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Summary

Mutations in TSPAN7—a member of the tetraspanin protein superfamily—are implicated in some forms of X-linked intellectual disability. Here we show that TSPAN7 overexpression promotes the formation of filopodia and dendritic spines in cultured hippocampal neurons from embryonic rats, whereas TSPAN7 silencing reduces head size and stability of spines and AMPA receptor currents. Via its C terminus, TSPAN7 interacts with the PDZ domain of protein interacting with C kinase 1 (PICK1), to regulate PICK1 and GluR2/3 association and AMPA receptor trafficking. These findings indicate that, in hippocampal neurons, TSPAN7 regulates AMPA receptor trafficking by limiting PICK1 accessibility to AMPA receptors and suggest an additional mechanism for the functional maturation of glutamatergic synapses, whose impairment is implicated in intellectual disability.

Highlights

► TSPAN7 is required for spine maturation in hippocampal neurons ► TSPAN7 knockdown impairs AMPAR currents ► TSPAN7 binds PICK1 and through this interaction regulates AMPAR trafficking

Mutations in TSPAN7 protein cause human intellectual disability. Bassani et al. now find that TSPAN7 regulates trafficking of essential receptor proteins to neuron surfaces and that absence impairs neuronal maturation in young animals, potentially underlying this intellectual disability.

Summary

PICK1 is a calcium-sensing, PDZ domain-containing protein that interacts with GluR2 and GluR3 AMPA receptor (AMPAR) subunits and regulates their trafficking. Although PICK1 has been principally implicated in long-term depression (LTD), PICK1 over expression in CA1 pyramidal neurons causes a CaMK- and PKC-dependent potentiation of AMPAR-mediated transmission and an increase in synaptic GluR2-lacking AMPARs, mechanisms associated with NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we directly tested whether PICK1 participates in both hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1 potentiation of AMPAR-mediated transmission is NMDAR-dependent and fully occludes LTP. Conversely, blockade of PICK1 PDZ interactions or lack of PICK1 prevents LTP. These observations demonstrate an important role for PICK1 in LTP. In addition, deletion of PICK1 or blockade of PICK1 PDZ binding prevented NMDAR-dependent LTD. Thus PICK1 plays a critical role in bidirectional NMDAR-dependent long-term synaptic plasticity in the hippocampus.

Trafficking of AMPA receptors is important for many forms of synaptic plasticity. However, the link between activity and resulting synaptic alterations is not fully understood. Here, we identified a direct interaction between NSF, an ATPase involved in membrane fusion events and stabilization of surface AMPARs, and Plk2, an activity-inducible kinase that homeostatically decreases excitatory synapse number and strength. Plk2 disrupted interaction of NSF with the GluA2 subunit of AMPARs, promoting extensive loss of surface GluA2 in rat hippocampal neurons, greater association of GluA2 with adapter proteins PICK1 and GRIP1, and decreased synaptic AMPAR current. Plk2 engagement of NSF, but not Plk2 kinase activity, was required for this mechanism and occurred through a novel motif within Plk2 independent from canonical polo box interaction sites. These data reveal that heightened synaptic activity, acting through Plk2, leads to homeostatic decreases in surface AMPAR expression via the direct dissociation of NSF from GluA2.

Receptor subunit composition is believed to play a major role in the synaptic trafficking of AMPA receptors (AMPARs), and thus in activity-dependent synaptic plasticity. To isolate a physiological role of GluA1-containing AMPARs in area CA3 of the hippocampus, pair recordings were performed in organotypic hippocampal slices taken from genetically modified mice lacking the GluA1 subunit. We report here that long-term potentiation (LTP) is impaired not only at active but also at silent synapses when the GluA1 subunit is absent. The GluA1 knockout mice also exhibited reduced AMPAR-mediated evoked currents between pairs of CA3 pyramidal neurons under baseline conditions suggesting a significant role for GluA1-containing AMPARs in regulating basal synaptic transmission. In two independent measures, however, long-term depression (LTD) was unaffected in tissue from these mice. These data provide a further demonstration of the fundamental role that GluA1-containing AMPARs play in activity-dependent increases in synaptic strength but do not support a GluA1-dependent mechanism for reductions in synaptic strength.

