Choquet
| Malinow | Molecular Methods | Quantum Dots | Choquet | AMPAR |
2003
Tardin, Cognet, Bats, Lounis, Choquet • 2003 • EMBO - PDF
In this study they tested the effects of glutamate application and calcium influx on AMPAR diffusion. Being one of their earlier studies aimed at measuring diffusion, they used antibodies instead of Qdots.
- Glutamate Effect
- Bath application of 100 uM Glutamate
- Calcium Influx Effect
- induced calcium influx with biccuculine, strychnine, glycine
- to mimic NMDAR stimulation?
- decreased number of mobile AMPARs
- increased (59%) AMPAR membrane expression
- Calcium Blocking Effect
- used BAPTA to block calcium influx
- increased number of mobile AMPARs
- Notes
- 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.
- Newly inserted receptors were found to be initially diffusive and then stabilized at synaptic sites.
- 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.
- Methods
- Anti-GluR2 antibodies were labeled with Cy5 or Alexa-647 molecules at low labeling ratio (mean labeling ratio of 0.4 dye per antibody) so that individual antibodies were labeled at most with one fluorophore. A small proportion of surface expressed AMPA receptors containing the GluR2 subunit were selectively labeled in live neurons through short incubations with these antibodies. We could thus image and resolve discrete fluorescence spots with an epifluorescence imaging setup
2004
Groc L, Heine M, Cognet L, Brickley K, Stephenson FA, Lounis B, Choquet D. • 2004 • Nature Neuroscience - - PDF
In this study they tried out Qdots for the first time, but didn't use the data. They simply made the claim that the results from Qdot-antibodies gave similar results as the Cy3-antibodies (but only in extrasynaptic space, Qdots were 5x slower in synaptic space). They found that AMPARs diffuse 4x faster than NMDARs when outside the synapse (10.0 vs 2.3 nm/s) and at similar speeds in the synapse (28.0 vs 21.0 nm/s). Note that diffusion was faster outside the synapse than inside the synapse (that doesn't make sense!). KCl was used to stimulate neurons - this caused extrasynaptic AMPARs to diffuse 5x faster than baseline; whereas AMPAR mobility didn't change much in the synapse (but lower N). They didn't report NMDAR changes from KCl. They stimulated PKC activity using TPA, resulting in significant mobility increases for both NMDAR and AMPAR in synaptic and extrasynaptic space.
- Abstract
- The basis for differences in activity-dependent trafficking of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) remains unclear. Using single-molecule tracking, we found different lateral mobilities for AMPARs and NMDARs: changes in neuronal activity modified AMPAR but not NMDAR mobility, whereas protein kinase C activation modified both. Differences in mobility were mainly detected for extrasynaptic AMPARs, suggesting that receptor diffusion between synaptic and extrasynaptic domains is involved in plasticity processes.
- Methods
- Here, we directly compared the lateral mobilities of AMPARs and NMDARs. For this, we measured the diffusion of GluR2 subunit–containing AMPAR and NR1 subunit–containing NMDAR at the surface of cultured hippocampal neurons at 9–11 days in vitro by single-molecule fluorescence microscopy tracking of receptors labeled with appropriate Cy3-coupled antibodies. We compared AMPAR diffusion obtained with Cy3-conjugated antibodies with quantum dot (QD)-coupled antibodies, because QDs are more photostable than organic dyes. In the extrasynaptic membrane, diffusion distributions measured using Cy3-conjugated and QD-coupled antibodies were similar, thus validating the diffusion estimates obtained with Cy3-conjugated antibodies. Distributions of synaptic receptor diffusions were significantly different, however, with QD-coupled receptor diffusion being five times slower. This effect could be due to limitation of receptor diffusion within the glutamatergic synaptic cleft by the bound QD (QD diameter >10–15 nm). Therefore, in the present study, AMPAR and NMDAR diffusions were estimated only from Cy3-conjugated antibodies.
