The cellular traffic of neurotransmitter receptors has captured a lot of attention over the last decade, mostly because synaptic receptor number is adjusted during synaptic development and plasticity. Although each neurotransmitter receptor family has its own trafficking characteristics, two main modes of receptor delivery to the synapse have emerged: endo-exocytotic cycling and surface diffusion [e.g., for glutamatergic receptors, see Bredt and Nicoll (2003) and Groc and Choquet (2006)]. Receptor cycling through endo-exocytotic processes can be measured by several experimental means, from biochemical to imaging assays. The use of fluorescent protein (XFP)-tag imaging provides a powerful approach to investigate the trafficking of receptor clusters between neuronal compartments (e.g., soma, dendrite, spine) (Kennedy and Ehlers, 2006). A disadvantage of the XFP-tag approach in live experiment is extreme difficulty in detecting XFP fluorescence signals from small nonclustered receptor pool (Cognet et al., 2002; Lippincott-Schwartz and Patterson, 2003). XFP-tagged neurotransmitter receptors are often present in several cellular compartments from the endoplasmic reticulum to the plasma membrane with various relative contents. For instance, surface XFP-tagged neurotransmitter receptors represent only a minor fraction of the total receptor population, precluding their specific detection. Alternative live-cell imaging approaches were thus required to specifically isolate surface receptors. Interestingly, a variant of the green fluorescent protein (GFP), ecliptic pHluorin, shows a reversible excitation ratio change between pH 7.5 and 5.5, and its absorbance decreases as the pH is lowered. Most neurotransmitter receptors, including the ionotropic glutamate ones, display an extracellular N-terminal region, implying that the N terminus will always be in an acidic environment inside the cell, whereas it will be exposed to a neutral pH after insertion into the plasma membrane. By this means, surface receptors can be specifically detected and tracked with live-imaging approaches (Ashby et al., 2004). Alternatively, surface receptors can be labeled and detected by immunocytochemical approaches using antibodies directed against receptor extracellular epitopes.
The purpose of this Toolbox is to outline currently available approaches to measure the surface trafficking of receptor in neurons, with a special emphasis on single-molecule (organic dye) and quantum dot (QDot) detection for neurotransmitter receptor tracking.
Exploring receptor surface trafficking: approaches
Investigation of receptor surface distribution and diffusion can be sorted in two groups. On the one hand, the average surface diffusion of labeled receptors is studied without distinction of individual behaviors (Fig. 1A). Surface receptors can be isolated by electrophysiologically tagged methods (Tovar and Westbrook, 2002; Adesnik et al., 2005; Thomas et al., 2005). Schematically, a subpopulation of surface receptors is irreversibly blocked (e.g., MK-801 for NMDA receptors) and the receptor surface diffusion is estimated from time-dependent functional recovery after receptor blockade. Another approach is based on the fluorescence labeling techniques coupled to live fluorescence microscopy, such as fluorescent recovery after photobleaching (FRAP) of pHluorin-tagged receptors (Rasse et al., 2005; Ashby et al., 2006; Kopec et al., 2006; Lober et al., 2006; Sharma et al., 2006). Note that both approaches provided equivalent characteristics of receptor surface diffusion [e.g., for AMPA receptor in hippocampal cultured neurons (Adesnik et al., 2005; Ashby et al., 2006)], indicating that ensemble surface receptor diffusion can efficiently be measured using either approaches.
Opposite to the ensemble methods, single-molecule detection methods retrieve the diffusion properties of individual labeled receptors over time (Fig. 1A). The typical outcome is the complete distribution of the behavior of surface neurotransmitter receptors, which have revealed non-Gaussian shapes and a variety of diffusion characteristics at a given time. To date, this approach has unraveled the surface trafficking of neurotransmitter receptors and channels, including glutamatergic metabotropic mGluR5, AMPA and NMDA receptors (Borgdorff and Choquet, 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 receptors (Bouzigues and Dahan, 2007), and Kv potassium channels (O'Connell et al., 2006). To label single molecules, there are essentially two possibilities: the use of a single dye (SD) or a single particle (SP) (e.g., nano-sized QDots). The SD tags are short lived but small size, whereas SP tags allow long observation times, but the size of the label can prevent its access to specific confined cellular areas.
Single dye tracking
SD tracking (SDT) first requires the attachment of a single tag (e.g., organic dye) to the molecular target through a specific high-affinity ligand that recognizes the extracellular domain of the molecular target in live cells (Fig. 1B). Labeling must be performed at low tag densities to be optically resolved (typically <1 molecule/μm3) and to avoid cross-linking in the case of multivalent ligands (e.g., antibody). The second obvious requirement is to obtain signals from an SD that are large enough to overcome both the background noise from the molecule environment and that of the detection system (Fig. 1B). Several dyes fulfill these requirements, such as cyanine dyes (Cy3 or Cy5) that can be detected with good signal-to-noise ratios in live cells. The main limitation of SDT is, however, photobleaching, i.e., the processes by which photochemical reactions transform the excited fluorophore into a nonfluorescent product. It limits imaging time of a SD to typically a few seconds. Note that the use of antioxidant, such as Trolox, provides an efficient way to significantly reduce photobleaching and thus improve photostability (Rasnik et al., 2006).
