Skip to main content
PMC home page
UKPMC Funders Author Manuscripts logoLink to UKPMC Funders Author Manuscripts
. Author manuscript; available in PMC: 2012 Mar 20.
Published in final edited form as: Neuroscience. 2008 Feb 16;158(1):19–24. doi: 10.1016/j.neuroscience.2008.01.075

SYNAPTIC RECEPTOR TRAFFICKING: THE LATERAL POINT OF VIEW

F JASKOLSKI 1, J M HENLEY 1,*
PMCID: PMC3309027  EMSID: UKMS40389  PMID: 18455319

Abstract

Activity dependent modification of receptors in the post-synaptic density is a key determinant in regulating the strength of synaptic transmission during development and plasticity. A major mechanism for this recruitment and removal of postsynaptic proteins is the lateral diffusion in the plane of the plasma membrane. Therefore, the processes that regulate this lateral mobility are of fundamental importance. In recent years significant progress has been achieved using optical approaches such as single particle tracking (SPT) and fluorescence recovery after photobleach (FRAP). Here, we provide an overview of the principles and methodology of these techniques and highlight the contributions they have made to current understanding of protein mobility in the plasma membrane.

Keywords: synapse, receptor, diffusion, FRAP, single particle tracking

In 1827, the botanist Robert Brown first described that pollen particles moved haphazardly in water under the microscope (Brown, 1828). This phenomenon, later termed brownian motion (for review see (Nelson, 2001)), is the erratic displacement of a particle due to random impacts with smaller particles (i.e. atoms and small molecules). More than a century later, the first description of a membrane protein (rhodopsin) undergoing lateral mobility was published (Poo and Cone, 1974). This work demonstrated that in the fluid matrix of the lipid bilayer proteins are constantly moving. More recently, using modern techniques in biophysics, it has been shown that both recombinant glycine receptors and metabotropic glutamate receptors diffuse in the plane of neuronal plasma membrane by oscillating between confined and free brownian motion (Meier et al., 2001, Serge et al., 2002). Subsequently, the first evidence of native neurotransmitter receptors (glycine receptors and AMPA receptors) laterally diffusing in and out of synapses was provided in 2003 (Dahan et al., 2003, Tardin et al., 2003).

Protein trafficking to post-synaptic sites is a key mechanism regulating synaptic efficiency and many studies have focused on the mechanisms governing protein trafficking to and from the plasma membrane via exocytosis and endocytosis (for examples see (Perestenko and Henley, 2003, Shi et al., 1999)). This can be viewed as a ‘vertical’ process since it involves the movement of proteins from inside the cell to the membrane and vice versa. Exocytosis and endocytosis have been used extensively to explain many aspects of protein recruitment and removal underlying synaptic plasticity (Sheng and Lee, 2001). However, there is still only rather limited evidence to support the hypothesis that exocytosis and/or endocytosis occurs within the spine (Blanpied et al., 2002, Lu et al., 2007, for comment see Jaskolski et al., 2007) or more specifically within the post-synaptic density (PSD) (Gerges et al., 2006). An attractive complementary hypothesis for the delivery of at least some, and most likely the majority, of membrane proteins to the post-synaptic site is the ‘horizontal’ process of lateral diffusion within the plane of the membrane (Triller and Choquet, 2005). Thus, although there is still no widely accepted consensus as to the details and precise locations of membrane insertion, this process most likely involves the exocytosis of membrane proteins destined for the postsynaptic compartment into the dendritic membrane in the vicinity of synapses, followed by their subsequent lateral diffusion to the PSD (Yudowski et al., 2007).

The emergence of lateral mobility as a key mechanism regulating synaptic protein targeting originates mainly from the application of recently developed imaging techniques. There is now a substantial body of evidence to support lateral diffusion and here we review the techniques that have been used to study the contribution of this phenomenon to neurotransmitter receptor trafficking.

SINGLE PARTICLE TRACKING (SPT)

SPT involves real time imaging of a small probe linked to a protein of interest. The development of high sensitivity detection devices that enable the detection of single particles was a significant advance that allowed detailed analysis of membrane lateral diffusion. For example, SPT has revealed the remarkable erratic displacement of individual receptors in the neuronal plasma membrane (Meier et al., 2001, Serge et al., 2002).