- Results
- We treated neurons with either potassium chloride increase neural activity, or tetrodotoxin to decrease neuronal activity. After 2 min of 40 mM KCl treatment, extrasynaptic AMPAR diffusion greatly increased (560% of control), owing to less immobile receptors (from 47% to 32%). Notably, the AMPAR diffusion measured after KCl treatment is comparable to the previously published ‘control’ value (median 0.11 μm2/s, n = 309) obtained after similar KCl treatment needed to stain synapses with FM1-43. When the spontaneous neuronal activity was blocked with TTX for only 10 min, no changes were observed, consistent with the low spontaneous basal activity of our cultured neurons. However, blocking neuronal activity for 48 h greatly decreased extrasynaptic AMPAR diffusion (12% of control), owing to more immobile receptors (from 47% to 67%).
- Activation of protein kinase C (PKC) induces rapid dispersal of NMDARs from a clustered to a uniform membrane distribution as well as endocytosis and redistribution of GluR2-containing AMPARs to synaptic sites. We thus investigated whether NMDAR and AMPAR mobilities were affected by the PKC agonist TPA. After TPA treatment, NMDAR diffusion was increased in both extrasynaptic (12-fold) and synaptic membranes (5-fold). This is consistent with the reported TPA-induced dispersion of NMDARs. Extrasynaptic and synaptic AMPAR diffusions were also significantly affected by TPA treatment (extrasynaptic +34%) (synaptic +333%).
2005
Triller, Choquet • 2005 • Trends in Neuroscience - PDF
Review Article
- Abstract
- Concentration of neurotransmitter receptors at synapses is thought to result from stable binding to subsynaptic scaffold proteins. Recent data on synaptic plasticity have shown that changes in synaptic strength derive partly from modification of postsynaptic receptor numbers. This has led to the notion of receptor trafficking into and out of synapses. The proposed underlying mechanisms have under-evaluated the role of extrasynaptic receptors. Recent technological advances have allowed imaging of receptor movements at the single-molecule level, and these experiments demonstrate that receptors switch at unexpected rates between extrasynaptic and synaptic localizations by lateral diffusion. Variation in receptor numbers at postsynaptic sites is therefore likely to depend on regulation of diffusion by modification of the structure of the membrane and/or by transient interactions with scaffolding proteins. This review is part of the TINS Synaptic Connectivity series.
Thoumine, et-al, Choquet • 2005 • Biophys - PDF
- Abstract
- To assess if membrane diffusion could affect the kinetics of receptor recruitment at adhesive contacts, we transfected neurons with green fluorescent protein-tagged immunoglobin cell adhesion molecules of varying length (25-180 kD), and measured the lateral mobility of single quantum dots bound to those receptors at the cell surface. The diffusion coefficient varied within a physiological range (0.1-0.5 microm(2)/s), and was inversely proportional to the size of the receptor. We then triggered adhesive contact formation by placing anti-green fluorescent protein-coated microspheres on growth cones using optical tweezers, and measured surface receptor recruitment around microspheres by time-lapse fluorescence imaging. The accumulation rate was rather insensitive to the type of receptor, suggesting that the long-range membrane diffusion of immunoglobin cell adhesion molecules is not a limiting step in the initiation of neuronal contacts.
2006
Thoumine, Lambert, Mège, Choquet • 2006 • Journal - PDF
- Abstract
- N-cadherin plays a key role in axonal outgrowth and synaptogenesis, but how neurons initiate and remodel N-cadherin-based adhesions remains unclear. We addressed this issue with a semiartificial system consisting of N-cadherin coated microspheres adhering to cultured neurons transfected for N-cadherin-GFP. Using optical tweezers, we show that growth cones are particularly reactive to N-cadherin coated microspheres, which they capture in a few seconds and drag rearward. Such strong coupling requires an intact connection between N-cadherin receptors and catenins. As they move to the basis of growth cones, microspheres slow down while gradually accumulating N-cadherin-GFP, demonstrating a clear delay between bead coupling to the actin flow and receptor recruitment. Using FRAP and photoactivation, N-cadherin receptors at bead-to-cell contacts were found to continuously recycle, consistently with a model of ligand-receptor reaction not limited by membrane diffusion. The use of N-cadherin-GFP receptors truncated or mutated in specific cytoplasmic regions show that N-cadherin turnover is exquisitely regulated by catenin partners. Turnover rates are considerably lower than those obtained previously in single molecule studies, demonstrating an active regulation of cadherin bond kinetics in intact cells. Finally, spontaneous neuronal contacts enriched in N-cadherin exhibited similar turnover rates, suggesting that such dynamics of N-cadherin may represent an intrinsic mechanism underlying the plasticity of neuronal adhesions.
