GluA1-containing AMPA Receptors Research Articles

A late phase of LTD in cultured cerebellar Purkinje cells requires persistent dynamin-mediated endocytosis

Long-term synaptic depression (LTD) of cerebellar parallel fiber-Purkinje cell synapses is a form of use-dependent synaptic plasticity that may be studied in cell culture. One form of LTD is induced postsynaptically through an mGlu1/Ca influx/protein kinase Cα (PKCα) cascade, and its initial expression requires phosphorylation of ser-880 in the COOH-terminal PDZ-ligand region of GluA2 and consequent binding of PICK1. This triggers postsynaptic clathrin/dynamin-mediated endocytosis of GluA2-containing surface AMPA receptors. Cerebellar LTD also has a late phase beginning 45-60 min after induction that is blocked by transcription or translation inhibitors. Here, I have sought to determine the expression mechanism of this late phase of LTD by applying various drugs and peptides after the late phase has been established. Neither bath application of mGluR1 antagonists (JNJ-16259685, LY-456236) nor the PKC inhibitor GF-109203X starting 60-70 min after LTD induction attenuated the late phase. Similarly, achieving the whole cell configuration with a second pipette loaded with the peptide PKC inhibitor PKC(19-36) starting 60 min postinduction also failed to alter the late phase. Late internal perfusion with peptides designed to disrupt PICK1-GLUA2 interaction or PICK1 dimerization failed to impact late phase LTD expression. However, late internal perfusion with two different blockers of dynamin, the drug dynasore and a dynamin inhibitory peptide (QVPSRPNRAP), produced rapid and complete reversal of cerebellar LTD expression. These findings suggest that the protein synthesis-dependent late phase of LTD requires persistent dynamin-mediated endocytosis, but not persistent PICK1-GluA2 binding nor persistent activation of the upstream mGluR1/PKCα signaling cascade.

GluA4 sustains sensing of sounds through stable, speedy, sumptuous, spineless synapses

Glutamatergic synapses are of immense importance in that they mediate most excitation in the vertebrate brain. They are remarkably diverse. Some produce large, precisely timed synaptic currents, while others produce small, slow currents. Some are indefatigable, signalling robustly and with high fidelity, independent of how rapidly they are driven while others are plastic, constantly strengthening or weakening as a function of whether they are driven rapidly or slowly. α-Amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptors report the presynaptic release of neurotransmitter to postsynaptic neurons. They are tetrameric ion channels formed as combinations of four α subunits, GluA1–4, each of which has the alternatively spliced forms, flip and flop. The subunits differ in the length and in the sequence of their cytoplasmic, carboxy terminal tails which mediate interactions with other molecular components of synapses that regulate receptor trafficking and tethering (Jackson & Nicoll, 2011). The receptor subunit composition determines the speed of synaptic signalling; its receptor number determines the strength of a synapse. It is essential for some functions of the brain that glutamatergic synapses not be altered by synaptic traffic. At the early stages of the auditory pathway, for example, acoustic information must be transmitted faithfully, without distortion by previous sounds, for that information to be useful for computing the location and meaning of sounds. Acoustic information from the cochlea is relayed through the cochlear nucleus and the contralateral medial nucleus of the trapezoid body (MNTB) to the contralateral lateral superior olive. The lateral superior olive compares the intensity of sounds at the two ears for localizing sound sources in the horizontal plane. MNTB neurons are excited through a single sumptuous, calyceal synapse that contacts the smooth surface of the target soma, the calyx of Held (Fig. 1A). This synapse has become a darling of synaptic physiologists because the presynaptic terminals are accessible to electrodes but has been largely ignored by cell biologists because it is boringly stable and not experimentally tractable. Figure 1 Glutamatergic, excitatory synapses are diverse As at upstream synapses, AMPA receptors in mature MNTB neurons are formed from GluA3 flop and/or GluA4 flop subunits in which GluA1 and GluA2 are not functionally evident (Joshi et al. 2004; Koike-Tani et al. 2005). In this issue of The Journal of Physiology, Yang et al. (2011) compare EPSCs in the MNTB of wild type mice with those in mice lacking GluA3 (GluA3 KO) and in mice lacking GluA4 (GluA4 KO). They find that in the MNTB of GluA4 KO mice, EPSCs are dramatically diminished in size and slowed whereas in GluA3 KO mice, EPSCs are only subtly slowed. GluA2 substituted to a small extent for the loss of GluA3 but not detectably for the loss of GluA4. The calyceal synapse does not rely entirely on GluA4, however, because the removal of GluA3 results in greater synaptic depression and desensitization than in the wild type. Subtle but detectable differences in presynaptic functions in KO mice serve as a reminder that differences between wild type and GluA4 KO mice reflect not only the removal of GluA4 at this synapse but can also reflect changes upstream in the auditory pathway as well as compensatory changes at all levels. These findings leave no doubt, however, that GluA4 is essential in mature, excitatory synapses in the MNTB, that GluA3 plays a minor role, and that GluA1 and 2 are almost undetectable in the MNTB. Cell biologists have been attracted by the dynamic glutamatergic synapses on spines in hippocampal pyramidal cells (Fig. 1B). They have studied the trafficking of AMPA receptors in hippocampal synapses where endogenous AMPA receptors are formed from either GluA1/2 or GluA2/3 subunits. Spines give neighbouring synapses biochemical privacy. The long, cytoplasmic, carboxy terminal tails of GluA1 subunits interact with proteins that drive AMPA receptors into synapses as a function of activity (Hayashi et al. 2000). GluA2/3 AMPA receptors cycle constitutively, replacing GluA1/2 receptors. The short cytoplasmic tails of GluA2 subunits regulate the interaction with a different group of synaptic proteins that promote the removal of GluA2-containing AMPA receptors (Kim et al. 2001). The relative rates of insertion and excision of receptors determines how many AMPA receptors are at a synapse and how much current it delivers at any moment. The findings of Yang et al. show that calyceal synapses of MNTB neurons manage without either GluA1 or GluA2, which are so important in the hippocampus, and do pretty well in the absence of GluA3. At these synapses fleets of the speedy GluA4 flop subunits seem to be coordinated by synaptic proteins in the communal postsynaptic environment of the soma.