As the movement of a protein in the plane of the plasma membrane is in the range of μm per second, SPT requires exquisitely accurate instrumentation. High-speed cameras are now readily available commercially but optical imaging still has intrinsic limitations in terms of spatial resolution at the μm level. Therefore, data acquired for motion capture in SPT are often subjected to a sub-pixel position calculation technique (Tardin et al., 2003). This post acquisition analysis of raw video frames allows the trajectories of individual particles to be tracked at very high resolution over time (Fig. 1A).

Fig. 1.

Fig. 1

Open in a new tab

SPT. (A) Sample traces of particles undergoing brownian motion in 2D oscillating between free motion (black, diffusion coefficient, D=0.5 μm2/s) and confined motion in a circular sub-domain (red, D=0.02 μm2/s). Generated by computer based Monte Carlo simulations of particles erratically exploring one of four possible positions at each time step (termed random walks). (B) Typical curves for MSD (area per time unit vs. time lag). For free motion the MSD grow linearly with time intervals, the slope is 4D where D is the microscopic diffusion coefficient. When a particle is confined, the MSD grow asymptotically to reach L2/3, where L2 is the area of the sub-domain. (C) Scale pictogram of various probes used in SPT, the latex bead diameter is 100 nm, 30 nm for the QD, 5 nm for the nano particle of gold (nGold) and 1–2 nm for the organic dye (oDye), the red scale bar=50 nm (average wide of the synaptic cleft).

To extract diffusion parameters from the trajectories of proteins of interest it is useful to compute the mean square displacement (MSD, for detailed formulas and fitting see (Kusumi et al., 1993, Bannai et al., 2006)). Briefly, the MSD can be described as the mean area covered by the particle during a given time of observation. For a particle freely moving in the plane of the membrane, the MSD increases in a linear manner with time and its slope is 4D, where D is the microscopic diffusion coefficient (Fig. 1B, area per time unit time). If the particle is confined to a sub-domain of the plasma membrane then the MSD grows asymptotically to reach a limit defined by the mean area of sub-domains (Fig. 1A/B, red trajectories) (Kusumi et al., 1993). In the case of confinement, the initial linear part of the curve can be used to calculate the microscopic diffusion coefficient as for free diffusion. For example, SPT experiments have been used to demonstrate that the metabotropic glutamate receptor (mGluR5) and the inhibitory glycine ligand-gated ion channel receptor are anchored by Homer and Gephyrin proteins respectively (Meier et al., 2001, Serge et al., 2002). These studies showed for the first time that neuronal membrane proteins can be confined in distinct subdomains.

When the computed MSD grows faster than 4D, the observed particle is undergoing facilitated rather than free diffusion. As yet, the only receptors which were shown to undergo driven motion is the mGluR5 and GABA-A receptors (Serge et al., 2003, Bouzigues et al., 2007) where microtubule polymerization has been proposed to generate the driving force. It has also been reported that AMPA receptor activation facilitates the diffusion of the membrane-anchored version of green fluorescent protein (mGFP) in dendritic spines (Richards et al., 2004) although the mechanisms underlying this facilitation remain unclear. In addition to demonstrating that neurotransmitter receptors move in the plane of the plasma membrane, SPT has also demonstrated that a significant proportion (30–50% for AMPA and NMDA receptors) of surface expressed receptors are immobile (Tardin et al., 2003, Groc et al., 2004, 2006). The transition between mobility and anchorage may be a key parameter regulating receptor stabilization in front of synaptic release sites. For example, it has been reported that calcium influx immobilizes AMPA receptors and may act to constrain receptors at the PSD during periods of synaptic activity (Borgdorff and Choquet, 2002).

Perhaps the most exciting potential application for SPT is the direct visualization of receptor entry into and exit from specialized plasma membrane domains such as the post synaptic density. To realize this potential, however, it is necessary to effectively and reliably stain synaptic contacts. This has been accomplished to some extent but notwithstanding the availability of a wide variety of both presynaptic and postsynaptic markers, no absolutely selective probes that do not stain other neuronal domains have been identified thus far. For example, mitochondria are highly enriched in presynaptic compartments and the scaffolding protein Homer 1C is a good marker for the postsynaptic density (Tardin et al., 2003, Bats et al., 2007) but labeling for both of these markers is present elsewhere in the neuron.