Cognet, Groc, Lounis, Choquet • 2006 • Science STKE - PDF
- Abstract
- Trafficking of glutamate receptors into and out of synapses is critically involved in the plasticity of excitatory synaptic transmission. Endocytosis and exocytosis of receptors have initially been thought to account alone for this trafficking. However, membrane proteins also traffic through surface lateral diffusion in the plasma membrane. We describe developments in electrophysiological and optical approaches that have allowed for the real-time measurement of glutamate receptor surface trafficking in live neurons. These include (i) specific imaging of surface receptors using a pH-sensitive fluorescent protein; (ii) design of a photoactivable drug to locally inactivate surface receptors and monitor electrophysiologically their recovery; and (iii) application of single-molecule fluorescence microscopy to directly track the movement of individual surface receptors with nanometer resolution inside and outside synapses. Together, these approaches have demonstrated that glutamate receptors diffuse at high rates in the neuronal membrane and suggest a key role for surface diffusion in the regulation of receptor numbers at synapses.
Tracking individual nano-objects in live cells during arbitrary long times is a ubiquitous need in modern biology. We present here a method for tracking individual 5-nm gold nanoparticles on live cells. It relies on the photothermal effect and the detection of the Laser Induced Scattering around a NanoAbsorber (LISNA). The key point for recording trajectories at video rate is the use of a triangulation procedure. The effectiveness of the method is tested against single fluorescent molecule tracking in live COS7 cells on subsecond timescales. We further demonstrate recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. Single Nanoparticle Photothermal Tracking has the unique potential to record arbitrary long trajectory of membrane proteins using nonfluorescent nanometer-sized labels.
2007
Groc, et-al, Choquet, Cognet • 2007 • J Neuro - PDF
Ehlers, Heine, Groc, Lee, Choquet • 2007 • Neuron - PDF
- Abstract
- Synaptic activity regulates the postsynaptic accumulation of AMPA receptors over timescales ranging from minutes to days. Indeed, the regulated trafficking and mobility of GluR1 AMPA receptors underlies many forms of synaptic potentiation at glutamatergic synapses throughout the brain. However, the basis for synapse-specific accumulation of GluR1 is unknown. Here we report that synaptic activity locally immobilizes GluR1 AMPA receptors at individual synapses. Using single-molecule tracking together with the silencing of individual presynaptic boutons, we demonstrate that local synaptic activity reduces diffusional exchange of GluR1 between synaptic and extraynaptic domains, resulting in postsynaptic accumulation of GluR1. At neighboring inactive synapses, GluR1 is highly mobile with individual receptors frequently escaping the synapse. Within the synapse, spontaneous activity confines the diffusional movement of GluR1 to restricted subregions of the postsynaptic membrane. Thus, local activity restricts GluR1 mobility on a submicron scale, defining an input-specific mechanism for regulating AMPA receptor composition and abundance.
Saglietti, et-al, Choquet, Sala, Sheng, Passafaro • 2007 • Neuron - PDF
- Abstract
- Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. Here we show that the first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. Our data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation.
Bats, Groc, Choquet • 2007 • Neuron - PDF
- Quantum Dot
- FRAP
- Live hippocampal neurons
- 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.
- AMPARs and Stargazin diffuse as complexes in and out synapses.
2008
Cognet, Lounis, Choquet • 2008 • CHS - PDF
- Abstract
- This article describes imaging techniques using single optical labels, ranging from fluorescent dyes to scattering particles, for the study of the movement of individual or small assemblies of membrane proteins. These techniques have been used to track the movements of different types of plasma membrane proteins, such as neurotransmitter receptors and adhesion proteins. They can be used to probe the degree of interaction between membrane proteins and cytoplasmic stabilizing elements in live cells.