Synaptic plasticity, the cellular basis of learning and memory, involves the dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses. One of the remaining key unanswered aspects of AMPAR trafficking is the mechanism by which synaptic strength is preserved in spite of protein turnover. In particular, the identity of AMPAR scaffolding molecule(s) involved in the maintenance of GluA2-containing AMPARs is completely unknown. Here we report that Synaptic scaffolding molecule (S-SCAM, also called membrane-associated guanylate kinase inverted-2 and atrophin interacting protein-1) plays the critical role of maintaining synaptic strength. Increasing S-SCAM levels in rat hippocampal neurons led to specific increases in the surface AMPAR levels, enhanced AMPAR-mediated synaptic transmission, and enlargement of dendritic spines, without significantly effecting GluN levels or NMDAR EPSC. Conversely, decreasing S-SCAM levels by RNA interference-mediated knockdown caused the loss of synaptic AMPARs, which was followed by a severe reduction in the dendritic spine density. Importantly, S-SCAM regulated synaptic AMPAR levels in a manner, dependent on GluA2 not GluA1, sensitive to NSF interaction, and independent of activity. Further, S-SCAM increased surface AMPAR levels in the absence of PSD-95, while PSD-95 was dependent on S-SCAM to increase surface AMPAR levels. Finally, S-SCAM overexpression hampered NMDA-induced internalization of AMPARs and prevented the induction of long term depression, while S-SCAM knockdown did not affect long term depression. Together, these results suggest that S-SCAM is an essential AMPAR scaffolding molecule for the GluA2-containing pool of AMPARs, which are involved in the constitutive pathway of maintaining synaptic strength.

NMDA receptor (NMDAR)-dependent LTD in the hippocampus is mediated primarily by the calcium-dependent removal of AMPA receptors (AMPARs) from the postsynaptic density. The AMPAR-binding, PDZ and BAR domain containing protein PICK1 has been implicated in the regulation of AMPAR trafficking underlying several forms of synaptic plasticity. Using a strategy involving shRNA-mediated knockdown of PICK1 and its replacement with recombinant PICK1, we performed a detailed structure-function analysis of the role of PICK1 in hippocampal synaptic plasticity and the underlying NMDAR-induced AMPAR trafficking. We found that PICK1 is not necessary for maintenance of the basal synaptic complement of AMPARs or expression of either mGluR-LTD or NMDAR-dependent LTP. Rather, PICK1 function is specific to NMDAR-dependent LTD and the underlying AMPAR trafficking. Furthermore, while PICK1 does not regulate the initial phase of NMDAR-induced AMPAR endocytosis, it is required for intracellular retention of internalized AMPARs. Detailed biophysical analysis of an N-terminal acidic motif indicated that it is involved in intramolecular electrostatic interactions that are disrupted by calcium. Mutations that interfered with the calcium-induced structural changes in PICK1 precluded LTD and the underlying NMDAR-induced intracellular retention of AMPARs. These findings support a model whereby calcium-induced modification of PICK1 structure is critical for its function in the retention of internalized AMPARs that underlies the expression of hippocampal NMDAR-dependent LTD.

PICK1 (protein interacting with C kinase-1) regulates the surface expression of the AMPA receptor (AMPAR) GluR2 subunit, however, the functional consequences of this interaction are not well understood. Previous work has suggested that PICK1 promotes the internalization of AMPARs. However, we found that when PICK1 is virally expressed in the CA1 region of hippocampal slices, it causes an increase in AMPAR-mediated EPSC amplitude. This effect is associated with increased AMPAR rectification and sensitivity to polyamine toxin. These effects are blocked by PKC or calcium/calmodulin-dependent protein kinase II inhibitors, indicating that the virally expressed PICK1 signals through an endogenous kinase cascade. In contrast, blockade of interactions with GluR2 at the N-ethylmaleimide-sensitive factor site did not cause a change in subunit composition, suggesting that the effects of PICK1 are not simply a nonspecific consequence of removing AMPARs from the surface. Immunocytochemical and biochemical analyses in dissociated cultured hippocampal neurons show that PICK1 causes a decrease in endogenous GluR2 surface expression but no change in GluR1 surface levels. To address the physiological role of PICK1, we virally expressed C-terminal GluR2 peptides. Blockade of endogenous PICK1 PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain interactions produced opposite effects on synaptic strength and AMPAR rectification to those observed with PICK1 expression. This demonstrates that AMPAR subunit composition is physiologically regulated through a mechanism involving PICK1 PDZ domain interactions. These findings suggest that PICK1 acts to downregulate the GluR2 content of AMPARs at hippocampal CA1 synapses, thereby increasing synaptic strength at resting membrane potentials.

