Dynamic extracellular interactions with AMPA receptors

Biochemistry, videomicroscopy, and immunocytochemistry reveal differential roles of GluA1/4.1N and GluA1/SAP97 interactions on GluA1 intracellular transport and exocytosis in basal transmission and during cLTP in cultured hippocampal neurons.

Routes, destinations and delays: recent advances in AMPA receptor trafficking

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

AMPA Receptor Trafficking and the Control of Synaptic Transmission

AMPA Receptor Subunit-Specific Regulation by a Distinct Family of Type II TARPs

SAP97 is a multidomain scaffold protein implicated in the forward trafficking and synaptic localization of NMDA- and AMPA-type glutamate receptors. Alternative splicing of SAP97 transcripts gives rise to palmitoylated αSAP97 and L27-domain containing βSAP97 isoforms that differentially regulate the subsynaptic localization of GluR1 subunits of AMPA receptors. Here, we examined whether SAP97 isoforms regulate the mechanisms underlying long-term potentiation (LTP) and depression (LTD) and find that both α- and β-forms of SAP97 impair LTP but enhance LTD via independent isoform-specific mechanisms. Live imaging of α- and βSAP97 revealed that the altered synaptic plasticity was not due to activity-dependent changes in SAP97 localization or exchange kinetics. However, by recording from pairs of synaptically coupled hippocampal neurons, we show that αSAP97 occludes LTP by enhancing the levels of postsynaptic AMPA receptors, while βSAP97 blocks LTP by reducing the synaptic localization of NMDA receptors. Examination of the surface pools of AMPA and NMDA receptors indicates that αSAP97 selectively regulates the synaptic pool of AMPA receptors, whereas βSAP97 regulates the extrasynaptic pools of both AMPA and NMDA receptors. Knockdown of βSAP97 increases the synaptic localization of both AMPA and NMDA receptors, showing that endogenous βSAP97 restricts glutamate receptor expression at excitatory synapses. This isoform-dependent differential regulation of synaptic versus extrasynaptic pools of glutamate receptors will determine how many receptors are available for the induction and the expression of synaptic plasticity. Our data support a model wherein SAP97 isoforms can regulate the ability of synapses to undergo plasticity by controlling the surface distribution of AMPA and NMDA receptors.

AMPA receptors (AMPARs) are glutamate-gated ion channels that mediate the majority of fast excitatory synaptic transmission throughout the brain. Changes in the properties and postsynaptic abundance of AMPARs are pivotal mechanisms in synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission. A wide range of neurodegenerative, neurodevelopmental and neuropsychiatric disorders, despite their extremely diverse etiology, pathogenesis and symptoms, exhibit brain region-specific and AMPAR subunit-specific aberrations in synaptic transmission or plasticity. These include abnormally enhanced or reduced AMPAR-mediated synaptic transmission or plasticity. Bidirectional reversal of these changes by targeting AMPAR subunits or trafficking ameliorates drug-seeking behavior, chronic pain, epileptic seizures, or cognitive deficits. This indicates that bidirectional dysregulation of AMPAR-mediated synaptic transmission or plasticity may contribute to the expression of many brain disorders and therefore serve as a therapeutic target. Here, we provide a synopsis of bidirectional AMPAR dysregulation in animal models of brain disorders and review the preclinical evidence on the therapeutic targeting of AMPARs.

Synaptic AMPA Receptor Plasticity and Behavior

Dendritic spines, the tiny and actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain that control synaptic activity and plasticity. Dendritic spines contain a specialized form of endoplasmic reticulum (ER), i.e., the spine apparatus, required for local calcium signaling and that is involved in regulating dendritic spine enlargement and synaptic plasticity. Many autism-linked genes have been shown to play critical roles in synaptic formation and plasticity. Among them, KLHL17 is known to control dendritic spine enlargement during development. As a brain-specific disease-associated gene, KLHL17 is expected to play a critical role in the brain, but it has not yet been well characterized. In this study, we report that KLHL17 expression in mice is strongly regulated by neuronal activity and KLHL17 modulates the synaptic distribution of synaptopodin (SYNPO), a marker of the spine apparatus. Both KLHL17 and SYNPO are F-actin-binding proteins linked to autism. SYNPO is known to maintain the structure of the spine apparatus in mature spines and contributes to synaptic plasticity. Our super-resolution imaging using expansion microscopy demonstrates that SYNPO is indeed embedded into the ER network of dendritic spines and that KLHL17 is closely adjacent to the ER/SYNPO complex. Using mouse genetic models, we further show that Klhl17 haploinsufficiency and knockout result in fewer dendritic spines containing ER clusters and an alteration of calcium events at dendritic spines. Accordingly, activity-dependent dendritic spine enlargement and neuronal activation (reflected by extracellular signal-regulated kinase (ERK) phosphorylation and C-FOS expression) are impaired. In addition, we show that the effect of disrupting the KLHL17 and SYNPO association is similar to the results of Klhl17 haploinsufficiency and knockout, further strengthening the evidence that KLHL17 and SYNPO act together to regulate synaptic plasticity. In conclusion, our findings unravel a role for KLHL17 in controlling synaptic plasticity via its regulation of SYNPO and synaptic ER clustering and imply that impaired synaptic plasticity contributes to the etiology of KLHL17-related disorders.

Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission

Neuromodulation of activity-dependent synaptic enhancement at crayfish neuromuscular junction

Synaptic transmission is a complex process that can be fine-tuned and tweaked by numerous mechanisms. Nowhere is that more true than at the mossy fibre to CA3 pyramidal cell synapse in the hippocampus. This specialized synapse, formed by the granule cell axons making en passant connections onto large multiheaded postsynaptic spines (named thorny excrescences), has a number of curious properties and is functionally regulated by several well-characterized mechanisms. Mossy fibre synapses are fundamentally different to other hippocampal synapses because the expression of long-term plasticity (both potentiation and depression) is presynaptic, requiring a long-lasting change in glutamate release probability. The almost universal acceptance that synaptic plasticity is, at least in part, a cellular correlate for learning and memory has provoked a slew of studies describing the molecules that play a role in both mediating and modulating long-term changes in synaptic efficacy. One of the primary modes by which mossy fibre synaptic transmission can be modulated is through the activation of presynaptic glutamate-activated autoreceptors. Two classes of glutamate receptor are known to be important for modulating mossy fibre synaptic transmission. Activation of the group II metabotropic glutamate receptor, mGluR2, depresses synaptic transmission and is required for the induction of mossy fibre long-term depression (LTD) (Yokoi et al. 1996). In contrast, activation of presynaptic ionotropic glutamate receptors of the kainate receptor subfamily can both facilitate and depress transmission, and contribute to mossy fibre long-term potentiation (LTP) (Contractor et al. 2001). These autoreceptors therefore represent convenient targets for modulating mossy fibre synaptic transmission and could be important for targeting hippocampal function in neurological diseases. In this issue of The Journal of Physiology Omrani and colleagues (Omrani et al. 2009) present evidence that regulating the amount of glutamate uptake can have a dramatic impact on the ability of mossy fibre synapses to express long-term depression (LTD). The authors make use of an interesting observation that some β-lactam antibiotics can transcriptionally up-regulate the expression of the glutamate transporter GLT-1, a process that might hold some promise for reducing excitotoxicity in certain neuropathologies (Rothstein et al. 2005). The authors demonstrate that the expression of GLT-1 is increased in the brains of rats which are treated with the β-lactam antibiotic ceftriaxone. GLT-1 is generally thought to be an astroglial transporter; however, more recently it has been demonstrated that it may also be expressed in neurons, and more importantly, in excitatory synaptic terminals in the hippocampus (Chen et al. 2004). In agreement with this, Omrani et al. found that immunogold particles labelled both astrocyte processes and mossy fibre terminals in the stratum lucidum. In animals that were treated with ceftriaxone the density of both cytoplasmic and membrane-associated particles was increased. The authors hypothesized that the elevated expression of glutamate transporters close to the active zones (where glutamate is released) could potentially alter the glutamate transient in the synaptic cleft during induction of synaptic plasticity and fail to activate presynaptic autoreceptors that are required for LTP or LTD. In particular, the authors focused on mossy fibre LTD and the activation of mGluR2. Indeed, the authors found that in recordings from slices from ceftriaxone-treated animals, a low-frequency stimulation protocol failed to induce LTD; however, LTD could be rescued by application of the GLT-1-selective antagonist dihydrokainate (DHK). These results indicate that it is likely that presynaptic mGluRs, which are thought to be localized somewhat distant from mossy fibre release sites, are not effectively engaged by low-frequency LTD induction protocols in ceftriaxone-treated slices probably because glutamate is more efficiently transported from the synaptic cleft (Fig. 1). Figure 1 Model of GLT-1 modulation of mossy fibre synaptic plasticity The present study describes an intriguing observation, and furthers our knowledge of mechanisms that regulate mossy fibre synaptic transmission. In particular, the novel demonstration of the contribution of glutamate transporters to mossy fibre synaptic plasticity provides a potential target for enhancing (or decreasing) synaptic plasticity. The study also raises several questions which the authors did not fully explore here. For instance, there are deficits in both frequency facilitation and mossy fibre LTP in ceftriaxone-treated animals. As stated above, ionotropic kainate receptors contribute to these forms of plasticity, so it would have been interesting to determine if, in fact, a reduction in the activation of kainate receptors underlay deficits in these forms of plasticity when GLT-1 is up-regulated. A broader question about the implication of these findings might be answered by performing behavioural studies in ceftriaxone-treated animals. Ceftriaxone is in clinical use as an antibiotic and has been proposed to be neuroprotective against excitotoxity in stroke. However, if synaptic plasticity mechanisms are also impaired by ceftriaxone one might potentially expect some impact on learning and memory. It will take future studies to work out these details, but the current cellular studies provide fascinating insight into glutamate transporters and their role in regulating synaptic transmission.

Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. All three ionotropic glutamate subfamilies (i.e. AMPA-type, kainate-type, and NMDA-type) assemble as tetramers of four homologous subunits. There is good evidence that both heteromeric AMPA and kainate receptors have a 2:2 subunit stoichiometry and an alternating subunit arrangement. Recent studies based on presumed structural homology have indicated that NMDA receptors adopt the same arrangement. Here, we use atomic force microscopy imaging of receptor-antibody complexes to show that whereas the GluA1/GluA2 AMPA receptor assembles with an alternating (i.e. 1/2/1/2) subunit arrangement, the GluN1/GluN2A NMDA receptor adopts an adjacent (i.e. 1/1/2/2) arrangement. We conclude that the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. This surprising finding necessitates a reassessment of the assembly of these important receptors.

Local Presynaptic Activity Gates Homeostatic Changes in Presynaptic Function Driven by Dendritic BDNF Synthesis

The AMPA Receptor Code of Synaptic Plasticity