluR1-S845. However, selective or simultaneous application of mGluR5 agonist (S)-3,5-dihydroxyphenylglycine (CHPG) and mGluR2/3 agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) did not produce detectable changes in GluR1-S845 phosphorylation. These results indicate that in the perirhinal cortex mGluR activation does not alter the phosphorylation state of GluR1-S845. Therefore, it is likely that the process involved in the modification of AMPA receptors in mGluR activation dependent LTD in the perirhinal cortex is mechanistically distinct from NMDA receptor-mediated LTD described in hippocampal neurons.     

Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors

Regulation of AMPA receptor (AMPAR) trafficking is important for neural plasticity. Here we examined the trafficking and synthesis of the GluR1 and GluR2 subunits using ReAsH-EDT(2) and FlAsH-EDT(2) staining. Activity blockade of rat cultured neurons increased dendritic GluR1, but not GluR2, levels. Examination of transected dendrites revealed that both AMPAR subunits were synthesized in dendrites and that activity blockade enhanced dendritic synthesis of GluR1 but not GluR2. In contrast, acute pharmacological manipulations increased dendritic synthesis of both subunits. AMPARs synthesized in dendrites were inserted into synaptic plasma membranes and, after activity blockade, the electrophysiological properties of native synaptic AMPARs changed in the manner predicted by the imaging experiments. In addition to providing a novel mechanism for synaptic modifications, these results point out the advantages of using FlAsH-EDT(2) and ReAsH-EDT(2) for studying the trafficking of newly synthesized proteins in local cellular compartments such as dendrites.     

Adenylyl cyclase I regulates AMPA receptor trafficking during mouse cortical 'barrel' map development

Cortical map formation requires the accurate targeting, synaptogenesis, elaboration and refinement of thalamocortical afferents. Here we demonstrate the role of Ca2+/calmodulin-activated type-I adenylyl cyclase (AC1) in regulating the strength of thalamocortical synapses through modulation of AMPA receptor (AMPAR) trafficking using barrelless mice, a mutant without AC1 activity or cortical 'barrel' maps. Barrelless synapses are stuck in an immature state that contains few functional AMPARs that are rarely silent (NMDAR-only). Long-term potentiation (LTP) and long-term depression (LTD) at thalamocortical synapses require postsynaptic protein kinase A (PKA) activity and are difficult to induce in barrelless mice, probably due to an inability to properly regulate synaptic AMPAR trafficking. Consistent with this, both the extent of PKA phosphorylation on AMPAR subunit GluR1 and the expression of surface GluR1 are reduced in barrelless neurons. These results suggest that activity-dependent mechanisms operate through an AC1/PKA signaling pathway to target some synapses for consolidation and others for elimination during barrel map formation.     

Reduced glycine transporter type 1 expression leads to major changes in glutamatergic neurotransmission of CA1 hippocampal neurones in mice

To investigate the effects of persistent elevation of synaptic glycine at Schaffer collateral–CA1 synapses of the hippocampus, we studied the glutamatergic synaptic transmission in acute brain slices from mice with reduced expression of glycine transporter type 1 (GlyT1+/−) as compared to wild type (WT) littermates using whole-cell patch-clamp recordings of CA1 pyramidal cells. We observed faster decay kinetics, reduced ifenprodil sensitivity and increased zinc-induced antagonism in N -methyl- d -aspartate receptor (NMDAR) currents of GlyT1+/− mice. Moreover, the ratio α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)/NMDAR was decreased in mutants compared to WT. Surprisingly, this change was associated with a reduction in the number of AMPARs expressed at the CA1 synapses in the mutants compared to WT. Overall, these findings highlight the importance of GlyT1 in regulating glutamatergic neurotransmission.     

PDZ binding of TARPγ-8 controls synaptic transmission but not synaptic plasticity

The reduction in synaptic transmission and plasticity in mice lacking the hippocampus-enriched AMPA receptor (AMPAR) auxiliary subunit TARPγ-8 could be a result of a reduction in AMPAR expression or of the direct action of γ-8. We generated TARPγ-8Δ4 knock-in mice lacking the C-terminal PDZ ligand. We found that synaptic transmission and AMPARs were reduced in the mutant mice, but extrasynaptic AMPAR expression and long-term potentiation (LTP) were unaltered. Our findings suggest that there are distinct TARP-dependent mechanisms for synaptic transmission and LTP.     