A common feature of all current SPT probes is their requirement to be coupled to an antibody, which either recognizes an epitope-tagged recombinant receptor or is directed against the native receptor of interest. Thus a significant limitation to SPT is the range of antibodies available to specifically bind the extracellular domain of a protein. Another critical and potentially limiting consideration for the interpretation of SPT is the size of the antibody-coupled probe used to monitor movement in the membrane.

Historically, the first probes used to track epitope-tagged recombinant receptors were latex beads cross-linked to antibodies (Meier et al., 2001, Serge et al., 2002). Latex beads can be conveniently imaged in phase contrast microscopy but suffer from the major drawback that they are very large (>100 nm) compared with the synaptic cleft, which is ~50 nm (Fig. 1C). The first alternatives to latex beads were the fluorescent organic dyes (e.g. Cy5). These probes are smaller (~30 nm with coupled antibody, Fig. 1C) but are limited by their short fluorescent lifetime due to rapid photobleaching. In addition, their relatively weak fluorescence signal requires the use of highly sensitive cameras (Tardin et al., 2003). The recent development of fluorescent semiconductors termed quantum dots (QDs) has provided a powerful new tool for tracking protein movement. These probes are both photostable and brighter than organic dyes with a reasonable size (30 nm) (Bannai et al., 2006).

The use of these last generation smaller probes led to the first clear demonstration that neurotransmitter receptors can enter and exit the post-synaptic site by lateral diffusion (Dahan et al., 2003; Tardin et al., 2003). The capability to image trajectories over long periods in and around synapses has provided new insights in the regulation of lateral diffusion. These include the discovery that AMPA receptor anchoring at synaptic sites depends both on presynaptic activity (Ehlers et al., 2007) and the interaction with PSD-95 via Stargazin (Bats et al., 2007). More recently the role of the extracellular matrix protein Reelin has been described in regulating NMDA receptor diffusion and synaptic recruitment (Groc et al., 2007). Taken together, these data show that synaptic activity, sub-membrane organization and extracellular environment are each important and illustrate the complex and subtle variety of mechanisms that act to regulate lateral diffusion of glutamate receptors.

Most recently, a new technique called single nanoparticle photothermal tracking (SNaPT) has been developed to image small probes in live cells. This method uses small gold particles (5 nm, coupled to an antibody, Fig. 1C) that can be detected by local thermal variation after light absorption (Lasne et al., 2006). This technique has a number of advantages compared with previous methods. For example, the stability of the probe allows long time-course tracking experiments and the small size of the gold particle reduces the potential for probe-related artifacts. It should be noted, however, that the imaging setup is highly sophisticated and is not, as yet, commercially available.

In summary, SPT is a very powerful technique and represents the only currently available method to clearly discriminate different types of protein movement in the membrane without population averaging effect. The main limitation with SPT is the technically demanding nature of the experiments and the need for highly sophisticated imaging apparatus and custom developed tracking analysis software (http://www.lkb.ens.fr/SINEMA), which restricts the accessibility of this approach to all but relatively few specialized laboratories.

FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING (FRAP)

Fluorescent molecules can absorb photons at a specific wavelength to emit another photon in response with a wavelength shift. This process is not unlimited however and the lifetime of a fluorescent molecule is dictated by the finite number of possible cycles of absorption/emission. Thus, sustained or intense excitation leads to loss of absorption. This is called photobleaching and has been used since 1970s to assess diffusion in cell membranes where photobleached molecules in a given area are replaced by non-photobleached ones by lateral diffusion (Fig. 2B) (Axelrod et al., 1976). This technique is called FRAP and the kinetics of the recovery depend on the diffusion properties of molecule studied. Briefly, living cells are imaged on a microscope with a strong excitation light source, usually a laser. Defined areas of membrane can be photobleached with high intensity light and the recovery over time is monitored by much lower levels of excitation with light at the same wavelength (Fig. 2B/C). Most often FRAP experiments are performed on recombinantly expressed proteins that incorporate a fluorescent tag (Fig. 2A). There is now a wide choice of fluorescent tags available many of which are based on green fluorescent protein (GFP) (Ashby et al., 2004, Giepmans et al., 2006).

Fig. 2.

