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AMPA Receptor

SAP

Tardin, Choquet et al. (2003)

Direct imaging of lateral movements of AMPA receptors inside synapses. The EMBO Journal, 22(18)

Statements

Trafficking of AMPAR in and out of synapses is crucial for synaptic plasticity. Protocols that induce plasticity of synaptic transmission in culture result in changes of AMPAR concentration at synapses and are thought to mimic at the molecular level the processes of LTP and LTD.

Membrane trafficking may occur outside of the synapse and accumulate at the PSD after a short delay (Passafaro et al. 2001). Altogether, a unified picture of the postsynaptic density could be one where receptors are immobilized for transient periods of time related to the receptor-scaffold affinity. This could also be true of NMDA receptors (Tovar and Westbrook, 2002).

Findings

Application of glutamate increased the diffusion rate of GluR2-containting AMPAR whereas a protocol designed to induce calcium influx (stimulation of NMDAR with glycine, glutamate) reduced the percentage of diffusible AMPARs at the PSD. Bath application of 100 uM glutamate caused an 85% increase in AMPAR endocytosis within 15 min (corresponding to a 22% drop in total membrane expression). Conversely , the calcium influx protocol (20 uM biccuculine, 1 uM strychnine, 200 uM glycine) caused a 59% increase in AMPAR membrane expression. Glutamate caused a 55% increase in AMPAR diffusion within synapses, but did not change diffusion outside synapses. Furthermore, glutamate decreased the number of completely immobile AMPARs by 30%. Interestingly, Glutamate causes endocytosis of AMPARs, and internal AMPARs are immobile. Therefore it seems like glutamate may be causing a general endocytotic episode at non-synaptic AMPARs, perhaps not even at the synapse that received the glutamate application. In a parallel effect, it was found that blocking calcium with BAPTA increased the % of mobile AMPARs. Newly inserted receptors were found to be initially diffusive and then stabilized at synaptic sites. In summary, they found that bath application of glutamate induces rapid depletion of AMPARs from PSDs increases synaptic diffusion rate, decreases % of completely immobile receptors, increases proportion of receptors in the area surrounding the synapse (juxtasynaptic region). Activation of NMDARs results in increased surface expression of AMPARs -- in the first few minutes there is mainly a decrease in the proportion of immobile synaptic receptors, but after 40 min, both diffusion rates and percentages of immobile synaptic receptors are back to control values and the proportion of juxtasynaptic receptors is decreased. This observation relates to the fate of newly exocytosed AMPARs: using cleavable extracellular tags, it was observed that at early times after exocytosis, new GluR1 containing AMPARs are diffusively distributed along dendrites. This is followed by their lateral translocation and accumulation into synapses (Passafaro et al., 2001). GluR2 subunits were addressed directly at synapses. In our experiments, we followed the movement of native GluR2 containing AMPARs, where the data suggests that at the level of synapses themselves, newly added receptors are initially diffusive and then stabilize over time.

AMPA receptors that lack edited GluA2 subunits have high single channel conductance, are permeable to Ca2+, are blocked by polyamines causing inward rectification at depolarized potentials.

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• 100 uM glutamate - within 15 min
• 85% increase in AMPAR endocytosis
• 22% drop in total membrane expression
• 55% increase in AMPAR diffusion rate within synapses
• 0% increase in AMPAR diffusion rate outside synapses
• 30% decrease in completely immobile AMPAR at PSD

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• Start: 100 AMPARs in PSD
• Usual endocytosis rate: -0.25% / min
• Add: 100 uM glutamate
• New endocytosis rate: -1.5% / min
• Time: 15 min
• Final: 77.5 AMPARs

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• calcium influx protocol (20 uM biccuculine, 1 uM strychnine, 200 uM glycine)
• 59% increase in AMPAR expression

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• Start: 100 AMPARs in PSD
• Usual endocytosis rate: -0.25% / min
• Add: NMDA antagonists above
• New exocytosis rate: +4% / min
• Time: 15 min
• Final: 160 AMPARs

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Rao-Ruiz, Spijker, et al.

Retrieval-specific endocytosis of GluA2-AMPARs underlies adaptive reconsolidation of contextual fear

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A consolidated memory returns to a transient destabilized state shortly after reactivation, necessitating a dynamic time-dependent process of reconsolidation to persist further. During this reconstruction, a memory is labile and subject to change. In general, a memory-recall causes internalization of AMPAR at activate synapses for ~2 hours. Then AMPAR repopulate these synapses and return to baseline levels, and sometimes even higher levels.

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GROUPS

• NS: No Shock
• US: Shock
• NR: No Retrieval (24h later)
• R: Retrieval (24h later)

Tissue collected 1 h after retrieval for western blot and ephys

RESULTS

Down-regulation of all AMPAR subtypes and smaller mEPSC amplitudes 1 h after retrieval. Increase at 7 h.