Probing TARP Modulation of AMPA Receptor Conductance with Polyamine Toxins

The properties of synaptic AMPA receptors (AMPARs) depend on their subunit composition and association with transmembrane AMPAR regulatory proteins (TARPs). Although both GluA2 incorporation and TARP association have been shown to influence AMPAR channel conductance, the manner in which different TARPs modulate the mean channel conductance of GluA2-containing AMPARs is unknown. Using ultrafast agonist application and nonstationary fluctuation analysis, we found that TARP subtypes differentially increase the mean channel conductance, but not the peak open probability, of recombinant GluA2-containing AMPARs. TARP γ-8, in particular, enhances mean channel conductance to a greater degree than γ-2, γ-3, or γ-4. We then examined the action of a use-dependent antagonist of GluA2-containing AMPARs, philanthotoxin-74 (PhTx-74), on recombinant AMPARs and on GluA2-containing AMPARs in cerebellar granule neurons from stargazer mice transfected with TARPs. We found that the rate and extent of channel block varies with TARP subtype, in a manner that correlates linearly with mean channel conductance. Furthermore, block of GluA2-containing AMPARs by polyamine toxins varied depending on whether channels were activated by the full agonist glutamate or the partial agonist kainate, consistent with conductance state-dependent block. Block of GluA2-lacking AMPARs by PhTx-433 is also modulated by TARP association and is a function of agonist efficacy. Our data indicate that channel block by polyamine toxins is sensitive to the mean channel conductance of AMPARs, which varies with TARP subtype and agonist efficacy. Furthermore, our results illustrate the utility of polyamine toxins as sensitive probes of AMPAR channel conductance and suggest the possibility that TARPs may influence their channel properties by selectively stabilizing specific channel conformations, rather than altering the pore structure.

Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3

SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Small microdeletions and point mutations in SHANK3 have been identified in a small subgroup of individuals with autism spectrum disorder (ASD) and intellectual disability. SHANK3 also plays a key role in the chromosome 22q13.3 microdeletion syndrome (Phelan-McDermid syndrome), which includes ASD and cognitive dysfunction as major clinical features. To evaluate the role of Shank3 in vivo, we disrupted major isoforms of the gene in mice by deleting exons 4-9. Isoform-specific Shank3(e4-9) homozygous mutant mice display abnormal social behaviors, communication patterns, repetitive behaviors and learning and memory. Shank3(e4-9) male mice display more severe impairments than females in motor coordination. Shank3(e4-9) mice have reduced levels of Homer1b/c, GKAP and GluA1 at the PSD, and show attenuated activity-dependent redistribution of GluA1-containing AMPA receptors. Subtle morphological alterations in dendritic spines are also observed. Although synaptic transmission is normal in CA1 hippocampus, long-term potentiation is deficient in Shank3(e4-9) mice. We conclude that loss of major Shank3 species produces biochemical, cellular and morphological changes, leading to behavioral abnormalities in mice that bear similarities to human ASD patients with SHANK3 mutations.

PICK1 Loss of Function Occludes Homeostatic Synaptic Scaling

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 knock-out mouse. In agreement with electrophysiological recordings, no defect of AMPAR trafficking is observed in PICK1 knock-out 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.

Long-Term Potentiation-Dependent Spine Enlargement Requires Synaptic Ca2+-Permeable AMPA Receptors Recruited by CaM-Kinase I

It is well established that long-term potentiation (LTP), a paradigm for learning and memory, results in a stable enlargement of potentiated spines associated with recruitment of additional GluA1-containing AMPA receptors (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 NMDA receptors (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 after GI-LTP and was mimicked by transfection with constitutively active CaMKI. Spine enlargement induced by transfection of GluA1 was associated with synaptic recruitment of Ca(2+)-permeable AMPARs (CP-AMPARs) as assessed by an increase in the rectification index of miniature EPSCs (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. 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.

Spatial working memory deficits in GluA1 AMPA receptor subunit knockout mice reflect impaired short-term habituation: Evidence for Wagner's dual-process memory model

Genetically modified mice, lacking the GluA1 AMPA receptor subunit, are impaired on spatial working memory tasks, but display normal acquisition of spatial reference memory tasks. One explanation for this dissociation is that working memory, win-shift performance engages a GluA1-dependent, non-associative, short-term memory process through which animals choose relatively novel arms in preference to relatively familiar options. In contrast, spatial reference memory, as exemplified by the Morris water maze task, reflects a GluA1-independent, associative, long-term memory mechanism. These results can be accommodated by Wagner's dual-process model of memory in which short and long-term memory mechanisms exist in parallel and, under certain circumstances, compete with each other. According to our analysis, GluA1−/− mice lack short-term memory for recently experienced spatial stimuli. One consequence of this impairment is that these stimuli should remain surprising and thus be better able to form long-term associative representations. Consistent with this hypothesis, we have recently shown that long-term spatial memory for recently visited locations is enhanced in GluA1−/− mice, despite impairments in hippocampal synaptic plasticity. Taken together, these results support a role for GluA1-containing AMPA receptors in short-term habituation, and in modulating the intensity or perceived salience of stimuli.