Fig. 2

Open in a new tab

FRAP. (A) The AMPA receptor subunit GluR2 fused at the extracellular N-terminus with a derivative (XFP) of the GFP. (B) Principles of FRAP. A volume containing fluorescent proteins, evenly distributed at t0, is photobleached in a given area (blank square). Particles diffusing will refill the photobleached area over time (t1, t2, …) leading to a recovery of fluorescence. (C) Sample images of a FRAP experiment in a dendritic spine (mGFP). (D) Typical trace of normalized fluorescence intensity recorded in the bleach area over time, with the corresponding fit in red. The half time to recovery is t1/2, i is the immobile fraction, m is the mobile one.

Two parameters can be determined from the fluorescence recovery curve in FRAP. First the difference between the pre-photobleach level and the recovered steady state level after bleaching reflects the immobile fraction of protein. That is the lower level of fluorescence after recovery from photobleach is accounted for by bleached fluorescent protein that does not diffuse away. For example, these immobile proteins may be directly or indirectly tethered to cytoskeletal elements or restricted in confined membrane compartments. Second, the half time to recovery indicates the mobility of the diffusible fraction of the protein under investigation (Fig. 2D).

Theoretical analyses have generated curve fits to describe these phenomena and indicated that the lack of full recovery in FRAP experiments is not necessarily due to protein immobility (Feder et al., 1996). For example, it has been proposed that confinement of protein in membrane micro-domains can produce anomalous sub-diffusion leading to very slow recovery. This hypothesis is consistent with electron tomography data for potassium channels that suggest an organization of the sub-membrane cytoskeleton that results in nonspecific ‘fences’ that can trap membrane protein (Morone et al., 2006, Tamkun et al., 2007).

Although some studies, e.g. (Sharma et al., 2006, Saglietti et al., 2007) have used the half time to recovery to compare various conditions, this parameter alone is not a reliable measure since it is dependent on the area of membrane that is photobleached. To rigorously quantitate and compare protein diffusion in different membrane regions or under various condition using FRAP it is necessary to compute the diffusion coefficient as:

D=Area4t1∕2

Where t1/2 is the half time to recovery, Area is the photobleached membrane area and D the diffusion coefficient (area units per unit time) (Weiss, 2004).

Precise determination of the membrane area that is actually subject to bleaching in neurons is difficult because of their morphological complexity. Dendrites are essentially cylindrical with multiple branches that in many neurons are decorated by spine structures, which themselves can have a wide range of different shapes and are constantly moving in space. The challenge in data interpretation is increased by the fact that fluorescence recovery is affected by membrane shape (Sbalzarini et al., 2005). A further consideration for FRAP of membrane proteins, particularly in small three dimensional dynamic structures like spines, is the need to obtain a fluorescent signal exclusively from the plasma membrane. One effective way to achieve this is the use of super ecliptic phluorin (SEP), a GFP mutant that is sensitive to pH (Miesenbock et al., 1998, Ashby et al., 2004). It is also necessary to take account the number of iterations required to photobleach the region of interest since this can affect the initial slope of the recovery and in consequence the half time to recovery measured (Weiss, 2004).

In summary, FRAP is a relatively straightforward and accessible approach to assess protein diffusion in the neuronal plasma membrane. As with many techniques, however, there are a number of technical issues that must be fully addressed.

OTHERS TECHNIQUES AND FUTURE ADVANCES

Fluorescence correlation spectroscopy (FCS)

FCS is another optical technique to assess protein and lipid diffusion in cell membranes. FCS is a correlation analysis of fluctuation of fluorescence intensity recorded from a single excited volume where fluctuations represent the variation in the mean number of fluorescent proteins within the volume (Lenne et al., 2006, Garcia-Saez and Schwille, 2007). Briefly, as proteins (or tagged lipids) enter and exit the light excited membrane domain they produce fluctuations in the fluorescence recorded from that domain. The more proteins diffuse the greater the signal fluctuates. This approach allows accurate determination of nanoscale cell membrane organization (Wawrezinieck et al., 2005). The capacity of FCS to measure diffusion in small volumes represents a significant advantage for the study of protein diffusion in dendritic spines which have dimensions close to the femtoliter volume illuminated by the laser. However, as yet, few studies have used FCS to study neuronal membrane proteins (Hegener et al., 2004).

