The Journal of Neuroscience, November 14, 2007 • 27(46):12433–12437 • 12433 Toolbox Editor’s Note: Toolboxes are intended to briefly highlight a new method or a resource of general use in neuroscience or to critically analyze existing approaches or methods. For more information, see http://www.jneurosci.org/misc/itoa.shtml. Surface Trafficking of Neurotransmitter Receptor: Comparison between Single-Molecule/Quantum Dot Strategies Laurent Groc,1 Mathieu Lafourcade,1 Martin Heine,1 Marianne Renner,1 Victor Racine,3 Jean-Baptiste Sibarita,3 Brahim Lounis,2 Daniel Choquet,1 and Laurent Cognet2 1Physiologie Cellulaire de la Synapse, Unite´ Mixte de Recherche (UMR) 5091, Centre National de la Recherche Scientifique (CNRS), Universite´ Bordeaux 2, 33077 Bordeaux, France, 2Centre de Physique Mole´culaire Optique et Hertzienne, UMR 5798, CNRS, Universite´ Bordeaux 1, 33405 Talence, France, and 3Curie Institute, Research Division, UMR 144-CNRS, 75005 Paris, France The cellular traffic of neurotransmitter re- XFP-tag approach in live experiment is antibodies directed against receptor ex- ceptors has captured a lot of attention extreme difficulty in detecting XFP fluo- tracellular epitopes. over the last decade, mostly because syn- rescence signals from small nonclustered The purpose of this Toolbox is to out- aptic receptor number is adjusted during receptor pool (Cognet et al., 2002; line currently available approaches to synaptic development and plasticity. Al- Lippincott-Schwartz and Patterson, measure the surface trafficking of receptor though each neurotransmitter receptor 2003). XFP-tagged neurotransmitter re- in neurons, with a special emphasis on family has its own trafficking characteris- ceptors are often present in several cellu- single-molecule (organic dye) and quan- tics, two main modes of receptor delivery lar compartments from the endoplasmic tum dot (QDot) detection for neurotrans- to the synapse have emerged: endo- reticulum to the plasma membrane with mitter receptor tracking. exocytotic cycling and surface diffusion various relative contents. For instance, [e.g., for glutamatergic receptors, see surface XFP-tagged neurotransmitter re- Exploring receptor surface Bredt and Nicoll (2003) and Groc and ceptors represent only a minor fraction of trafficking: approaches Choquet (2006)]. Receptor cycling the total receptor population, precluding Investigation of receptor surface distribu- through endo-exocytotic processes can be their specific detection. Alternative live- tion and diffusion can be sorted in two measured by several experimental means, cell imaging approaches were thus re- groups. On the one hand, the average sur- from biochemical to imaging assays. The quired to specifically isolate surface recep- face diffusion of labeled receptors is stud- use of fluorescent protein (XFP)-tag im- tors. Interestingly, a variant of the green ied without distinction of individual be- aging provides a powerful approach to in- fluorescent protein (GFP), ecliptic pHlu- haviors (Fig. 1A). Surface receptors can be vestigate the trafficking of receptor clus- orin, shows a reversible excitation ratio isolated by electrophysiologically tagged ters between neuronal compartments change between pH 7.5 and 5.5, and its methods (Tovar and Westbrook, 2002; (e.g., soma, dendrite, spine) (Kennedy absorbance decreases as the pH is low- Adesnik et al., 2005; Thomas et al., 2005). and Ehlers, 2006). A disadvantage of the ered. Most neurotransmitter receptors, Schematically, a subpopulation of surface including the ionotropic glutamate ones, receptors is irreversibly blocked (e.g., Received April 11, 2007; revised Sept. 18, 2007; accepted Oct. 2, 2007. display an extracellular N-terminal re- MK-801 for NMDA receptors) and the re- This work was supported by grants from the Centre National de la Re- gion, implying that the N terminus will ceptor surface diffusion is estimated from cherche Scientifique, Conseil Re´gional d’Aquitaine, Ministe`re de la Recher- always be in an acidic environment inside time-dependent functional recovery after che, and Fondation pour la Recherche Me´dicale and by a European Com- the cell, whereas it will be exposed to a receptor blockade. Another approach is munity Grant (GRIPANT, CT-2005-005320). We thank Richard Huganir for the cDNA BGG construct and Christelle Breillat, Beatrice Teissier, and Del- neutral pH after insertion into the plasma based on the fluorescence labeling tech- phine Bouchet for technical assistance. membrane. By this means, surface recep- niques coupled to live fluorescence mi- Correspondence should be addressed to Laurent Groc, Unite´ Mixte de tors can be specifically detected and croscopy, such as fluorescent recovery af- Recherche 5091, Centre National de la Recherche