Distinct triggering and expression mechanisms underlie LTD of AMPA and NMDA synaptic responses

Although long-term depression (LTD) of AMPA receptor-mediated postsynaptic currents (AMPAR EPSCs) has been extensively examined, little is known about the mechanisms responsible for LTD of NMDA receptor (NMDAR)-mediated EPSCs. Here we show differences in the intracellular signaling cascades that mediate LTD of AMPAR EPSCs versus NMDAR EPSCs in rat hippocampus. Both forms of LTD were blocked by inhibitors of protein phosphatase 1, but only LTD of AMPAR EPSCs was affected by inhibition of calcineurin. Notably, in contrast to LTD of AMPAR EPSCs, LTD of NMDAR EPSCs was unaffected by endocytosis inhibitors. A role for calcium-dependent actin depolymerization in LTD of NMDAR EPSCs was supported by the findings that the actin stabilizer phalloidin and a cofilin inhibitory peptide each blocked LTD of NMDAR EPSCs but not AMPAR EPSCs. These results suggest that the same pattern of afferent activity elicits depression of AMPAR- and NMDAR-mediated synaptic responses by means of distinct triggering and expression mechanisms.     

Cocaine triggered AMPA receptor redistribution is reversed in vivo by mGluR-dependent long-term depression

Drugs of abuse induce long-lasting changes in neural circuits that may underlie core components of addiction. Here we focus on glutamatergic synapses onto dopamine (DA) neurons of the ventral tegmental area (VTA). Using an 'ex vivo' approach in mice, we show that a single injection of cocaine caused strong rectification and conferred sensitivity to the polyamine Joro spider toxin (JST) of AMPAR-mediated excitatory postsynaptic currents (AMPAR EPSCs), indicating the recruitment of receptors that lack GluR2. This qualitative change in transmission was paralleled by an increase in the AMPAR:NMDAR ratio and was prevented by interfering with the protein interacting with C kinase-1 (PICK1) in vivo. Activation of metabotropic glutamate receptors (mGluR1s) by intraperitoneal injection of a positive modulator depotentiated synapses and abolished rectification in slices of cocaine-treated mice, revealing a mechanism to reverse cocaine-induced synaptic plasticity in vivo.     

Potentiation of synaptic AMPA receptors induced by the deletion of NMDA receptors requires the GluA2 subunit

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

alpha-Isoform of calcium-calmodulin-dependent protein kinase II and postsynaptic density protein 95 differentially regulate synaptic expression of NR2A- and NR2B-containing N-methyl-d-aspartate receptors in hippocampus

N-methyl-d-aspartate receptors (NMDARs) are critical determinants of bidirectional synaptic plasticity, however, studies of NMDAR function have been based primarily on pharmacological and electrophysiological manipulations, and it is still debated whether there are subunit-selective forms of long-term potentiation (LTP) and long-term depression (LTD). Here we provide ultrastructural analyses of axospinous synapses in cornu ammonis field 1 of hippocampus (CA1) stratum radiatum of transgenic mice with mutations to two key underlying postsynaptic density (PSD) proteins, postsynaptic density protein 95 (PSD-95) and the alpha-isoform of calcium-calmodulin-dependent protein kinase II (alphaCaMKII). Distribution profiles of synaptic proteins in these mice reveal very different patterns of subunit-specific NMDAR localization, which may be related to the divergent phenotypes of the two mutants. In PSD-95, Dlg, ZO-1/Dlg-homologous region (PDZ) 3-truncated mutant mice in which LTD could not be induced but LTP was found to be enhanced, we found a subtle, yet preferential displacement of synaptic N-methyl-d-aspartate receptor subunit 2B (NR2B) subunits in lateral regions of the synapse without affecting changes in the localization of N-methyl-d-aspartate receptor subunit 2A (NR2A) subunits. In persistent inhibitory alphaCaMKII Thr305 substituted with Asp in alpha-isoform of calcium-calmodulin kinase II (T305D) mutant mice with severely impaired LTP but stable LTD expression, we found a selective reduction of NR2A subunits at both the synapse and throughout the cytoplasm of the spine without any effect on the NR2B subunit. In an experiment of mutual exclusivity, neither PSD-95 nor alphaCaMKII localization was found to be affected by mutations to the corresponding PSD protein suggesting that they are functionally independent of the other in the regulation of NR2A- and NR2B-containing NMDARs preceding synaptic activity. Consequently, there may exist at least two distinct PSD-95 and alphaCaMKII-specific NMDAR complexes involved in mediating LTP and LTD through opposing signal transduction pathways in synapses of the hippocampus. The contrasting phenotypes of the PSD-95 and alphaCaMKII mutant mice further establish the prospect of an independent and, possibly, competing mechanism for the regulation of NMDAR-dependent bidirectional synaptic plasticity.     