Regulated trafficking controls AMPA receptor (AMPAR) number at the postsynaptic membrane to modify the efficiency of synaptic transmission. The PDZ proteins GRIP1 and the related ABP-L/GRIP2 bind AMPAR subunit GluA2, and have been proposed to play a role in AMPAR trafficking associated with Long Term Depression (LTD) of synaptic transmission. Both GRIP1 and ABP-L/GRIP2 exist in different splice isoforms, including alternative 18 amino acid domains at the extreme N-terminus, which determine whether the protein can be palmitoylated. The implications of this differential splicing for AMPAR trafficking is unknown. Here, we use surface biotinylation and quantitative Western blotting to show that the N-terminal splice variants GRIP1a and GRIP1b have differential effects in NMDA-induced AMPAR internalization in cultured hippocampal neurons. GRIP1a inhibits, but GRIP1b enhances this trafficking event. We further demonstrate that GRIP1a and GRIP1b have dramatically different subcellular distributions in cultured neurons and exhibit NMDA-dependent colocalisation with early endosomes. We propose that GRIP1 palmitoylation modulates NMDA-induced AMPAR internalisation by differential regulation of the early endosomal system.

Background

Specific delivery to synapses of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors with long-tailed subunits is believed to be a key event in many forms of activity-dependent changes in synaptic strength. GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97). In GluA4, another long-tailed subunit implicated in synaptic plasticity, the PDZ motif is blocked by a single proline residue. This feature is highly conserved in vertebrates, whereas the closest invertebrate homologs of GluA4 have a canonical class I PDZ binding motif. In this work, we have examined the role of GluA4 in PDZ interactions.

Methodology/Principal Findings

Deletion of the carboxy-terminal proline residue of recombinant GluA4 conferred avid binding to SAP97 in cultured cells as shown by coimmunoprecipitation, whereas wild-type GluA4 did not associate with SAP97. Native GluA4 and SAP97 coimmunoprecipitated from mouse brain independently of the GluA1 subunit, supporting the possibility of in vivo PDZ interaction. To obtain evidence for or against the exposure of the PDZ motif by carboxyterminal processing of native GluA4 receptors, we generated an antibody reagent specific for proline-deleted GluA4 C-terminus. Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent.

Conclusion/Significance

We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.

Homeostatic synaptic scaling calibrates neuronal excitability by adjusting synaptic strengths during prolonged changes in synaptic activity. The molecular mechanisms that regulate the trafficking of AMPA receptors (AMPARs) during synaptic scaling are largely unknown. Here we show that chronic activity blockade reduces PICK1 protein level on a time scale that coincides with the accumulation of surface AMPARs. PICK1 loss of function alters the subunit composition and the abundance of GluA2-containing AMPARs. Due to aberrant trafficking of these receptors, the increase in synaptic strength in response to synaptic inactivity is occluded in neurons generated from PICK1 knockout mouse. In agreement with electrophysiological recordings, no defect of AMPAR trafficking is observed in PICK1 knockout neurons in response to elevated neuronal activity. Overall, our data reveal an important role of PICK1 in inactivity-induced synaptic scaling by regulating the subunit composition, abundance and trafficking of GluA2-containing AMPARs.

Stress and glucocorticoids (GCs) can facilitate memory formation. However, the molecular mechanisms mediating their effects are largely unknown. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) trafficking has been implicated in the changes in synaptic strength at central glutamatergic synapses associated with memory formation. In cell cultures, corticosterone has been shown to condition the synaptic trafficking of the AMPAR GluA2 subunit. In this study, we investigated the involvement of GluA2 trafficking in the facilitation of learning by stress. Using the water maze spatial task involving different stress levels, mice trained under more stressful conditions (water at 22°C) showed better learning and memory, and higher post-training corticosterone levels, than mice trained under lower stress (water at 30°C). Strikingly, this facilitated learning by stress was accompanied by enhanced synaptic expression of GluA2 AMPARs that was not observed in mice trained under less stressful conditions. Interfering with GC actions by injecting the GC synthesis inhibitor, metyrapone, blocked both the memory facilitation and the enhanced GluA2 trafficking induced by stressful learning. Intracerebroventricular infusion of the peptide, pep2m, that blocks GluA2 synaptic trafficking by interfering with the interaction between N-ethylmaleimide-sensitive factor and GluA2, impaired immediate performance at learning as well as long-term memory retrieval, supporting a causal role for GluA2 trafficking in stress-induced facilitation of spatial learning and memory. Evidence for the involvement of the neural cell adhesion molecule N-cadherin in interaction with GluA2 is also provided. These findings underscore a new mechanism whereby stress can improve memory function.

