The Journal of Neuroscience, November 14, 2007 • 27(46):12433–12437 • 12433

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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 in the icantly reduce photobleaching and thus jectories can be constructed using a Vogel case of multivalent ligands (e.g., anti- improve photostability (Rasnik et al., algorithm. This algorithm performs a cor- body). The second obvious requirement is 2006). relation analysis between the positions of to obtain signals from an SD that are large In practice, single molecules are iden- the SD found in consecutive images enough to overcome both the background tified by their diffraction-limited signals, (Schmidt et al., 1995) (supplemental Box noise from the molecule environment and their well defined intensities, and the one- 2, available at www.jneurosci.org as sup- that of the detection system (Fig. 1B). Sev- step photobleaching behavior (supple- plemental material). eral dyes fulfill these requirements, such mental Box 1, available at www. as cyanine dyes (Cy3 or Cy5) that can be QDot tracking detected with good signal-to-noise ratios jneurosci.org as supplemental material). QDots are passivated semiconductor in live cells. The main limitation of SDT is, Two-dimensional fitting by a Gaussian nanocrystals that are water soluble and however, photobleaching, i.e., the pro- approximating the true point spread functionalized for biological applications. cesses by which photochemical reactions function of the microscope allows sub- In addition to their superior brightness transform the excited fluorophore into a wavelength localization of the labels compared with SD, QDots have a larger nonfluorescent product. It limits imaging (Cheezum et al., 2001), typically Ͻ50 nm absorption cross section, such that they time of a SD to typically a few seconds. for SDs in live cells (Tardin et al., 2003). can be excited with a mercury lamp. This Note that the use of antioxidant, such as Finally, once SDs are identified in each allows recording large fields of view and Groc et al. • Toolbox J. Neurosci., November 14, 2007 • 27(46):12433–12437 • 12435

(maximum distance between reconnected events and maximum “dark” time) are adjusted according to the diffusion time of the molecules and the image acquisi- tion rate. To prevent false reconnections, the surface density of QDots must be low enough that parameters of reconnection can be found without mixing the trajecto- ries of different QDots. In practice, the maximum distance of reconnection is on the order of the mean QDot image-to- image steps and at least 10 times smaller than the mean distance between different QDots. It should be mentioned that other analytical approaches have been devel- oped to overcome the problem of QDot blinking in trajectory reconstructions (Bonneau et al., 2005; Bachir et al., 2006). Interestingly, QDot blinking provides a criterion to identify individual QDots, be- Figure 2. Tracking of a BBS-GFP-tagged GluR2 subunit using three different single-molecule/particle approaches. A, Sche- cause fluorescence changes between “on” maticrepresentationoftheBGGandthesingle-molecule/particlecomplexes:DotcoupledtoantibodydirectedagainstGFP(left), and “off” states for a single Dot alternate cyanine5coupledtoanantibodydirectedagainstGFP(center),andcyanine5coupledtobungarotoxin(right).Theinsertionofthe only in two levels. BGG into the surface membrane was observed by incubating neurons transfected with BGG with bungarotoxin coupled to Alexa 568 (data not shown). Hippocampal neurons from 18-d-old rat embryos were cultured on glass coverslips following the Banker ␮ technique.ForBGGexpression,hippocampalneuronscultured8–11dinvitroweretransfectedwith1 gofBGGcDNAfor24–36 Does the size and valence of single tag h before the experiment using Lipofectamine 2000 reagent. Surface BGGs were detected by immunocytochemistry after live complexes influence surface diffusion? incubation with Alexa568-bungarotoxin (data not shown). Because cultured neurons can express low levels of endogenous At the scale of the nano-sized probes, nicotinicacetylcholinereceptors,neuronswereincubatedwiththenicotinergicreceptorantagonistmethyllycaconitine(10nM,20 min,37°C)duringsingletagtracking.Finally,forsingle-moleculetracking,cyanine3orcyanine5wascoupledtobungarotoxinor movements of surface extrasynaptic re- to the rabbit polyclonal anti-GFP antibody. For quantum dot (QD) tracking, QD 655 goat F(AbЈ)2 anti-rabbit IgGs (0.1 ␮M) were ceptors are not governed by the mass of coupled with the polyclonal antibodies against GFP (1 ␮g). B, Representative extrasynaptic trajectories from the three different the probe, but instead by the motion of surface single-molecule/particle complexes. Scale bar, 600 nm. Note the difference in trajectory length as a result of the photo- the membrane protein. Indeed, the vis- stability of single Dot when compared with