Choquet

Malinow

Molecular Methods

Quantum Dots

Choquet

AMPAR

Things Choquet has already done

Bats, Groc, Choquet • 2007 • Cell Press - Neuron

[http://www.ncbi.nlm.nih.gov/pubmed/17329211 The Interaction between Stargazin and PSD-95 Regulates AMPA Receptor Surface Trafficking {{{5}}}]

We show, using single quantum dot and FRAP imaging in live hippocampal neurons, that exchange of AMPAR by lateral diffusion between extrasynaptic and synaptic sites mostly depends on the interaction of Stargazin with PSD-95 and not upon the GluR2 AMPAR subunit C terminus. Disruption of interactions between Stargazin and PSD-95 strongly increases AMPAR surface diffusion, preventing AMPAR accumulation at postsynaptic sites. Furthermore, AMPARs and Stargazin diffuse as complexes in and out synapses. These results propose a model in which the Stargazin- PSD-95 interaction plays a key role to trap and transiently stabilize diffusing AMPARs in the postsynaptic density.

Tardin, Cognet, Bats, Lounis, Choquet • 2003 • The EMBO Journal

imaging of lateral movements of AMPA receptors inside synapses {{{5}}}

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.

Studies

Tardin • Cognet • Bats • Lounis • Choquet • 2003 • The EMBO Journal

Direct imaging of lateral movements of AMPA receptors inside synapses

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.

---

• 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

--

• 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

---

---

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

--

• 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

---

Choquet Email

Hi Roberto,

I hope you’re doing well, haven’t seen each other in a while. As far as receptor tracking in slices go, we’ve not progressed much. As you’ve done, we routinely use FRAP of phluorin-tagged receptors to evaluate mobility in slices, and this works well, except for the over-expression issue. As for quantum dot tracking in slices, our own trials have been quite unsuccessful, most QDs being generally too sticky and not diffusing well in tissue. Thus, as for tracking endogenous receptors, I think it’s quite hopeless. I do have seen in a few other labs people using GFP tagged proteins and managing to track them with anti-GFP coated QDs, but I have no direct experience with this approach as if I’m to use a tagged receptor, I prefer then to use FRAP in slice as it’s less prone to artifacts I think. Sorry I can’t help more, sure I’d wish we could do that……

All the best and see you in the near future

Best

Daniel