Lamina I of the spinal cord contains many projection neurons that express the neurokinin 1 receptor (NK1r). It has been reported that these cells can undergo long-term potentiation (LTP), which may result from insertion of AMPA-type glutamate receptors (AMPArs) containing GluA1 or GluA4 subunits. We therefore investigated synaptic AMPAr expression on these cells with immunocytochemistry following antigen-retrieval. We also examined their density of glutamatergic input (by analysing AMPAr synaptic puncta and contacts from glutamatergic boutons), and phosphorylation of extracellular signal-regulated kinases (pERKs) following noxious stimulation. Our results indicate that there are two populations of NK1r-expressing projection neurons: large GluA4+/GluA1− cells with a high density of glutamatergic input and small GluA1+/GluA4− cells with a much lower input density. Results from pERK experiments suggested that the two groups may not differ in the types of noxious stimulus that activate them. Glutamatergic synapses on distal dendrites of the large cells were significantly longer than those on proximal dendrites, which presumably compensates for the greater attenuation of distally-generated excitatory postsynaptic currents (EPSCs). Both types of cell received contacts from peptidergic primary afferents, however, on the large cells these appeared to constitute over half of the glutamatergic synapses, and were often associated with elongated AMPAr puncta. This suggests that these afferents, which probably contain substance P, provide a powerful, secure synaptic input to large NK1r-expressing projection neurons. These results demonstrate the importance of GluA4-containing AMPArs in nociceptive transmission and raise the possibility that different forms of LTP in lamina I projection neurons may be related to differential expression of GluA1/GluA4.

Purpose

Mutations in tetraspanin 12 (TSPAN12) have recently been identified as a cause of autosomal dominant familial exudative vitreoretinopathy (FEVR). The purpose of this study was to detect TSPAN12 mutations in Chinese patients with FEVR and to describe the associated phenotypes.

Methods

Sanger sequencing was used to analyze the seven coding exons and their adjacent regions of TSPAN12 in 49 unrelated FEVR patients. Clinical phenotypes of the patients with TSPAN12 mutations were documented.

Results

Three novel heterozygous mutations in TSPAN12 were identified in three patients from unrelated families: c.146C>T (p.Thr49Met), c.313T>C (p.Cys105Arg), and c.601delC (p.Leu201PhefsX14). All three mutations involved highly conserved residues and were not present in 180 normal individuals. Ocular phenotypes included retinal folds, inferotemporal dragging of the optic disc and macula, increased vessels in the equatorial region, and a peripheral avascular zone. A father and his daughter had the same mutation but the father only had mild peripheral fundus changes while his daughter had obvious dragged disc and macular ectopia.

Conclusions

Our results suggest that TSPAN12 mutations are responsible for FEVR. Similar to patients with mutations in NDP, LRP5, or FZD4, the phenotypes associated with TSPAN12 mutations showed great variations between different individuals within a family and between the two eyes in individual patients.

Ionotropic glutamate receptors, which underlie a majority of excitatory synaptic transmission in the CNS, associate with transmembrane proteins that modify their intracellular trafficking and channel gating. For AMPA-type glutamate receptors (AMPARs), significant advances have been made in our understanding of their regulation by transmembrane AMPAR regulatory proteins (TARPs). Less is known about the functional influence of cornichons – unrelated AMPAR-interacting proteins, identified by proteomic analysis. Here we confirm that cornichon homologs 2 and 3 (CNIH-2 and CNIH-3), but not CNIH-1, slow the deactivation and desensitization of both GluA2-containing calcium-impermeable (CI-) and GluA2-lacking calcium-permeable (CP-) AMPARs expressed in tsA201 cells. CNIH-2 and -3 also enhanced the glutamate sensitivity, single-channel conductance and calcium permeability of CP-AMPARs, while decreasing their block by intracellular polyamines. We examined the potential effects of CNIHs on native AMPARs by recording from rat optic nerve oligodendrocyte precursor cells (OPCs), known to express a significant population of CP-AMPARs. These glial cells exhibited surface labelling with an anti-CNIH-2/3 antibody. Two features of their AMPAR-mediated currents – the relative efficacy of the partial agonist kainate (IKA/IGlu ratio 0.4), and a greater than five-fold potentiation of kainate responses by cyclothiazide – suggest AMPAR association with CNIHs. Additionally, overexpression of CNIH-3 in OPCs markedly slowed AMPAR desensitization. Together, our experiments support the view that CNIHs are capable of altering key properties of AMPARs and suggest that they may do so in glia.