Heine, Groc, Huganir, Cognet, Choquet • 2008 • Science - PDF
- Abstract
- AMPA glutamate receptors (AMPARs) mediate fast excitatory synaptic transmission. Upon fast consecutive synaptic stimulation, transmission can be depressed. Recuperation from fast synaptic depression has been attributed solely to recovery of transmitter release and/or AMPAR desensitization. We show that AMPAR lateral diffusion, observed in both intact hippocampi and cultured neurons, allows fast exchange of desensitized receptors with naïve functional ones within or near the postsynaptic density. Recovery from depression in the tens of millisecond time range can be explained in part by this fast receptor exchange. Preventing AMPAR surface movements through cross-linking, endogenous clustering, or calcium rise all slow recovery from depression. Physiological regulation of postsynaptic receptor mobility affects the fidelity of synaptic transmission by shaping the frequency dependence of synaptic responses.
Groc, Choquet • 2008 • Mol Membr Biol - PDF
- Abstract
- Neurotransmitter receptor trafficking in and out synapses has emerged as a key process to regulate synaptic transmission during synaptic development and plasticity both at excitatory and inhibitory synapses. Lateral diffusion of surface neurotransmitter receptors has recently emerged as a key pathway to regulate receptor trafficking to and from synapses. Receptors enter and exit synapses mainly by lateral diffusion within the plane of the membrane while their retrieval and addition from and to the plasma membrane by endo and exocytotic processes occur largely at extrasynaptic sites. As a consequence, regulation of receptor surface trafficking is likely to be a major process to regulate receptor numbers at synapses. Measurement of receptor surface diffusion has required the development of new experimental approaches to specifically label and track surface receptor with appropriate time- and space-resolutions. In this review, we first discuss the approaches that have been used to measure receptor surface diffusion, such as the ensemble approach that measure average diffusion of a defined surface receptor population and the single molecule/particle approaches that measure the surface diffusion of isolated receptors. To date, surface diffusion has been described for a variety of neurotransmitter receptors that exhibit common as well as specific features. These points are discussed in a comparative manner and emerging rules of surface trafficking as well as potential interplay between receptor classes are further commented. Because our knowledge on neurotransmitter receptor surface diffusion is fairly recent, open questions and experimental challenges facing the field are highlighted throughout the review.
Triller, Choquet • 2008 • Neuron - PDF
- Abstract
- Single-molecule approaches give access to the full distribution of molecule behaviors and overcome the averaging intrinsic to bulk measurement methods. They allow access to complex processes where a given molecule can have heterogeneous properties over time. Recent developments in single-molecule imaging technologies have been followed by their wide application in cellular biology and are leading to the unraveling of new mechanisms related to molecular movements. They are shaping new concepts in the dynamic equilibria of complex biological macromolecular assemblies such as synapses. These advances were made possible thanks to improvements in visualization approaches combined with new strategies to label proteins with nanoprobes. In this primer, we will review the different approaches used to track single molecules in live neurons, compare them to bulk measurements, and discuss the different concepts that have emerged from their application to synaptic biology.
2009
2010
2011
2012
Czöndör, Mondin, Garcia, Heine, Frischknecht, Choquet, Sibarita, Thoumine • 2012 • PNAS - PDF
Trafficking of AMPA receptors (AMPARs) plays a key role in synaptic transmission. However, a general framework integrating the two major mechanisms regulating AMPAR delivery at postsynapses (i.e., surface diffusion and internal recycling) is lacking. To this aim, we built a model based on numerical trajectories of individual AMPARs, including free diffusion in the extrasynaptic space, confinement in the synapse, and trapping at the postsynaptic density (PSD) through reversible interactions with scaffold proteins. The AMPAR/scaffold kinetic rates were adjusted by comparing computer simulations to single-particle tracking and fluorescence recovery after photobleaching experiments in primary neurons, in different conditions of synapse density and maturation. The model predicts that the steady-state AMPAR number at synapses is bidirectionally controlled by AMPAR/scaffold binding affinity and PSD size. To reveal the impact of recycling processes in basal conditions and upon synaptic potentiation or depression, spatially and temporally defined exocytic and endocytic events were introduced. The model predicts that local recycling of AMPARs close to the PSD, coupled to short-range surface diffusion, provides rapid control of AMPAR number at synapses. In contrast, because of long-range diffusion limitations, extrasynaptic recycling is intrinsically slower and less synapse-specific. Thus, by discriminating the relative contributions of AMPAR diffusion, trapping, and recycling events on spatial and temporal bases, this model provides unique insights on the dynamic regulation of synaptic strength.
2013
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
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