cyanine 5 fluorophores. C, Cumulative distributions of the instantaneous diffusion cosity of membranes is 100- to 1000-fold coefficient (bin size ϭ 0.075 ␮m 2/s) ( p Ͼ 0.05 for all comparisons, Kolmogorov–Smirnov test). The median values were not greater than that of extracellular medium. Ϫ3 2 2 Ϫ3 significantly different (QDot-Ab ϭ 1 ϫ 10 ␮m /s, IQR ϭ 0–0.049 ␮m /s, n ϭ 1498 trajectories; Cy-Ab ϭ 2 ϫ 10 In restricted environments, however, like ␮ 2 ϭ ϫ Ϫ4 ␮ 2 ϭ ϭ ϫ Ϫ3 ␮ 2 ϭ ␮ 2 ϭ Ͼ m /s,IQR 1 10 –0.049 m /s,n 919;Cy-BTx 4 10 m /s,IQR 0–0.025 m /s,n 1387;p 0.05, in the synaptic cleft, the size and valence of Mann–Whitney U test). The percentage of immobile receptor (first point of the cumulative distribution) and the instantaneous a single complex can impact surface re- diffusion coefficient (Inst. Diff. Coeff.) of the mobile molecules/particles (set to D Ͼ 0.0075 ␮m 2/s) were also not significantly different between complexes. All trajectories (each condition) were obtained from 30–60 dendritic fields and three to five ceptor diffusion. The impact of different differentculturepreparations.D,LabelingofsynapsesusingMitotracker(1minat1nM).Synaptictrajectories(green)aredefined labeling strategies on SD/QDot measure- by their colocalization with synaptic labeling, the trajectory outside synapse being considered as extrasynaptic (yellow). Note the ments in nonconfined cellular environ- change in surface diffusion from a free diffusion behavior (extrasynaptic, yellow) to a confined one (synaptic, green). E, Compar- ments (the extrasynaptic membrane) and Ͻ ison of the instantaneous diffusion coefficient of mobile molecules/particles ( p 0.05, Mann-Whitney U test between Cy-BTx confined ones (synapses labeled with Mi- and the two other complexes). Note the subset of highly diffusing receptor detected with Cy-BTx. However, the instantaneous Ϫ Ϫ totracker) (Tardin et al., 2003; Groc et al., diffusion coefficient of all trajectories (QDot-Ab ϭ 5 ϫ 10 3 ␮m 2/s, IQR ϭ 4 ϫ 10 4–0.023 ␮m 2/s, n ϭ 45; Cy-Ab ϭ 5 ϫ 10 Ϫ3 ␮m 2/s,IQRϭ0–0.024␮m 2/s,nϭ98;Cy-BTxϭ10ϫ10 Ϫ3 ␮m 2/s,IQRϭ7ϫ10 Ϫ4–0.035␮m 2/s,nϭ51;pϾ 2004) was measured. Cultured hippocam- 0.05, Kruskal–Wallis test) and the percentage of immobile receptors ( p Ͼ 0.05) were not significantly different between pal neurons were transfected with the complexes. F, Percentage of dyes/QDots detected within the synapse (QDot-Ab ϭ 5.4 Ϯ 1.3%, n ϭ 9 dendritic areas; Cy-Ab ϭ GluR2 AMPA receptor subunit tagged with 9.4 Ϯ 2.3%, n ϭ 33; Cy-BTx ϭ 20.3 Ϯ 5.2%, n ϭ 35; p Ͻ 0.01, ANOVA followed by Tukey’s post hoc test). Note the relative a bungarotoxin binding site (BBS; 13 aa) increase toward smaller complexes. and a GFP on an extracellular loop (Sekine- Aizawa and Huganir, 2004). The BBS-GFP- tagged GluR2 (BGG) subunits were then gaining parallelism in data acquisition. identify the QDots through their mor- tracked at the neuronal surface using three Importantly, QDots are much more pho- phology and not their intensity. This algo- different single-molecule complexes that tostable than SDs, although they are sub- rithm provides minimal time and mem- have different sizes (QDot-Ab Ͼ Cy5-Ab Ͼ ject to blinking (Michalet et al., 2005). ory consumption and provides clear Cy5-BTx) and valences (QDot-Ab/Cy5-Ab/ However, these nanoparticles are still big- advantages when recording large images Cy5-BTx: 2/2/1), where QDot-Ab stands for ger than organic dyes and rather bulky and very long QDot-based trajectories QDots coupled to anti-GFP antibody (Ab, (ϳ10–30 nm). (supplemental Box 2, available at www. ϳ150 kDa); Cy5-Ab for a cyanine 5 (Cy5) Because of the larger signals obtained jneurosci.org as supplemental material). coupled to anti-GFP antibody; and Cy5- from QDots than from SDs, a faster detec- To account for the blinking of lumines- BTx for a Cy5 coupled to bungarotoxin tion algorithm than the two-dimensional cent QDots, an algorithm was further (BTx, 8 kDa) (Fig. 2A). The comparison of Gaussian fitting of the luminescent signal used to reconnect iteratively portions of the diffusion of the three complexes in the was developed. It relies on wavelet trans- trajectories originating from the same extrasynaptic membrane revealed that the forms that filter the image frames and QDots. The parameters of reconnection cumulative distributions of instantaneous 12436 • J. Neurosci., November 14, 2007 • 27(46):12433–12437 Groc et al. • Toolbox diffusion coefficients (Fig. 2B,C) and the Do the different single-tagged aptic cleft (results above). The engineer- median values of the total and mobile recep- complexes penetrate equally the ing of smaller QDots, directly coupled to tors were not significantly different. synaptic cleft? the detection probes (e.g., antibody or Within synapses (Fig. 2D), the median To answer this question, surface tag com- synthetic peptide), will surely improve the values of instantaneous diffusion coeffi- plexes