Sustained postsynaptic kainate receptor activation downregulates AMPA receptor surface expression and induces hippocampal LTD.
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ABSTRACT:
SUBMITTER: Nair JD
PROVIDER: S-EPMC8441151 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The decrease in number of AMPA-type glutamate receptor (AMPAR) at excitatory synapses causes LTD, a cellular basis of learning and memory. The number of postsynaptic AMPARs is regulated by the balance of exocytosis and endocytosis, and enhanced endocytosis of AMPAR has been suggested to underlie the LTD expression. However, it remains unclear how endocytosis and exocytosis of AMPAR change during LTD. In this study, we addressed this question by analyzing exocytosis and endocytosis of AMPAR by imaging super-ecliptic pHlorin (SEP)-tagged AMPAR around postsynaptic structure formed directly on the glass surface in the hippocampal culture prepared from rat embryos of both sexes. Contrary to a prevailing view on the LTD expression by endocytosis enhancement, the LTD induction by NMDA application only transiently enhanced endocytosis of SEP-tagged GluA1 subunits of AMPAR, which was counteracted by simultaneous augmentation of exocytosis. As a result, soon after the start of the LTD induction (∼1 min), the surface AMPAR did not markedly decrease. Thereafter, the surface GluA1-SEP gradually decreased (2-5 min) and kept at a low level until the end of observation (>30 min). Surprisingly, this gradual and sustained decrease of surface AMPAR was accompanied not by the enhanced endocytic events of GluA1, but by the suppression of exocytosis. Together, our data highlight an unprecedented mechanism for the LTD expression by attenuation of exocytosis of AMPAR, but not by enhanced endocytosis, together with a reduction of postsynaptic AMPAR scaffolding protein PSD95.SIGNIFICANCE STATEMENT It has been generally assumed that LTD is expressed by enhancement of AMPAR endocytosis. Previous studies reported that endocytosis-related protein was involved in LTD and that significant amount of cell-surface AMPAR moved into intracellular compartments during LTD. Here, we report changes of cell-surface amount of AMPAR, and where and when individual exocytosis and endocytosis occurred during LTD. Cell-surface AMPAR gradually decreased in synchrony with suppression of exocytosis but not with enhancement of endocytosis. These results suggest that the decrease of cell-surface AMPAR amount during LTD was caused not by enhancement of endocytosis but rather by suppression of exocytosis, which revises current understanding of the expression mechanism of LTD.
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Project description:Naturally occurring glutamate analogs, such as kainate and domoate, which cause excitotoxic shellfish poisoning, induce nondesensitizing responses at neuronal alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In addition to acting on AMPA receptors, kainate and domoate also activate high-affinity kainate-type glutamate receptors. The receptor type that mediates their neurotoxicity remains uncertain. Here, we show that the transmembrane AMPA receptor-associated protein (TARP) gamma-2 (or stargazin) and the related TARP gamma-8 augment responses to kainate and domoate by making these neurotoxins more potent and more efficacious AMPA receptor agonists. Genetic deletion of hippocampal enriched gamma-8 selectively abolishes sustained depolarizations in hippocampus mediated by kainate activation of AMPA receptors. gamma-8 knockout mice display typical kainate-induced seizures; however, the associated neuronal cell death in the hippocampus is attenuated in mice lacking gamma-8. This work decisively demonstrates that TARP-associated AMPA receptors mediate kainate neurotoxicity and identifies TARPs as targets for modulating neurotoxic properties of AMPA receptors.
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Project description:Synaptic adhesion molecules regulate synapse development through trans-synaptic adhesion and assembly of diverse synaptic proteins. Many synaptic adhesion molecules positively regulate synapse development; some, however, exert negative regulation, although such cases are relatively rare. In addition, synaptic adhesion molecules regulate the amplitude of post-synaptic receptor responses, but whether adhesion molecules can regulate the kinetic properties of post-synaptic receptors remains unclear. Here we report that Clmp, a homophilic adhesion molecule of the Ig domain superfamily that is abundantly expressed in the brain, reaches peak expression at a neonatal stage (week 1) and associates with subunits of AMPA receptors (AMPARs) and kainate receptors (KARs). Clmp deletion in mice increased the frequency and amplitude of AMPAR-mediated miniature excitatory post-synaptic currents (mEPSCs) and the frequency, amplitude, and decay time constant of KAR-mediated mEPSCs in hippocampal CA3 neurons. Clmp deletion had minimal impacts on evoked excitatory synaptic currents at mossy fiber-CA3 synapses but increased extrasynaptic KAR, but not AMPAR, currents, suggesting that Clmp distinctly inhibits AMPAR and KAR responses. Behaviorally, Clmp deletion enhanced novel object recognition and susceptibility to kainate-induced seizures, without affecting contextual or auditory cued fear conditioning or pattern completion-based contextual fear conditioning. These results suggest that Clmp negatively regulates hippocampal excitatory synapse development and AMPAR and KAR responses in the neonatal hippocampal CA3 as well as object recognition and kainate seizure susceptibility in mice.