Summary

Fast synaptic transmission in the brain is mediated by activation of AMPA-type glutamate receptors (AMPARs). AMPARs are comprised of four pore forming subunits (GluAs) as well as auxiliary subunits referred to as transmembrane AMPA receptor regulatory proteins (TARPs). TARPs control the trafficking and gating of AMPARs. However, the number of TARP molecules that assemble within an individual AMPAR complex is unknown. Here, we covalently link GluAs to TARPs to investigate the properties of TARP/AMPAR complexes with known stoichiometry in HEK cells. We find that AMPARs are functional when associated with either four, two, or no TARPs, and that the efficacy of the partial agonist kainate varies across these conditions, providing a sensitive assay for TARP/AMPAR stoichiometry. By comparing these results with data obtained from hippocampal neurons, we show that native AMPARs are normally associated with multiple TARP molecules, and that native TARP/AMPAR stoichiometry varies with the expression level of endogenous and exogenous TARPs. Interestingly, AMPARs in hippocampal pyramidal cells contain more TARP molecules than those in dentate gyrus granule cells, suggesting a cell type-specific regulatory mechanism for TARP/AMPAR stoichiometry.

AMPA receptors (AMPARs) are postsynaptic glutamate-gated ion channels that mediate fast excitatory neurotransmission in the mammalian brain. Synaptic activity modulates the density of synaptic AMPARs, thereby affecting synaptic function, learning and memory. Consequently, there is intense interest in defining the molecular mechanisms regulating AMPAR trafficking. Protein expression in the postsynaptic density of excitatory synapses is tightly regulated by ubiquitination, a post-translational modification that dynamically regulates protein trafficking and degradation in response to synaptic activity. Surprisingly, the ubiquitination of mammalian AMPARs has not been reported. In this study, we demonstrate that increasing synaptic activity, via treatment with the GABA(A) receptor antagonist bicuculline, rapidly and robustly induces ubiquitination of the GluA2 AMPAR subunit. Similarly, treatment with AMPAR agonists results in GluA2 ubiquitination, which suggests that ligand-binding plays a critical role. Finally, we find that clathrin- and dynamin-dependent endocytosis of AMPARs is required for activity-dependent GluA2 ubiquitination. Our findings that GluA2 undergoes activity-dependent ubiquitination expand our understanding of how ubiquitination regulates synaptic plasticity.

SUMMARY

The cytoplasmic polyadenylation element-binding protein 3 (CPEB3), a regulator of local protein synthesis, is the mouse homologue of ApCPEB, a functional prion protein in Aplysia. Here, we provide evidence that CPEB3 is activated by Neuralized1, an E3 ubiquitin ligase. In hippocampal cultures, CPEB3 activated by Neuralized1-mediated ubiquitination leads both to the growth of new dendritic spines and to an increase of the GluA1 and GluA2 subunits of AMPA receptors, two CPEB3 targets essential for synaptic plasticity. Conditional overexpression of Neuralized1 similarly increases GluA1 and GluA2 and the number of spines and functional synapses in the hippocampus, and is reflected in enhanced hippocampal-dependent memory and synaptic plasticity. By contrast, inhibition of Neuralized1 reduces GluA1 and GluA2 levels and impairs hippocampal-dependent memory and synaptic plasticity. These results suggest a model whereby Neuralized1-dependent ubiquitination facilitates hippocampal plasticity and hippocampal-dependent memory storage by modulating the activity of CPEB3 and CPEB3-dependent protein synthesis and synapse formation.