Start at 100% baseline expression

• Post-Recall: 1 h 4 h 7 h
• GluR1 70% 100% 100%
• GluR2 85% 85% 130%
• GluR3 50% 50% 100%

Blocking GluR2 internalization with 3Y peptide (3A is a control peptide) 1 h before or 1 h after recall prevented a subsequent GluR2 increase at 7 hours. The narrow decay time found at 7 hours in 3A controls suggests that synapses are composed more of GluR2 than GluR1 or GluR3 receptors which are permeable to calcium. Furthermore, 3Y increased fear memory when tested 2 h (RT2) after a recall event (RT1) or 24 h (RT3) after recall.

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Start at 100% baseline expression

• Post-Recall: 1 h 4 h 7 h
• GluR1 70% 100% 100%
• GluR2 85% 85% 130%
• GluR3 50% 50% 100%

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Bassani · Folci · Zapata · Passafaro 2013

Abstract

Glutamate ionotropic AMPA receptors (AMPAR) mediate most fast excitatory synaptic transmission in the central nervous system. The content and composition of AMPARs in postsynaptic membranes (which determine synaptic strength) are dependent on the regulated trafficking of AMPAR subunits in and out of the membranes. AMPAR trafficking is a key mechanism that drives nascent synapse development, and is the main determinant of both Hebbian and homeostatic plasticity in mature synapses. Hebbian plasticity seems to be the biological substrate of at least some forms of learning and memory; while homeostatic plasticity (also known as synaptic scaling) keeps neuronal circuits stable by maintaining changes within a physiological range

Introduction

Glutamatergic synapses mediate excitatory transmission in the central nervous system. AMPARs and NMDARs are the two principal types of ionotropic glutamate receptors in glutamatergic synapses, and changes in the trafficking, subunit composition, and signaling of these receptors are fundamental processes underlying synapse strength. AMPARs are tetrameric (four subunit) cation channels that mediate fast excitatory synaptic transmission. NMDARs are also tetrameric cation channels; their activation requires glutamate (ligand-gating) and also membrane depolarization (voltage dependence), which removes the Mg2+ normally blocking the channel. Activated NMDARs allow Ca2+ to enter the neuron; the magnitude of the Ca2+ signal in the postsynaptic neuron largely determines whether long-term potentiation (LTP) or long-term depression (LTD) of AMPAR currents occurs.

In mature synapses, AMPAR trafficking is a major determinant of both Hebbian and homeostatic plasticity. Hebbian plasticity is a mechanism by which long-lasting modifications in synaptic strength occur [4 ]. Synaptic modifications resulting from Hebbian plasticity give rise to LTP and LTD and are largely determined by the delivery and removal of postsynaptic AMPARs. There is good evidence that LTP and LTD are substrates for at least some aspects of learning, memory, and cognition [5 ]; and some diseases characterized by defective learning and cognitive function are directly related to alterations in AMPAR trafficking [6 ]. Homeostatic synaptic plasticity on the other hand is a mechanism ensuring that changes in synaptic activity occur within a limited range, thereby preserving the stability of neuronal circuits and functional integrity of the brain [7 ].

AMPAR structure and subunit composition

AMPARs are homo or hetero tetramers assembled from GluA1-A4 subunits, encoded by genes GRIA1-4 [8 ]. The four subunits that make up each receptor combine in various stoichiometries to form receptor subtypes with distinct channel properties [9 ]. Most AMPARs in adult hippocampus and cortex appear to consist of the GluA1 plus GluA2, or GluA2 plus GluA3 subunits [10 ]. Since GluA3 is expressed at relatively low levels, over 70 % of GluA2 is associated with GluA1 [11 ]. All AMPAR subunits consist of an extracellular amino terminal domain (ATD or NTD), a ligand-binding domain (LBD) (S1 and S2), three membrane-spanning domains (M1, M2 and M3), one cytoplasmic re-entrant loop (P), and a carboxy-terminal intracellular region (Fig. 1) [9, 12, 13]. The extracellular and transmembrane regions of all GluAs are highly homologous and the four GluAs differ from each other mainly in terms of their intracellular cytoplasmic tails. The function of the NTD in AMPARs is to control the initial dimerization of subunits and to prevent heteromerization between AMPAR and kainate receptor subunits [14]. Subsequent tetramerization of dimers is mediated by associations at the ligand binding and membrane domains (M1–M3 and P) and also depends on Q/R editing of GluA2 (see below) [15]. GluA1, GluA4, and GluA2L (alternative splice form of GluA2) have long cytoplasmic tails; GluA2, GluA3, and GluA4S (alternative splice form of GluA4) have short C-terminal tails [16, 17, 18]. Alternative splicing also generates the flop (short) and flip (long) variants encoded by exons 14 and 15, respectively, that differ by a 38-amino-acid insertion into a region that forms part of the extracellular LBD and is localized before the M3 domain [19, 20]. The flip variants of all subunits are prominently expressed before birth and their expression, as determined by in situ hybridization, remains largely unchanged during postnatal development and in the adult; whereas the expression of flop variants increases throughout development, and reaches adult levels by postnatal day 14 in the rat [21]. Flop versions are less responsive to AMPAR potentiators [21, 22] and generally de-sensitize more rapidly in response to glutamate than receptors containing flip variant [19].