Scientifique, Universite´ tracked with live-imaging approaches ter photobleaching (FRAP) of pHluorin- Bordeaux 2, 146 rue Le´o Saignat, 33077 Bordeaux, France. E-mail: [email protected]. (Ashby et al., 2004). Alternatively, surface tagged receptors (Rasse et al., 2005; Ashby DOI:10.1523/JNEUROSCI.3349-07.2007 receptors can be labeled and detected by et al., 2006; Kopec et al., 2006; Lober et al., Copyright©2007SocietyforNeuroscience 0270-6474/07/2712433-05$15.00/0 immunocytochemical approaches using 2006; Sharma et al., 2006). Note that both 12434 • J. Neurosci., November 14, 2007 • 27(46):12433–12437 Groc et al. • Toolbox approaches provided equivalent charac- teristics of receptor surface diffusion [e.g., for AMPA receptor in hippocampal cul- tured neurons (Adesnik et al., 2005; Ashby et al., 2006)], indicating that en- semble surface receptor diffusion can effi- ciently be measured using either approaches. Opposite to the ensemble methods, single-molecule detection methods re- trieve the diffusion properties of individ- ual labeled receptors over time (Fig. 1A). The typical outcome is the complete dis- tribution of the behavior of surface neu- rotransmitter receptors, which have re- vealed non-Gaussian shapes and a variety of diffusion characteristics at a given time. To date, this approach has unraveled the surface trafficking of neurotransmitter re- ceptors and channels, including glutama- tergic metabotropic mGluR5, AMPA and NMDA receptors (Borgdorff and Cho- quet, 2002; Serge et al., 2002; Tardin et al., 2003; Groc et al., 2004; Howarth et al., 2005; Groc et al., 2006), glycine (Meier et al., 2001; Dahan et al., 2003), GABA re- ceptors (Bouzigues and Dahan, 2007), and Kv potassium channels (O’Connell et Figure1. Schematicdescriptionofsingle-molecule/particleexperimentforthetrackingofsurfacereceptorsinneurons.A,The al., 2006). To label single molecules, there first choice to make when planning to study receptors at the surface of neurons (left, hippocampal neuron expressing GFP-Homer are essentially two possibilities: the use of 1C) is the type of experimental approach to setup. Right, There are schematically two ways to examine surface trafficking of receptors: ensemble or single-molecule approaches. The first one measures the surface behavior of the ensemble of labeled a single dye (SD) or a single particle (SP) receptors using, for instance, FRAP. The second one measures the surface behavior of one (or possibly two if using antibody) (e.g., nano-sized QDots). The SD tags are receptor using single-molecule or Dot tracking. Although both approaches provide good measurements of receptor surface short lived but small size, whereas SP tags diffusion, differences in time and spatial resolutions exist. For instance, the spatial resolution in the type of microscopy used for allow long observation times, but the size FRAPandDotexperimentsisdefinedbythediffractionlimit,typicallyontheorderof200–300nm.Thesingle-moleculetechnique of the label can prevent its access to spe- provides, however, a 10 nm pointing accuracy, which provides a unique way to collect the number of points necessary to build a cific confined cellular areas. trajectory in submicrometer membrane domains (e.g., synapse, lipid raft). B, Experimental procedure to run single-molecule or Dottracking.First,thelabelusedtodetectthemoleculeofinterest(e.g.,antibody)iscoupledtothetag(singledye,singleDot)in Single dye tracking the ratio 1:1 (left). Second, live neurons are incubated with the single tag complexes (high dilution, ϳ1 ng/ml) for several SD tracking (SDT) first requires the at- minutes. Third, single tag complexes are detected and tracked (see Single dye tracking and QDot tracking) over time as exempli- tachment of a single tag (e.g., organic dye) fiedbythedetectionofasingleDotona30msacquisitionimageusingaCCDcamera.Scalebar,600nm.Toensurethatthetracked complexes are in majority at the surface, rates of endocytosis of the receptor of interest should be first measured. SDT can then be to the molecular target through a specific performed during a time window in which only a low percentage (classically set as Ͻ15–20%) of receptors are internalized. For high-affinity ligand that recognizes the example, AMPAR surface tracking is performed only during 20 min after labeling, ensuring that Ͻ10–20% of labeled receptors extracellular domain of the molecular tar- are internalized. Note that the newly internalized AMPARs are mostly immobile and represent a small fraction of the single- get in live cells (Fig. 1B). Labeling must be molecule signals (Tardin et al., 2003). performed at low tag densities to be opti- cally resolved (typically Ͻ1 molecule/ Trolox, provides an efficient way to signif- image frame, their two-dimensional tra- ␮ 3 m ) and to avoid cross-linking