Synaptic refinement, a developmental process that consists of selective elimination and strengthening of immature synapses, is essential for the formation of precise neuronal circuits and proper brain function. At glutamatergic synapses in the brain, activity-dependent recruitment of AMPA receptors (AMPAR) is a key mechanism underlying the strengthening of immature synapses. Studies using receptor over-expression have shown that the recruitment of AMPARs is subunit specific. With the notable exception of hippocampal CA3-CA1 synapses, however, little is known about how native receptors behave or the roles of specific AMPAR subunits in synaptic refinement in vivo. Using patch-clamp recordings in acute slices, we examined developmental refinement of whisker relay (lemniscal) synapses in the thalamus in mice deficient of AMPAR subunits. Deletion of GluA3 or GluA4 caused significant reductions of synaptic AMPAR currents in thalamic neurons at P16-17, with a greater reduction observed in GluA3-deficient mice. Deletions of both GluA3 and GluA4 abolished synaptic AMPAR responses in the majority of thalamic neurons, indicating that at thalamic relay synapses AMPARs are composed primarily of GluA3 and GluA4. Surprisingly, deletions of GluA3 or GluA4 or both had no effect on the elimination of relay inputs: the majority of thalamic neurons in these knockout mice—as in wild type mice—receive a single relay input. However, experience-dependent strengthening of thalamic relay synapses was impaired in GluA3 knockout mice. Together these findings suggest that the elimination of immature glutamatergic synapses proceeds normally in the absence of synaptic strengthening, and highlight the role of GluA3-containing AMPARs in experience-dependent synaptic plasticity.

The impact of conductive hearing loss (CHL), the second most common form of hearing loss, on neuronal plasticity in the central auditory pathway is unknown. After short-term (1 day) monaural earplugging, the GluA3 subunits of the AMPA receptor (AMPAR) are upregulated at auditory nerve synapses on the projection neurons of the cochlear nucleus; glycine receptor α1 (GlyRα1) subunits are downregulated at inhibitory synapses in the same neuronal population. These data suggest that CHL affects receptor trafficking at synapses. We examined the impact of 7 days of CHL on the general expression of excitatory and inhibitory receptors by quantitative biochemistry and immunohistochemistry, using specific antibodies to detect AMPAR subunits (GluA1, GluA2, GluA2/3, and GluA4), GlyRα1, and the GABAA receptor subunit β2/3. Following monaural earplugging and an elevation of the hearing threshold by approximately 35 dB, the immunolabeling of the antibody for the GluA2/3 subunits but not the GluA2 subunit increased on bushy cells (BCs) and fusiform cells (FCs) of the ipsilateral ventral and dorsal cochlear nuclei. These same cell types showed a downregulation of the GlyRα1 subunit. Similar results were observed in the contralateral nuclei. The expression levels of GABAA β2/3 were unchanged. These findings suggest that, following longer periods of monaural conductive hearing loss, the synthesis and subsequent composition of specific glutamate and glycine receptors in projection neurons and their synapses are altered; these changes may contribute to abnormal auditory processing.

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.

RNA trafficking to dendrites and local translation are crucial processes for superior neuronal functions. To date, several α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) mRNAs have been detected in dendrites and are subject to local protein synthesis. Here, we report the presence of all AMPAR GluA1-4 mRNAs in hippocampal and cortical rat synaptic spines by synaptoneurosomes analysis. In particular, we showed that dendritic AMPAR mRNAs are present in the Flip versions in the cortex and hippocampus. To further confirm these data, we demonstrate, using in situ hybridization, the dendritic localization of the GluA2 Flip isoform in vitro and in vivo, whereas the Flop variant is restricted mainly to the soma. In addition, we report that dendritic AMPA mRNAs are edited at low levels at their R/G sites; this result was also supported with transfection experiments using chimeric GluA2 DNA vectors, showing that transcripts carrying an unedited nucleotide at the R/G site, in combination with the Flip exon, are more efficiently targeted to dendrites when compared with the edited-Flip versions. Our data show that post-transcriptional regulations such as RNA splicing, editing and trafficking might be mutually coordinated and that the localization of different AMPAR isoforms in dendrites might play a functional role in the regulation of neuronal transmission.

It is well-established that LTP, a paradigm for learning and memory, results in a stable enlargement of potentiated spines associated with recruitment of additional GluA1-containing AMPARs. Although regulation of the actin cytoskeleton is involved, the detailed signaling mechanisms responsible for this spine expansion are unclear. Here we used cultured mature hippocampal neurons stimulated with a Glycine-Induced, synapse-specific form of chemical LTP (GI-LTP). We report that the stable structural plasticity (i.e., spine head enlargement and spine length shortening) that accompanies GI-LTP was blocked by inhibitors of NMDARs (APV) or CaM-kinase kinase (STO-609), the upstream activator of CaM-kinase I (CaMKI), as well as by transfection with dominant-negative (dn) CaMKI but not dnCaMKIV. Recruitment of GluA1 to the spine surface occurred following GI-LTP and was mimicked by transfection with constitutively-active CaMKI. Spine enlargement induced by transfection of GluA1 was associated with synaptic recruitment of Ca2+-permeable AMPARs (CP-AMPARs) as assessed by an increase in the rectification index of mEPSCs and their sensitivity to IEM-1460, a selective antagonist of CP-AMPARs. Furthermore, the increase in spine size and mEPSC amplitude resulting from GI-LTP itself was blocked by IEM-1460, demonstrating involvement of CP-AMPARs. Downstream signaling effectors of CP-AMPARs, identified by suppression of their activation by IEM-1460, included the Rac/PAK/LIM-kinase pathway that regulates spine actin dynamics.