Metabotropic receptor-dependent long-term depression persists in the absence of protein synthesis in the mouse model of fragile X syndrome

Fragile X syndrome (FXS), a form of human mental retardation, is caused by loss of function mutations in the fragile X mental retardation gene (FMR1). The protein product of FMR1, fragile X mental retardation protein (FMRP) is an RNA-binding protein and may function as a translational suppressor. Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) in hippocampal area CA1 is a form of synaptic plasticity that relies on dendritic protein synthesis. mGluR-LTD is enhanced in the mouse model of FXS, Fmr1 knockout (KO) mice, suggesting that FMRP negatively regulates translation of proteins required for LTD. Here we examine the synaptic and cellular mechanisms of mGluR-LTD in KO mice and find that mGluR-LTD no longer requires new protein synthesis, in contrast to wild-type (WT) mice. We further show that mGluR-LTD in KO and WT mice is associated with decreases in AMPA receptor (AMPAR) surface expression, indicating a similar postsynaptic expression mechanism. However, like LTD, mGluR-induced decreases in AMPAR surface expression in KO mice persist in protein synthesis inhibitors. These results are consistent with recent findings of elevated protein synthesis rates and synaptic protein levels in Fmr1 KO mice and suggest that these elevated levels of synaptic proteins are available to increase the persistence of LTD without de novo protein synthesis.     

AMPA receptor-dependent, light-evoked D-serine release acts on retinal ganglion cell NMDA receptors

NMDA receptor (NMDAR) activation requires coincident binding of the excitatory neurotransmitter glutamate and a coagonist, either glycine or d-serine. Changes in NMDAR currents during neural transmission are typically attributed to glutamate release against a steady background of coagonist, excluding the possibility of coagonist release. AMPA receptor (AMPAR) stimulation evokes d-serine release, but it is unknown whether this is a physiological phenomenon capable of influencing synaptic responses. In this study, we utilized the intact retina to determine whether light-evoked synaptic activity in retinal ganglion cells (RGCs) is shaped by a dynamic pool of coagonist. The application of AMPAR antagonist abolished light-evoked NMDAR currents, which were rescued by adding coagonist to the bath. When NMDA was globally applied to RGCs via bath or picospritzing, the coagonist occupancy was also dependent on AMPARs but to a lesser extent than that observed during light responses, suggesting a difference in extrasynaptic coagonist regulation. By saturating the glutamate binding site of NMDARs, we were able to detect released coagonist reaching RGCs during light-evoked responses. Mutant mice lacking the d-serine-synthesizing enzyme serine racemase were deficient in coagonist release. Coagonist release in wild-type retinas was notably greater in ON than in OFF responses and depended on AMPARs. These findings suggest activity-dependent modulation of coagonist availability, particularly d-serine, and may add an extra dimension to NMDAR coincidence detection in the retina.     