Photoinactivation

As an alternative to optical imaging, an interesting approach has been developed using photoactivable antagonists coupled to patch clamp electrophysiology. ANQX (6-azido-7-nitro-1,4-dihydroquinoxaline-2,3-dione), a photoreactive derivative of the AMPA receptor selective antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione), has been used to inactivate surface expressed AMPA receptors (Adesnik et al., 2005). That study suggested that AMPA receptors are inserted into the plasma membrane at the cell body and diffuse laterally throughout the dendritic tree to synapses. While this conclusion remains controversial since it is not readily reconciled with many other studies suggesting that AMPA receptors are transported intracellularly and exocytosed to the plasma membrane in dendrites (for example see (Perestenko and Henley, 2003, Yudowski et al., 2007)). Nonetheless, the strategy of photoinactivating receptors has considerable potential to assess their membrane dynamics. The technique requires irreversible antagonists that must be shown to be membrane impermeable to ensure that the antagonist does not inactivate intracellular pools of receptors. This has not yet been conclusively demonstrated for ANQX although it is referred to in the original description (Chambers et al., 2004). Notwithstanding these essential controls, it seems likely that this potentially elegant technique hold promise for future studies.

Mathematical modeling

Modeling approaches are being increasingly applied to cellular neuroscience. At the scale of synapses, protein diffusion is a matter of entering and exiting nanometer scale domains. Due to the resolution limit in optical imaging some of these events cannot be visualized. Thus, biophysicists have found a convenient way to partly circumvent this limitation by mathematically modeling particle diffusion. While the methodology is not easily accessible to all experimental neurobiologists, this approach is an emerging field of cellular neuroscience especially for the study the regulated trafficking of synaptic proteins such as glutamate receptors.

It has been proposed that the dendritic tree, with projecting spines, may act as a percolation system to trap receptors in spines (Bressloff and Earnshaw, 2007). Modeling has also predicted changes in the number and composition of AMPA type glutamate receptors during synaptic plasticity (Earnshaw and Bressloff, 2006). These studies, albeit interesting, rely on the assumption that exocytosis and endocytosis evenly occur in spines and dendritic shaft independently of synaptic activity or receptor composition. These assumptions are still a matter of considerable debate in the field but the general consensus is that this is in fact unlikely. For example, it has recently been reported that there is active dynamin-3 dependent AMPAR receptor recycling in the vicinity of the PSD (Lu et al., 2007).

By considering dendritic spines and the PSD as compartments with small openings it has been predicted that the time for a receptor to reach the PSD by diffusion depends on the length of the spine neck (Holcman and Triller, 2006). In generating this model, however, no account was taken of the spine morphology (Ashby et al., 2006) or membrane curvature (Yoshigaki, 2007), both of which may affect receptor diffusion in dendritic spines.

CONCLUSION

There has been impressive progress in the study of the mechanisms and dynamics of lateral diffusion of protein in the plasma membrane using the techniques discussed above. This area is important because it is a key determinant of receptor delivery to the postsynapse and therefore controls the efficiency of synaptic transmission and plasticity. There is, as yet, no single ultimate tool that can address all of the outstanding questions. For example, the major limitation with optical imaging is the limited resolution, especially with respect to the three-dimensional membrane topology of dendritic shafts and spines. Future approaches will make use of recent advances such as bifocal microscopy (Toprak et al., 2007) and 4D particle tracking (Anthony et al., 2006). We expect that, in combination with existing techniques, these emergent technologies are likely to provide new insights that will deepen our understanding of the fundamental rules that govern basal and activity-dependent membrane protein recruitment to synapses.

Acknowledgments

We are grateful to the MRC, the Wellcome Trust and the EU (GRIPPANT; PL 005320) for financial support. Frédéric Jaskolski is an EMBO postdoctoral fellow.