Taken together, our results suggest that synaptic recruitment of CP-AMPARs via CaMKI may provide a mechanistic link between NMDAR activation in LTP and regulation of a signaling pathway that drives spine enlargement via actin polymerization.

AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesoaccumbal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization we have examined phosphorylation of GluA1 at key residues serine (S)845 and S831, as well as GluA1 cell surface levels in the nucleus accumbens (NAc) of cocaine pre-exposed mice and the role of brain-specific Cav1.2 and Cav1.3 L-type Ca2+ channels (LTCCs), therein. We find higher basal levels of S845 phospho-GluA1 (P-GluA1) and cell surface GluA1 in the NAc following protracted withdrawal from cocaine exposure, changes that occur independently of LTCCs. In contrast, we find that a cocaine challenge that elicits expression of the cocaine sensitized response increases S831 P-GluA1 that further increases surface GluA1 beyond the higher basal levels. Intra-NAc pharmacological manipulations indicate that the Cav1.2-activated CaM kinase II (CaMKII) mediates cocaine-induced increase in S831 P-GluA1 and that both Cav1.2-activated CaMKII and extracellular signal-regulated kinase 2 (ERK2) mediate the increase in GluA1 cell surface levels specific to the sensitized response. Experiments using adenoassociated viral vectors (AAV) expressing Cav1.3 and ERK2 siRNA further indicate that recruitment of the Cav1.2 pathway in the NAc is dependent on ventral tegmental area (VTA) Cav1.3 LTCCs and ERK2. Taken together, these results identify candidate pathways that mediate cocaine-induced AMPAR plasticity in the NAc and provide a mechanism linking dopamine, LTCCs and GluA1 plasticity to cocaine-induced persistent behavioral changes.

Long-term potentiation (LTP) at hippocampal CA3–CA1 synapses is thought to be mediated, at least in part, by an increase in the postsynaptic surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors induced by N-methyl-d-aspartate (NMDA) receptor activation. While this process was originally attributed to the regulated synaptic insertion of GluA1 (GluR-A) subunit-containing AMPA receptors, recent evidence suggests that regulated synaptic trafficking of GluA2 subunits might also contribute to one or several phases of potentiation. However, it has so far been difficult to separate these two mechanisms experimentally. Here we used genetically modified mice lacking the GluA1 subunit (Gria1−/− mice) to investigate GluA1-independent mechanisms of LTP at CA3–CA1 synapses in transverse hippocampal slices. An extracellular, paired theta-burst stimulation paradigm induced a robust GluA1-independent form of LTP lacking the early, rapidly decaying component characteristic of LTP in wild-type mice. This GluA1-independent form of LTP was attenuated by inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC), two enzymes known to regulate GluA2 surface expression. Furthermore, the induction of GluA1-independent potentiation required the activation of GluN2B (NR2B) subunit-containing NMDA receptors. Our findings support and extend the evidence that LTP at hippocampal CA3–CA1 synapses comprises a rapidly decaying, GluA1-dependent component and a more sustained, GluA1-independent component, induced and expressed via a separate mechanism involving GluN2B-containing NMDA receptors, neuronal nitric oxide synthase and PKC.

Summary

We identified four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, as interactors with the kainate receptor (KAR) subunits GluR52b, GluR52c, and GluR6. Of these, we show that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKCα can phosphorylate ct-GluR52b at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. We propose that PICK1 targets PKCα to phosphorylate KARs, causing their stabilization at the synapse by an interaction with GRIP. Importantly, this mechanism is not involved in the constitutive recycling of AMPA receptors since blockade of PDZ interactions can simultaneously increase AMPAR- and decrease KAR-mediated synaptic transmission at the same population of synapses.