Double dissociation between long-term depression and dendritic spine morphology in cerebellar Purkinje cells

Experiments in hippocampal area CA1 suggest that long-term potentiation could be associated with spine addition and enlargement, and long-term depression (LTD) with spine shrinkage and loss. Is this a general principle of synaptic plasticity? We used two-photon microscopy to measure dendritic spines in rat cerebellar Purkinje cells. Neither local synaptic induction of LTD nor global chemical induction of LTD changed spine number or size. Conversely, a manipulation that evoked persistent dendritic spine retraction did not alter parallel fiber-evoked excitatory postsynaptic currents.     

Chemical induction of mGluR5- and protein synthesis--dependent long-term depression in hippocampal area CA1.

Recent work has demonstrated that specific patterns of synaptic stimulation can induce long-term depression (LTD) in area CA1 that depends on activation of metabotropic glutamate receptors (mGluRs) and rapid protein synthesis. Here we show that the same form of synaptic modification can be induced by brief application of the selective mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG). DHPG-LTD 1) is a saturable form of synaptic plasticity, 2) requires mGluR5, 3) is mechanistically distinct from N-methyl-D-aspartate receptor (NMDAR)--dependent LTD, and 4) shares a common expression mechanism with protein synthesis-dependent LTD evoked using synaptic stimulation. DHPG-LTD should be useful for biochemical analysis of mGluR5- and protein synthesis-dependent synaptic modification.     

Distribution of AMPA receptor subunits and TARPs in synaptic and extrasynaptic membranes of the adult rat nucleus accumbens

We characterized the distribution of AMPA receptor (AMPAR) subunits and the transmembrane AMPA receptor regulatory proteins (TARPs) γ-2 and γ-4 in adult rat nucleus accumbens (NAc) using a method that separates plasma membranes into synaptic membrane-enriched and extrasynaptic membrane-enriched fractions. We also measured GluA1 phosphorylated at serine 845 (pS845 GluA1) and serine 831 (pS831 GluA1). GluA1-3 protein levels and pS831 GluA1/total GluA1 were higher in synaptic membranes. However, pS845 GluA1/total GluA1 was higher in extrasynaptic membranes, consistent with a role for S845 phosphorylation in GluA1 insertion at extrasynaptic sites. Homeric GluA1 receptors were detected in extrasynaptic membranes, consistent with evidence for extrasynaptic Ca(2+)-permeable AMPARs in other systems. The TARP γ-2 was enriched in synaptic membranes, whereas γ-4 was mainly found in extrasynaptic membranes, suggesting distinct roles for these proteins in the NAc. These experiments provide fundamental information that will aid in the interpretation of studies on AMPAR-related plasticity in the NAc.     

Coexistence of muscarinic long-term depression with electrically induced long-term potentiation and depression at CA3-CA1 synapses

Our laboratory recently characterized a form of long-term depression (LTD) at CA3-CA1 synapses mediated by M1 muscarinic receptors (mAChRs), termed muscarinic LTD (mLTD). mLTD is both activity and NMDAR dependent, characteristics shared by forms of synaptic plasticity thought to be relevant to learning and memory, including long-term potentiation (LTP) induced by high-frequency stimulation (HFS-LTP) and long-term depression induced by low-frequency stimulation (LFS-LTD). However, it remains unclear whether mLTD can occur sequentially with these electrically induced forms of hippocampal plasticity or whether mLTD might interact with them. The first goal of this study was to examine the interplay of mLTD and HFS-LTP. We report that mLTD expression does not alter subsequent induction of HFS-LTP and, further, at synapses expressing HFS-LTP, mLTD can mediate a novel form of depotentiation. The second goal was to determine whether mLTD would alter LFS-LTD induction and/or expression. Although we show that mLTD is occluded by saturation of LFS-LTD, suggesting mechanistic similarity between these two plasticities, saturation of mLTD does not occlude LFS-LTD. Surprisingly, however, the LFS-LTD that follows cholinergic receptor activation is NMDAR independent, indicating that application of muscarinic agonist induces a change in the induction mechanism required for LFS-LTD. These data demonstrate that mLTD can coexist with electrically induced forms of synaptic plasticity and support the hypothesis that mLTD is one of the mechanisms by which the cholinergic system modulates hippocampal function.