In practice, single molecules are identified by their diffraction-limited signals, their well defined intensities, and the one-step photobleaching behavior (supplemental Box 1, available at www.jneurosci.org as supplemental material). Two-dimensional fitting by a Gaussian approximating the true point spread function of the microscope allows subwavelength localization of the labels (Cheezum et al., 2001), typically <50 nm for SDs in live cells (Tardin et al., 2003). Finally, once SDs are identified in each image frame, their two-dimensional trajectories can be constructed using a Vogel algorithm. This algorithm performs a correlation analysis between the positions of the SD found in consecutive images (Schmidt et al., 1995) (supplemental Box 2, available at www.jneurosci.org as supplemental material).
QDots are passivated semiconductor nanocrystals that are water soluble and functionalized for biological applications. In addition to their superior brightness compared with SD, QDots have a larger absorption cross section, such that they can be excited with a mercury lamp. This allows recording large fields of view and gaining parallelism in data acquisition. Importantly, QDots are much more photostable than SDs, although they are subject to blinking (Michalet et al., 2005). However, these nanoparticles are still bigger than organic dyes and rather bulky (∼10–30 nm).
Because of the larger signals obtained from QDots than from SDs, a faster detection algorithm than the two-dimensional Gaussian fitting of the luminescent signal was developed. It relies on wavelet transforms that filter the image frames and identify the QDots through their morphology and not their intensity. This algorithm provides minimal time and memory consumption and provides clear advantages when recording large images and very long QDot-based trajectories (supplemental Box 2, available at www.jneurosci.org as supplemental material). To account for the blinking of luminescent QDots, an algorithm was further used to reconnect iteratively portions of trajectories originating from the same QDots. The parameters of reconnection (maximum distance between reconnected events and maximum “dark” time) are adjusted according to the diffusion time of the molecules and the image acquisition rate. To prevent false reconnections, the surface density of QDots must be low enough that parameters of reconnection can be found without mixing the trajectories 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 developed 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, because fluorescence changes between “on” and “off” states for a single Dot alternate only in two levels.
Does the size and valence of single tag complexes influence surface diffusion?
At the scale of the nano-sized probes, movements of surface extrasynaptic receptors are not governed by the mass of the probe, but instead by the motion of the membrane protein. Indeed, the viscosity of membranes is 100- to 1000-fold greater than that of extracellular medium. In restricted environments, however, like in the synaptic cleft, the size and valence of a single complex can impact surface receptor diffusion. The impact of different labeling strategies on SD/QDot measurements in nonconfined cellular environments (the extrasynaptic membrane) and confined ones (synapses labeled with Mitotracker) (Tardin et al., 2003; Groc et al., 2004) was measured. Cultured hippocampal neurons were transfected with the GluR2 AMPA receptor subunit tagged with a bungarotoxin binding site (BBS; 13 aa) and a GFP on an extracellular loop (Sekine-Aizawa and Huganir, 2004). The BBS-GFP-tagged GluR2 (BGG) subunits were then tracked at the neuronal surface using three different single-molecule complexes that have different sizes (QDot-Ab > Cy5-Ab > Cy5-BTx) and valences (QDot-Ab/Cy5-Ab/Cy5-BTx: 2/2/1), where QDot-Ab stands for QDots coupled to anti-GFP antibody (Ab, ∼150 kDa); Cy5-Ab for a cyanine 5 (Cy5) coupled to anti-GFP antibody; and Cy5-BTx for a Cy5 coupled to bungarotoxin (BTx, 8 kDa) (Fig. 2A). The comparison of the diffusion of the three complexes in the extrasynaptic membrane revealed that the cumulative distributions of instantaneous diffusion coefficients (Fig. 2B,C) and the median values of the total and mobile receptors were not significantly different.
Within synapses (Fig. 2D), the median values of instantaneous diffusion coefficients for all trajectories were also not significantly different, and neither were the percentage of immobile receptors (Fig. 2E). Interestingly, the distributions of the instantaneous diffusion coefficient for the only mobile receptors were different: Cy5-BTx complexes displayed a broader range than QDot-Ab and Cy5-Ab (Fig. 2E). Consistently, the median value of the Cy5-BTx distribution was significantly higher than the one of QDot-Ab and Cy5-Ab. Together, these results indicate that the three antibody-based complexes provide an equivalent picture of the proportion of diffusing synaptic receptors, although the smaller complex allow the additional visualization of a subset of highly diffusing receptors.