Abbreviations

ANQX

6-azido-7-nitro-1,4-dihydroquinoxaline-2,3-dione

FCS

fluorescence correlation spectroscopy

FRAP

fluorescence recovery after photobleaching

GFP

green fluorescent protein

mGFP

membrane-anchored version of green fluorescent protein

mGluR5

metabotropic glutamate receptor

MSD

mean square displacement

PSD

post-synaptic density

QD

quantum dot

SPT

single particle tracking

REFERENCES

  1. Adesnik H, Nicoll RA, England PM. Photoinactivation of native AMPA receptors reveals their real-time trafficking. Neuron. 2005;48:977–985. doi: 10.1016/j.neuron.2005.11.030. [DOI] [PubMed] [Google Scholar]
  2. Anthony SM, Hong L, Kim M, Granick S. Single-particle colloid tracking in four dimensions. Langmuir. 2006;22:9812–9815. doi: 10.1021/la062094h. [DOI] [PubMed] [Google Scholar]
  3. Ashby MC, Ibaraki K, Henley JM. It’s green outside: tracking cell surface proteins with pH-sensitive GFP. Trends Neurosci. 2004;27:257–261. doi: 10.1016/j.tins.2004.03.010. [DOI] [PubMed] [Google Scholar]
  4. Ashby MC, Maier SR, Nishimune A, Henley JM. Lateral diffusion drives constitutive exchange of AMPA receptors at dendritic spines and is regulated by spine morphology. J Neurosci. 2006;26:7046–7055. doi: 10.1523/JNEUROSCI.1235-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 1976;16:1055–1069. doi: 10.1016/S0006-3495(76)85755-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bannai H, Levi S, Schweizer C, Dahan M, Triller A. Imaging the lateral diffusion of membrane molecules with quantum dots. Nat Protoc. 2006;1:2628–2634. doi: 10.1038/nprot.2006.429. [DOI] [PubMed] [Google Scholar]
  7. Bats C, Groc L, Choquet D. The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking. Neuron. 2007;53:719–734. doi: 10.1016/j.neuron.2007.01.030. [DOI] [PubMed] [Google Scholar]
  8. Blanpied TA, Scott DB, Ehlers MD. Dynamics and regulation of clathrin coats at specialized endocytic zones of dendrites and spines. Neuron. 2002;36:435–449. doi: 10.1016/s0896-6273(02)00979-0. [DOI] [PubMed] [Google Scholar]
  9. Borgdorff AJ, Choquet D. Regulation of AMPA receptor lateral movements. Nature. 2002;417:649–653. doi: 10.1038/nature00780. [DOI] [PubMed] [Google Scholar]
  10. Bouzigues C, Morel M, Triller A, Dahan M. Asymmetric redistribution of GABA receptors during GABA gradient sensing by nerve growth cones analyzed by single quantum dot imaging. Proc Natl Acad Sci U S A. 2007;104:11251–11256. doi: 10.1073/pnas.0702536104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bressloff PC, Earnshaw BA. Diffusion-trapping model of receptor trafficking in dendrites. Phys Rev E Stat Nonlin Soft Matter Phys. 2007;75:041915. doi: 10.1103/PhysRevE.75.041915. [DOI] [PubMed] [Google Scholar]
  12. Brown R. Microscopical observations on the particles contained in the pollen of plants. Edinburgh New Philos J. 1828:258–371. [Google Scholar]
  13. Chambers JJ, Gouda H, Young DM, Kuntz ID, England PM. Photochemically knocking out glutamate receptors in vivo. J Am Chem Soc. 2004;126:13886–13887. doi: 10.1021/ja048331p. [DOI] [PubMed] [Google Scholar]
  14. Dahan M, Levi S, Luccardini C, Rostaing P, Riveau B, Triller A. Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science. 2003;302:442–445. doi: 10.1126/science.1088525. [DOI] [PubMed] [Google Scholar]
  15. Earnshaw BA, Bressloff PC. Biophysical model of AMPA receptor trafficking and its regulation during long-term potentiation/long-term depression. J Neurosci. 2006;26:12362–12373. doi: 10.1523/JNEUROSCI.3601-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ehlers MD, Heine M, Groc L, Lee MC, Choquet D. Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity. Neuron. 2007;54:447–460. doi: 10.1016/j.neuron.2007.04.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Feder TJ, Brust-Mascher I, Slattery JP, Baird B, Webb WW. Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys J. 1996;70:2767–2773. doi: 10.1016/S0006-3495(96)79846-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Garcia-Saez AJ, Schwille P. Single molecule techniques for the study of membrane proteins. Appl Microbiol Biotechnol. 2007;76:257–266. doi: 10.1007/s00253-007-1007-8. [DOI] [PubMed] [Google Scholar]