were counted for each image series Dot-based complexes (Pinaud et al., 2004; cients for all trajectories were also not sig- and were affected as extrasynaptic, synap- Michalet et al., 2005). The tracking of in- nificantly different, and neither were the tic, or juxtasynaptic (300–500 nm around dividual few-nanometer gold particles in percentage of immobile receptors (Fig. the labeled synapses). In total, 150,584 live neurons using a photothermal tech- 2E). Interestingly, the distributions of the molecules/particles were analyzed. The nique offers a promising alternative be- instantaneous diffusion coefficient for the percentage of tag complexes localized cause it has the unique potential to record only mobile receptors were different: within synapses (synaptic over total mol- long trajectories (no theoretical limit) Cy5-BTx complexes displayed a broader ecule number) averaged ϳ20% (range from very small probes (5 nm wide) range than QDot-Ab and Cy5-Ab (Fig. 0–26%). Interestingly, the synaptic con- (Lasne et al., 2006). As for the ligands, the 2E). Consistently, the median value of the tent of Cy5-Ab and Cy5-BTx molecules engineering of small probes (ϽϽantibody Cy5-BTx distribution was significantly was higher than that of QDot-Ab (Fig. or streptavidin) with high affinity will of- higher than the one of QDot-Ab and Cy5- 2F). However, in the juxtasynaptic mem- fer alternative ways, although the effects of Ab. Together, these results indicate that brane, this difference vanished, indicating recombinant subunit overexpression on the three antibody-based complexes pro- that the size of single complexes is likely to cellular trafficking have to be carefully as- vide an equivalent picture of the propor- affect the accessibility of the complexes to sayed. Finally, one aim of the cellular neu- tion of diffusing synaptic receptors, al- the highly confined synaptic cleft. roscience field is to track receptors di- though the smaller complex allow the rectly from live brains. Current additional visualization of a subset of Are surface diffusions of endogenous roadblocks of this approach are based on highly diffusing receptors. and transfected receptor similar? in vivo imaging (multiphoton micros- Regarding receptor diffusion within Recombinant receptor subunits are, and copy) of XFP-tag receptors in neurons synaptic areas, two points should be dis- will be, widely used to study receptor traf- that have been previously infected by vi- cussed. First, synapses could be labeled ficking during various paradigms of neu- rus (e.g., Sindbis virus, lentivirus) con- using various markers, either presynap- ronal function. The simple but important taining the XFP-tag receptor constructs question of whether endogenous and (Svoboda and Yasuda, 2006). Whether tic (e.g., Mitotracker, synaptotagmin) transfected receptors behave similarly at pHluorin-XFP for ensemble measure- or postsynaptic (e.g., PSD-95, Homer the neuronal surface appeared. The sur- ments or QDots, organic molecules, and 1c) (Tardin et al., 2003; Groc et al., 2004; face diffusion of both endogenous and nanogold particles for single particle ap- Bats et al., 2007; Ehlers et al., 2007) transfected recombinant glutamatergic proaches will be the appropriate probes (Figs. 1, 2). Comparing diffusion of subunits were then compared using data for receptor surface tracking in vivo re- AMPA receptors within synapses la- collected from published (Groc et al., mains an open and exciting question. beled with either Mitotracker (presyn- 2006; Bats et al., 2007) and unpublished aptic) or Homer 1c (postsynaptic) (supplemental Box 3, available at www. shows almost similar diffusion charac- References jneurosci.org as supplemental material) Adesnik H, Nicoll RA, England PM (2005) Pho- teristics (indistinguishable percentage studies. 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Such comparison strongly sug- exchange of AMPA receptors at dendritic distributions of synaptic GluR2 subunit gests that pertinent controls (e.g., spines and is regulated by spine morphology. J Neurosci 26:7046–7055. are slower when measured with QDots comparison between endogenous and re- coated with protein A (ϳ50 kDa) [me- Bachir A, Durisic N, Grutter P, Wiseman P combinant subunit surface trafficking) (2006) Characterization of blinking dynam- ϭ ␮ 2 dian 0.005 m /s, interquartile range should automatically be performed. ics in quantum dot ensembles using image (IQR; 25–75%) ϭ 0.0005–0.03, n ϭ 59] correlation spectroscopy. J Appl Phys (Groc et al., 2004) compared with Future directions 99:064503. QDots coated with Fab fragment (20 What are the “ideal” probes for future single- Bats C, Groc L, Choquet D (2007) The interac- tion between Stargazin and PSD-95 regulates kDa) (QDot-Fab: median ϭ 0.025 molecule/particle tracking? The obvious and ␮ 2 ϭ ϭ AMPA receptor surface trafficking. 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