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Project description:Kainate receptors (KARs) play fundamentally important roles in controlling synaptic function and regulating neuronal excitability. Postsynaptic KARs contribute to excitatory neurotransmission but the molecular mechanisms underlying their activity-dependent surface expression are not well understood. Strong activation of KARs in cultured hippocampal neurons leads to the downregulation of postsynaptic KARs via endocytosis and degradation. In contrast, low-level activation augments postsynaptic KAR surface expression. Here, we show that this increase in KARs is due to enhanced recycling via the recruitment of Rab11-dependent, transferrin-positive endosomes into spines. Dominant-negative Rab11 or the recycling inhibitor primaquine prevents the kainate-evoked increase in surface KARs. Moreover, we show that the increase in surface expression is mediated via a metabotropic KAR signalling pathway, which is blocked by the protein kinase C inhibitor chelerythrine, the calcium chelator BAPTA and the G-protein inhibitor pertussis toxin. Thus, we report a previously uncharacterized positive feedback system that increases postsynaptic KARs in response to low- or moderate-level agonist activation and can provide additional flexibility to synaptic regulation.
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Project description:Kainate receptors (KARs) are crucial for the regulation of both excitatory and inhibitory neurotransmission, but little is known regarding the mechanisms controlling KAR surface expression. We used super ecliptic pHluorin (SEP)-tagged KAR subunit GluR6a to investigate real-time changes in KAR surface expression in hippocampal neurons. Sindbis virus-expressed SEP-GluR6 subunits efficiently co-assembled with native KAR subunits to form heteromeric receptors. Diffuse surface-expressed dendritic SEP-GluR6 is rapidly internalized following either N-methyl-d-aspartate or kainate application. Sustained kainate or transient N-methyl-d-aspartate application resulted in a slow decrease of base-line surface KAR levels. Surprisingly, however, following the initial loss of surface receptors, a short kainate application caused a long lasting increase in surface-expressed KARs to levels significantly greater than those prior to the agonist challenge. These data suggest that after initial endocytosis, transient agonist activation evokes increased KAR exocytosis and reveal that KAR surface expression is bidirectionally regulated. This process may provide a mechanism for hippocampal neurons to differentially adapt their physiological responses to changes in synaptic activation and extrasynaptic glutamate concentration.
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Project description:AMPA receptor (AMPA-R) complexes consist of channel-forming subunits, GluA1-4, and auxiliary proteins, including TARPs, CNIHs, synDIG1, and CKAMP44, which can modulate AMPA-R function in specific ways. The combinatorial effects of four GluA subunits binding to various auxiliary subunits amplify the functional diversity of AMPA-Rs. The significance and magnitude of molecular diversity, however, remain elusive. To gain insight into the molecular complexity of AMPA and kainate receptors, we compared the proteins that copurify with each receptor type in the rat brain. This interactome study identified the majority of known interacting proteins and, more importantly, provides candidates for additional studies. We validate the claudin homolog GSG1L as a newly identified binding protein and unique modulator of AMPA-R gating, as determined by detailed molecular, cellular, electrophysiological, and biochemical experiments. GSG1L extends the functional variety of AMPA-R complexes, and further investigation of other candidates may reveal additional complexity of ionotropic glutamate receptor function.
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Project description:Kainate receptors (KARs) contribute to postsynaptic excitation in only a select subset of neurons. To define the parameters that specify the postsynaptic expression of KARs, we examined the contribution of KARs to EPSCs on hippocampal interneurons in area CA1. Interneurons in stratum radiatum/lacunosum-moleculare express KARs both with and without the GluR5 subunit, but KAR-mediated EPSCs are generated mainly, if not entirely, by GluR5-containing KARs. Extrasynaptic glutamate spillover profoundly recruits AMPA receptors (AMPARs) with little effect on KARs, indicating that KARs are targeted at the synapse more precisely than AMPARs. However, spontaneous EPSCs with a conventional AMPAR component did not have a resolvable contribution of KARs, suggesting that the KARs that contribute to the evoked EPSCs are at a distinct set of synapses. GluR5-containing KARs on interneurons in stratum oriens do not contribute substantially to the EPSC. We conclude that KARs are localized to synapses by cell type-, synapse-, and subunit-selective mechanisms.
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Project description:Long-term potentiation (LTP) is a compelling synaptic correlate of learning and memory. LTP induction requires NMDA receptor (NMDAR) activation, which triggers SNARE-dependent exocytosis of AMPA receptors (AMPARs). However, the molecular mechanisms mediating AMPAR exocytosis induced by NMDAR activation remain largely unknown. Here, we show that complexin, a protein that regulates neurotransmitter release via binding to SNARE complexes, is essential for AMPAR exocytosis during LTP but not for the constitutive AMPAR exocytosis that maintains basal synaptic strength. The regulated postsynaptic AMPAR exocytosis during LTP requires binding of complexin to SNARE complexes. In hippocampal neurons, presynaptic complexin acts together with synaptotagmin-1 to mediate neurotransmitter release. However, postsynaptic synaptotagmin-1 is not required for complexin-dependent AMPAR exocytosis during LTP. These results suggest a complexin-dependent molecular mechanism for regulating AMPAR delivery to synapses, a mechanism that is surprisingly similar to presynaptic exocytosis but controlled by regulators other than synaptotagmin-1.
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