  19. Gerges NZ, Backos DS, Rupasinghe CN, Spaller MR, Esteban JA. Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane. EMBO J. 2006;25:1623–1634. doi: 10.1038/sj.emboj.7601065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Giepmans BN, Adams SR, Ellisman MH, Tsien RY. The fluorescent toolbox for assessing protein location and function. Science. 2006;312:217–224. doi: 10.1126/science.1124618. [DOI] [PubMed] [Google Scholar]
  21. Groc L, Choquet D, Stephenson FA, Verrier D, Manzoni OJ, Chavis P. NMDA receptor surface trafficking and synaptic subunit composition are developmentally regulated by the extracellular matrix protein Reelin. J Neurosci. 2007;27:10165–10175. doi: 10.1523/JNEUROSCI.1772-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Groc L, Heine M, Cognet L, Brickley K, Stephenson FA, Lounis B, Choquet D. Differential activity-dependent regulation of the lateral mobilities of AMPA and NMDA receptors. Nat Neurosci. 2004;7:695–696. doi: 10.1038/nn1270. [DOI] [PubMed] [Google Scholar]
  23. Groc L, Heine M, Cousins SL, Stephenson FA, Lounis B, Cognet L, Choquet D. NMDA receptor surface mobility depends on NR2A-2B subunits. Proc Natl Acad Sci U S A. 2006;103:18769–18774. doi: 10.1073/pnas.0605238103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hegener O, Jordan R, Haberlein H. Dye-labeled benzodiazepines: development of small ligands for receptor binding studies using fluorescence correlation spectroscopy. J Med Chem. 2004;47:3600–3605. doi: 10.1021/jm021009+. [DOI] [PubMed] [Google Scholar]
  25. Holcman D, Triller A. Modeling synaptic dynamics driven by receptor lateral diffusion. Biophys J. 2006;91:2405–2415. doi: 10.1529/biophysj.106.081935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jaskolski F, Martin S, Henley JM. Retaining synaptic AMPARs. Neuron. 2007;55:825–827. doi: 10.1016/j.neuron.2007.09.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kusumi A, Sako Y, Yamamoto M. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys J. 1993;65:2021–2040. doi: 10.1016/S0006-3495(93)81253-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lasne D, Blab GA, Berciaud S, Heine M, Groc L, Choquet D, Cognet L, Lounis B. Single nanoparticle photothermal tracking (SNaPT) of 5-nm gold beads in live cells. Biophys J. 2006;91:4598–4604. doi: 10.1529/biophysj.106.089771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lenne PF, Wawrezinieck L, Conchonaud F, Wurtz O, Boned A, Guo XJ, Rigneault H, He HT, Marguet D. Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork. EMBO J. 2006;25:3245–3256. doi: 10.1038/sj.emboj.7601214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lu J, Helton TD, Blanpied TA, Racz B, Newpher TM, Weinberg RJ, Ehlers MD. Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to homer. Neuron. 2007;55:874–889. doi: 10.1016/j.neuron.2007.06.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meier J, Vannier C, Serge A, Triller A, Choquet D. Fast and reversible trapping of surface glycine receptors by gephyrin. Nat Neurosci. 2001;4:253–260. doi: 10.1038/85099. [DOI] [PubMed] [Google Scholar]
  32. Miesenbock G, De Angelis DA, Rothman JE. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature. 1998;394:192–195. doi: 10.1038/28190. [DOI] [PubMed] [Google Scholar]
  33. Morone N, Fujiwara T, Murase K, Kasai RS, Ike H, Yuasa S, Usukura J, Kusumi A. Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography. J Cell Biol. 2006;174:851–862. doi: 10.1083/jcb.200606007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nelson E. In: Dynamical theories of brownian motion. 2nd edition Nelson E, editor. Princeton University Press; 2001. [Google Scholar]
  35. Perestenko P, Henley JM. Characterization of the intracellular transport of GluR1 and GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits in hippocampal neurons. J Biol Chem. 2003;44:43525–43532. doi: 10.1074/jbc.M306206200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Poo M, Cone RA. Lateral diffusion of rhodopsin in the photoreceptor membrane. Nature. 1974;247:438–441. doi: 10.1038/247438a0. [DOI] [PubMed] [Google Scholar]