Regarding receptor diffusion within synaptic areas, two points should be discussed. First, synapses could be labeled using various markers, either presynaptic (e.g., Mitotracker, synaptotagmin) or postsynaptic (e.g., PSD-95, Homer 1c) (Tardin et al., 2003; Groc et al., 2004; Bats et al., 2007; Ehlers et al., 2007) (Figs. 1, 2). Comparing diffusion of AMPA receptors within synapses labeled with either Mitotracker (presynaptic) or Homer 1c (postsynaptic) shows almost similar diffusion characteristics (indistinguishable percentage of immobile receptors and exchange rate between extrasynaptic and synaptic membrane but a slight difference in the diffusion constant) (our unpublished data). Second, Dot functionalizing strategies can influence synaptic diffusion likely through difference in size and steric properties. For instance, diffusion distributions of synaptic GluR2 subunit are slower when measured with QDots coated with protein A (∼50 kDa) [median = 0.005 μm2/s, interquartile range (IQR; 25–75%) = 0.0005–0.03, n = 59] (Groc et al., 2004) compared with QDots coated with Fab fragment (20 kDa) (QDot-Fab: median = 0.025 μm2/s, IQR = 0.0012–0.11, n = 366). In conclusion, all examined probes allow the tracking of synaptic receptors, but it appears that the smaller the probe, the better the tracking of fast diffusing synaptic receptors. This further encourages the development of very small probes (only few nanometers) for confined environments.
Do the different single-tagged complexes penetrate equally the synaptic cleft?
To answer this question, surface tag complexes were counted for each image series and were affected as extrasynaptic, synaptic, or juxtasynaptic (300–500 nm around the labeled synapses). In total, 150,584 molecules/particles were analyzed. The percentage of tag complexes localized within synapses (synaptic over total molecule number) averaged ∼20% (range 0–26%). Interestingly, the synaptic content of Cy5-Ab and Cy5-BTx molecules was higher than that of QDot-Ab (Fig. 2F). However, in the juxtasynaptic membrane, this difference vanished, indicating that the size of single complexes is likely to affect the accessibility of the complexes to the highly confined synaptic cleft.
Are surface diffusions of endogenous and transfected receptor similar?
Recombinant receptor subunits are, and will be, widely used to study receptor trafficking during various paradigms of neuronal function. The simple but important question of whether endogenous and transfected receptors behave similarly at the neuronal surface appeared. The surface diffusion of both endogenous and transfected recombinant glutamatergic subunits were then compared using data collected from published (Groc et al., 2006; Bats et al., 2007) and unpublished (supplemental Box 3, available at www.jneurosci.org as supplemental material) studies. It emerged that overexpressed GluR or NR glutamatergic subunits exhibit either similar or different surface trafficking, respectively, when compared with endogenous ones. These results are only indicative for specific experimental designs and may vary with different transfection method, subunit types, or neuronal stages. Such comparison strongly suggests that pertinent controls (e.g., comparison between endogenous and recombinant subunit surface trafficking) should automatically be performed.
What are the “ideal” probes for future single-molecule/particle tracking? The obvious and straightforward answer is to develop probes that are as small as possible, photoresistant, and with very limited blinking. Although the QDots are photoresistant and exhibit blinking, their actual multilayer water-stabilizing structure and coupling with antibodies (or streptavidin) results in tens-of-nanometers-size Dot complexes that equal the size of the synaptic cleft (results above). The engineering of smaller QDots, directly coupled to the detection probes (e.g., antibody or synthetic peptide), will surely improve the Dot-based complexes (Pinaud et al., 2004; Michalet et al., 2005). The tracking of individual few-nanometer gold particles in live neurons using a photothermal technique offers a promising alternative because it has the unique potential to record long trajectories (no theoretical limit) from very small probes (5 nm wide) (Lasne et al., 2006). As for the ligands, the engineering of small probes (≪antibody or streptavidin) with high affinity will offer alternative ways, although the effects of recombinant subunit overexpression on cellular trafficking have to be carefully assayed. Finally, one aim of the cellular neuroscience field is to track receptors directly from live brains. Current roadblocks of this approach are based on in vivo imaging (multiphoton microscopy) of XFP-tag receptors in neurons that have been previously infected by virus (e.g., Sindbis virus, lentivirus) containing the XFP-tag receptor constructs (Svoboda and Yasuda, 2006). Whether pHluorin-XFP for ensemble measurements or QDots, organic molecules, and nanogold particles for single particle approaches will be the appropriate probes for receptor surface tracking in vivo remains an open and exciting question.
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.
This work was supported by grants from the Centre National de la Recherche Scientifique, Conseil Régional d'Aquitaine, Ministère de la Recherche, and Fondation pour la Recherche Médicale and by a European Community Grant (GRIPANT, CT-2005-005320). We thank Richard Huganir for the cDNA BGG construct and Christelle Breillat, Beatrice Teissier, and Delphine Bouchet for technical assistance.
- Correspondence should be addressed to Laurent Groc, Unité Mixte de Recherche 5091, Centre National de la Recherche Scientifique, Université Bordeaux 2, 146 rue Léo Saignat, 33077 Bordeaux, France.