  37. Richards DA, De Paola V, Caroni P, Gahwiler BH, McKinney RA. AMPA-receptor activation regulates the diffusion of a membrane marker in parallel with dendritic spine motility in the mouse hippocampus. J Physiol. 2004;558:503–512. doi: 10.1113/jphysiol.2004.062091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Saglietti L, Dequidt C, Kamieniarz K, Rousset MC, Valnegri P, Thoumine O, Beretta F, Fagni L, Choquet D, Sala C, Sheng M, Passafaro M. Extracellular interactions between GluR2 and N-cadherin in spine regulation. Neuron. 2007;54:461–477. doi: 10.1016/j.neuron.2007.04.012. [DOI] [PubMed] [Google Scholar]
  39. Sbalzarini IF, Mezzacasa A, Helenius A, Koumoutsakos P. Effects of organelle shape on fluorescence recovery after photobleaching. Biophys J. 2005;89:1482–1492. doi: 10.1529/biophysj.104.057885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Serge A, Fourgeaud L, Hemar A, Choquet D. Receptor activation and homer differentially control the lateral mobility of metabotropic glutamate receptor 5 in the neuronal membrane. J Neurosci. 2002;22:3910–3920. doi: 10.1523/JNEUROSCI.22-10-03910.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Serge A, Fourgeaud L, Hemar A, Choquet D. Active surface transport of metabotropic glutamate receptors through binding to microtubules and actin flow. J Cell Sci. 2003;116:5015–5022. doi: 10.1242/jcs.00822. [DOI] [PubMed] [Google Scholar]
  42. Sharma K, Fong DK, Craig AM. Postsynaptic protein mobility in dendritic spines: long-term regulation by synaptic NMDA receptor activation. Mol Cell Neurosci. 2006;31:702–712. doi: 10.1016/j.mcn.2006.01.010. [DOI] [PubMed] [Google Scholar]
  43. Sheng M, Lee SH. AMPA receptor trafficking and the control of synaptic transmission. Cell. 2001;105:825–828. doi: 10.1016/s0092-8674(01)00406-8. [DOI] [PubMed] [Google Scholar]
  44. Shi SH, Hayashi Y, Petralia RS, Zaman SH, Wenthold RJ, Svoboda K, Malinow R. Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science. 1999;284:1811–1816. doi: 10.1126/science.284.5421.1811. [DOI] [PubMed] [Google Scholar]
  45. Tamkun MM, O’Connell KM, Rolig AS. A cytoskeletal-based perimeter fence selectively corrals a sub-population of cell surface Kv2.1 channels. J Cell Sci. 2007;120:2413–2423. doi: 10.1242/jcs.007351. [DOI] [PubMed] [Google Scholar]
  46. Tardin C, Cognet L, Bats C, Lounis B, Choquet D. Direct imaging of lateral movements of AMPA receptors inside synapses. EMBO J. 2003;22:4656–4665. doi: 10.1093/emboj/cdg463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Toprak E, Balci H, Blehm BH, Selvin PR. Three-dimensional particle tracking via bifocal imaging. Nano Lett. 2007;7:2043–2045. doi: 10.1021/nl0709120. [DOI] [PubMed] [Google Scholar]
  48. Triller A, Choquet D. Surface trafficking of receptors between synaptic and extrasynaptic membranes: and yet they do move! Trends Neurosci. 2005;28:133–139. doi: 10.1016/j.tins.2005.01.001. [DOI] [PubMed] [Google Scholar]
  49. Wawrezinieck L, Rigneault H, Marguet D, Lenne PF. Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization. Biophys J. 2005;89:4029–4042. doi: 10.1529/biophysj.105.067959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weiss M. Challenges and artifacts in quantitative photobleaching experiments. Traffic. 2004;5:662–671. doi: 10.1111/j.1600-0854.2004.00215.x. [DOI] [PubMed] [Google Scholar]
  51. Yoshigaki T. Theoretically predicted effects of Gaussian curvature on lateral diffusion of membrane molecules. Phys Rev E Stat Nonlin Soft Matter Phys. 2007;75:041901. doi: 10.1103/PhysRevE.75.041901. [DOI] [PubMed] [Google Scholar]
  52. Yudowski GA, Puthenveedu MA, Leonoudakis D, Panicker S, Thorn KS, Beattie EC, von Zastrow M. Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors. J Neurosci. 2007;27:11112–11121. doi: 10.1523/JNEUROSCI.2465-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES