Brain extracellular matrix affects AMPA receptor lateralmobility and short-term synaptic plasticity

Renato Frischknecht1,4, Martin Heine2–4, David Perrais2, Constanze I Seidenbecher1, Daniel Choquet2 &Eckart D Gundelfinger1

Many synapses in the mature CNS are wrapped by a dense extracellular matrix (ECM). Using single-particle tracking and

fluorescence recovery after photobleaching, we found that this net-like ECM formed surface compartments on rat primary neurons

that acted as lateral diffusion barriers for AMPA-type glutamate receptors. Enzymatic removal of the ECM increased extrasynaptic

receptor diffusion and the exchange of synaptic AMPA receptors. Whole-cell patch-clamp recording revealed an increased paired-

pulse ratio as a functional consequence of ECM removal. These results suggest that the surface compartments formed by the ECM

hinder lateral diffusion of AMPA receptors and may therefore modulate short-term synaptic plasticity.

Brain synapses are wrapped by a dense meshwork of ECM, whichconsists of glycoproteins and proteoglycans of glial and neuronalorigin. A notable feature of the brain ECM is its net-like appearanceon neuronal surfaces, which is particularly prominent on GABAergicinterneurons1,2, but can be observed on virtually all neurons3. This net-like structure is assembled during the first 3–5 weeks of postnataldevelopment, reaching its adult expression pattern after the maturesynaptic circuitry is established3,4. Because of its known inhibitoryeffect on structural rearrangements and axonal outgrowth, the ECMhas been implicated in synaptic stabilization and the maintenance ofsynaptic networks in the adult CNS2,5.

Cell surface proteins, including neurotransmitter receptors, arehighly mobile in the neuronal plasma membrane as a result of lateraldiffusion6–8. The exchange of receptors between synaptic and extra-synaptic membranes contributes to the regulation of receptor numbersat synapses. The physiological importance of lateral diffusion becameevident by our finding that fast movements of AMPA-type glutamatereceptors (AMPARs) are involved in the modulation of synaptictransmission; that is, AMPAR diffusion accounts for a fast exchangeof desensitized receptors for naive functional ones in or near the post-synaptic density, thus increasing synaptic fidelity during fast repetitivestimulation9. However, synaptic exchange may be limited, as planarmovement is confined by anchored membrane proteins that hinder freelateral diffusion10–12. Here we propose that, as a result of its net-likestructure, the ECM may compartmentalize the neuronal surface andthereby restrict the surface mobility of integral membrane proteins.These compartments seem to constrain AMPAR mobility and thuscontrol the availability of naive receptors for synapses. This mayrepresent an additional determinant for synaptic plasticity introducedlate during CNS development.

RESULTS

Mature primary hippocampal neurons are surrounded by ECM

We used dissociated rat hippocampal neurons in culture to study theinfluence of the ECM on receptor mobility, as lateral diffusion ofsurface molecules13 and ECM formation is well described in thissystem3,14. The ECM in culture, as in the brain, incorporates glyco-proteins and proteoglycans of neuronal and glial origin that arecoordinated by the glycosaminoglycan hyaluronic acid as a corecomponent2,15. In initial experiments, we established a live stainingprocedure for the ECM and determined a time point that is suitablefor receptor mobility studies, when most neurons are interconnectedand embedded in the ECM. To this end, we labeled the core componentof the ECM by an overnight incubation of living neurons withhyaluronic acid–binding protein coupled to Alexa568 (HABP-568).The specificity of labeling was controlled by co-staining with antibodyto brevican (Fig. 1a), an established marker of the adult ECM16.Labeled ECM structures were considered to be mature when hyaluronicacid and brevican were present on neuronal surfaces. Only 2% ofneurons contained both brevican and hyaluronic acid at 10 d in vitro(DIV). These cells were most likely inhibitory interneurons, whichexpress a very prominent ECM form that is classically referred to asperineuronal nets17. At this stage, brevican was merely found at theaxon initial segments of neurons, where it interacts with Neurofas-cin186 and represents an immature form of the ECM3,18.

At 21 DIV or older, almost all (97 ± 2%) neurons were positive forboth HABP-568 and brevican, confirming the presence of ubiquitousnet-like ECM on dissociated hippocampal neurons at this stage(Fig. 1a). A more detailed view of dendritic segments reveals theheterogeneous appearance of the ECM. Most dendritic spines arewrapped by ECM, whereas synaptic contacts are devoid of HABP

Received 6 February; accepted 31 March; published online 31 May 2009; doi:10.1038/nn.2338

1Leibniz Institute for Neurobiology, Magdeburg, Germany. 2Centre National de la Recherche Scientifique, Universite de Bordeaux, UMR 5091, Bordeaux, France.3Present address: Leibniz Institute for Neurobiology, Magdeburg, Germany. 4These authors contributed equally to this work. Correspondence should be addressedto D.C. (dchoquet@u-bordeaux2.fr) or E.D.G. (gundelfi@ifn-magdeburg.de).

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staining (Fig. 1b,c and Supplementary Fig. 1 online). An overnightincubation of the cells with the hyaluronic acid–degrading enzymehyaluronidase abolished HABP-568 staining (Fig. 1b) and brevicanlabeling was only found in the axon initial segment (SupplementaryFig. 2 online), as in young neurons. Other components of the matureECM, including tenascin-R, were also removed by hyaluronidasetreatment (Supplementary Fig. 2). Spine length, spine density andsynapse size were unaffected by hyaluronidase incubation (Supple-mentary Fig. 2). In addition, the surface expression of endogenousGluR1 (Fig. 1d) and the organization of the actin cytoskeleton(Supplementary Fig. 2) were unchanged after enzyme treatment.Thus, hyaluronidase treatment provides a powerful tool for removingthe mature ECM from the cell surface without changing synapsestructure or AMPAR surface expression.

ECM removal increases AMPA receptor surface mobility

The lateral diffusion of endogenous GluR1-containing receptors in themembrane of hippocampal neurons can be tracked with quantum dotscoupled to antibody to GluR1 (refs. 9,19,20). We acquired images ofGluR1 quantum dots at a frequency of 33 Hz and reconstructedtrajectories using single-particle tracking (SPT)10,13,21. A correlationof receptor trajectories with HABP-568 labeling revealed that fast-moving receptors were preferentially found in areas devoid of ECMand that the diffusion constant of such receptors dropped markedlywhen they approached or entered strongly HABP-568–labeled areas(Fig. 2a,b). However, the fuzzy appearance of the ECM, with its largevariance in staining intensity, did not allow a reliable definition of theborders between ECM-covered and ECM-free areas. For a quantitativeassessment of receptor mobility, we therefore compared only clearly

net-localized receptors with the total pool of mobile receptors. Themean square displacement (msd) versus time plot allowed us tomeasure the instantaneous diffusion from its initial slope and todistinguish between free and confined diffusion (Fig. 2c). In the caseof unrestricted diffusion, the msd plot is linear, whereas it rectifies to aquasi-maximum if receptor mobility is confined22. Receptors are moreconfined in the ECM as compared with the total receptor population,as indicated by the curvatures of the msd plots (Fig. 2c). Thedistribution of instantaneous diffusion coefficients (Dinst) of GluR1in the ECM was restricted to lower values (Fig. 2d), whereas the Dinst

distribution of the total receptor population had a subpopulation athigher values (Fig. 2d). When the ECM was removed from neuronsusing hyaluronidase, the Dinst distribution was shifted to significantlyhigher rates (Kruskal-Wallis test followed by a Dunn’s test, Po 0.0001;Fig. 2d). Of note, the instantaneous diffusion on hyaluronidase-treatedcells did not exceed the maximal values on ECM-bearing cells. Thefraction of immobile receptors (Dinst ¼ 10�4 mm2 s�1) did not changesignificantly after hyaluronidase treatment (control, 14.4 ± 2%; hyal-uronidase, 9.7 ± 1%, P 4 0.05). These data suggest that GluR1mobility is slowed and restricted by the ECM.

A trivial explanation for the observed differences in mobility wouldbe that the antibody–quantum dot complex, B20 nm in size, stericallyhinders and thereby alters receptor diffusion in the ECM13. To excludethis possibility, we carried out fluorescence recovery after photobleach-ing (FRAP) experiments on neurons transfected with the AMPARsubunits GluR1 or GluR2, which were tagged at their N termini withthe much smaller tag super-ecliptic pHluorin (pHGFP) (Fig. 3). Theextent of fluorescence recovery in bleached areas provides informationabout the bulk mobility of surface receptors. pHGFP restricts FRAP tosurface receptors and excludes intracellular AMPAR pools from obser-vation23. Up to six separate extrasynaptic dendritic regions on a trans-fected neuron were bleached by single laser pulses of 5 ms, resulting inan average bleached area of 1.8 ± 0.1 mm2. To exclude crosstalk of thefluorescence recoveries from adjacent bleached areas, we kept a mini-mal spacing of 30 mm. The recovery at 300 s post-bleach was comparedfor receptors on ECM-bearing and hyaluronidase-treated neurons.

Overnight incubation with hyaluronidase did not change the surfaceexpression of GluR1-pHGFP or GluR2-pHGFP (Fig. 1d and Supple-mentary Fig. 3 online), but it did cause an increase in fluorescencerecovery at extrasynaptic sites after 300 s from 60.6 ± 2% in controlcells (n¼ 16) to 76 ± 2% in ECM-free cells (n¼ 21) for GluR1-pHGFP(Fig. 3b,c). For GluR2-pHGFP, the recovery was 64 ± 3% and 75 ± 3%in 300 s for control (n ¼ 18 cells) and hyaluronidase-treated cultures(n ¼ 22 cells), respectively (Fig. 3d). Thus, both types of AMPARsshowed significantly higher fluorescence recoveries after ECM removal(Student’s t test, Po 0.0001). The degree of FRAP after hyaluronidase

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Figure 1 Development of ECM structures in cultured hippocampal neurons.

(a) Neurons at 10 and 24 DIV were stained with antibodies to brevican

(green) and HABP-568 (magenta). At 10 DIV, brevican was mainly localized

at the axon initial segment (arrows), whereas HABP-568 (magenta) yields no

signal. At 24 DIV, virtually all cells were strongly labeled for brevican and

HABP-568, confirming the presence of mature ECM. Scale bars represent

20 mm. (b) Cells at DIV 24 were live-stained for GluR1 (green) and HABP-

568 (magenta). Note that HABP-568 enwrapped many spine necks (arrow;see also Supplementary Fig. 1). When cells were treated with hyaluronidase

(Hy), most of the HABP-568 fluorescence was eliminated (bottom). Scale

bars represent 2 mm. (c) Line scan of the fluorescence profile of HABP-568

(magenta) and GluR1 (green) at the position depicted in b (white bar).

Note that HABP-568 fluorescence was clearly reduced at the synapse.

(d) Hyaluronidase treatment did not change surface expression of GluR1 on

dendrites (Con den versus Hy den) or at synapses (Con syn versus Hy syn).

Error bars represent s.e.m.

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treatment at 24 DIV was similar to the recovery at 10 DIV in neurons,when no adult ECM was present (Fig. 3c,d). At this stage, hyaluroni-dase treatment affected neither FRAP of GluR1-pHGFP nor of GluR2-pHGFP (Fig. 3c,d). To confirm these data in a more physiologicalexperimental setting, we transfected hippocampal slice cultures from9-d-old mice with GluR1-pHGFP using a gene gun. FRAP experimentswere performed at 12 DIV on control cells and cells treated withhyaluronidase overnight. At 300 s, post-bleach FRAP increased from52.2 ± 5% in control slices (n ¼ 17 cells from 12 slices) to 77 ± 6% inhyaluronidase-treated slices (n ¼ 15 cells from 10 slices).

Next, we tested whether the ECM specifically affects AMPAR mobilityor represents a more general diffusion constraint. To this end, weexpressed either a GFP-tagged deletion mutant of the cell adhesionmolecule NrCAM (NrCAMDcytDfn-GFP) that lacked its intracellularpart and the fibronectin type-III domains24 or GFP-GPI, a small globu-lar molecule anchored to the outer leaflet of the cell membrane via a

glycolipid anchor, in hippocampal neurons. Both recombinant mole-cules cannot interact with intracellular scaffolds, cytoskeleton or otherobstacles25. Because of the smaller size and the higher mobility in themembrane of these molecules25, we assessed recovery rates after 7 and70 s for GFP-GPI and after 80 s for NrCAMDcytDfn-GFP. Fluorescencerecovery of NrCAMDcytDfn-GFP after 80 s and recovery of GFP-GPIafter 7 s was increased after hyaluronidase treatment (SupplementaryFig. 4 online). GPI-GFP showed no significant differences in FRAP after70 s (Student’s t test, P¼ 0.8467). Thus, the ECM probably represents apassive diffusion barrier that acts in a size-dependent manner on abroad range of diffusive surface proteins.

ECM removal expands surface area explored by AMPA receptors

Our SPT experiments suggested that lateral diffusion of receptors fromECM-free areas was slowed and more confined when entering theECM-covered areas. It is therefore conceivable that receptors aretrapped in domains delineated by the ECM. We tested this hypothesis

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Figure 2 Mobility of GluR1-containing AMPA receptors labeled with

antibodies to GluR1 coupled to quantum dots outside and inside of ECM

structures. (a) Two example trajectories of quantum dot–labeled mobile

GluR1-containing AMPARs were correlated to HABP-568–labeled ECM

(blue). Red circles mark the start and red triangles mark the ending of the

trajectories. Scale bar represents 1 mm. (b) Instantaneous diffusion constants

(Dinst) of the trajectories were plotted against time. Blue areas indicate the

colocalization of the trajectory with the ECM. Note the low Dinst of trajectory1, which moves exclusively inside ECM (top), compared with trajectory

2 (bottom), which is mainly in ECM-free areas. In the latter, Dinst decreased

when GluR1 entered ECM-covered areas. (c) Plot of the msd versus time

of receptors in the matrix (intranet, ntrajectories ¼ 35) and total receptor

population (total, ntrajectories ¼ 206). (d) Normalized distribution of Dinst for

total GluR1 population, GluR1 on ECM-covered areas and GluR1 after

hyaluronidase treatment. Dinst of the mobile receptors was different for all

three populations (Kruskal-Wallis test followed by a Dunns test, P o 0.0001;

total receptor population (black line) mobile fraction: 0.014 mm2 s�1, IQR

0.004/0.06, ntrajectories ¼ 3,091; median of the subpopulation which

colocalized with HABP-568 (blue line) mobile fraction: 0.009 mm2 s�1, IQR

0.004/0.025, ntrajectories ¼ 974; total receptor population of hyaluronidase-

treated cells (red line) mobile fraction: 0.04 mm2 s�1, IQR 0.01/0.12,

ntrajectories ¼ 4,055). Data were recorded and analyzed from 28 experiments

out of nine different hippocampal neuron cultures.

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Figure 3 FRAP experiments reveal different mobilities of AMPAR before and

after ECM removal. (a) Examples of GluR1-pHGFP transfected dendrite

before and after local photobleaching at three different time points. We used

a 5-ms laser pulse (yellow circle at 0 s) to bleach 70–90% of the initial

fluorescence (o0 s) at dendrites. Fluorescence recovery was measured 300 s

post-bleach. (b) Examples of normalized recovery curves over time of GluR1-

pHGFP from a control (gray squares) and a hyaluronidase-treated cell (red

dots). The lines represent fitted curves as described in the Online Methods.

(c) Fluorescence recovery at 300 s after photobleaching for GluR1-pHGFP

at 10 and 24 DIV before (gray bars) and after (red bars) hyaluronidase

treatment. At 24 DIV, we observed a significant increase of FRAP, from

60.0 ± 2% to 75.9 ± 2% (Student’s t test, P o 0.001). (d) Fluorescencerecovery 300 s after photobleaching for GluR2-pHGFP at 10 and 24 DIV

before and after hyaluronidase. At 24 DIV, we observed a significant increase

of FRAP from 64.1 ± 3% to 75.1 ± 3% (Student’s t test, P o 0.01).

The numbers of cells used are given in the graphs; on each cell, four

regions were photobleached. For each construct, four different hippocampal

neuron cultures were used. ** P o 0.01, *** P o 0.001. Error bars

represent s.e.m.

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by employing SPT to follow the trajectories of individual receptorsbefore and after ECM removal. To do so, we labeled GluR1-pHGFPwith quantum dots coupled to antibody to GFP and imaged at afrequency of 33 Hz for 1 min. The ECM was then removed by a 10-minhyaluronidase incubation in the imaging chamber, as monitored byHABP-568 labeling. Trajectories represent the averaged explored area ofindividual GluR1-pHGFP molecules in 1 min. Under control condi-tions, receptors were confined to distinct areas of variable size on thedendritic surface (Fig. 4a). This observation was confirmed by longeracquisition at lower frequency (4 Hz, 4 min, data not shown). AfterECM removal, nearly all of the previously recorded quantum dotscould be monitored again in the same dendritic segment (total numberof quantum dots: control, n ¼ 497; after hyaluronidase, n ¼ 486). Thedirect comparison of trajectories from individual receptors before andafter hyaluronidase treatment showed a 20–150% increase of exploredsurfaces after ECM removal (Fig. 4b,c). The plot of msd over timereveals a less confined diffusion after hyaluronidase treatment (Fig. 4c).The median of the instantaneous diffusion coefficient of mobilereceptors shifted from 0.038 mm2 s�1 to 0.065 mm2 s�1 (P o 0.0001,Kruskal-Wallis test) after hyaluronidase treatment (Fig. 4d). Theseresults were confirmed using chondroitinase ABC, a well-establishedECM-degrading enzyme5, instead of hyaluronidase in the same experi-mental setup (Supplementary Fig. 5 online). Thus, individual recep-tors diffuse more widely and with higher mobility after ECM removal.

To confirm the hypothesis that removal of the ECM increases theability of receptors to diffuse over longer distances, we carried outfluorescence loss in photobleaching (FLIP) experiments on neurons

expressing GluR1-pHGFP. We bleached a 2-mm2 area continuously for300 s and compared the degree of fluorescence loss on control andhyaluronidase-treated neurons at various distances from the bleachedareas. In close vicinity to the bleaching point, we found no differencebetween control and hyaluronidase-treated cells (data not shown).However, at a distance of 3 mm from the bleaching point, the loss offluorescence was higher in hyaluronidase-digested cells than in controlcells, confirming a higher lateral mobility of AMPARs after ECMremoval (Fig. 4e,f). We then calculated the bleaching time needed fora 50% loss of the initial fluorescence at a 3-mm distance from thebleaching point (t50%; Fig. 4g). As expected, t50% was significantlyshorter in hyaluronidase-treated cells (t50% ¼ 62.6 ± 10.5 s) than incontrol cells (t50% ¼ 197.6 ± 56.4 s) (Student’s t test, P ¼ 0.0431). Thedifference in the variance of the data was even more notable; data fromhyaluronidase-treated cells varied much less than data from control cells(Fig. 4g). These results indicate that the ECM forms compartments onthe neuronal surface that restrict GluR1 lateral mobility and attenuatelong-distance diffusion.

ECM tunes synaptic accessibility of mobile receptors

Alterations in receptor diffusion at synapses can influence fast synaptictransmission9. Therefore, we wondered whether synaptic receptor diffu-sion is constrained by the perisynaptic ECM. To this end, we carried outFRAP experiments on spine synapses on neurons expressing GluR1-pHGFP or GluR2-pHGFP (Fig. 5a). Postsynaptic densities were visua-lized by co-transfection with Homer1c-dsRed, which is known not tointerfere with synaptic function19. Consistent with previously publisheddata19,23, fluorescence recovery was lower at spines than at dendriticshafts. However, a significantly increased FRAP was observed at spinesynapses after ECM removal for both GluR1-pHGFP (control, 46 ± 2%recovery after 300 s; hyaluronidase, 57 ± 2%; n¼ 30) and GluR2-pHGFP(control, 39 ± 2%; hyaluronidase, 49 ± 2%; n ¼ 17) (Student’s t test,P ¼ 0.0005; Fig. 5b,c). For comparison, we explored the mobility ofNMDA receptors. In particular, NR2A-containing receptors are knownto be immobile in mature synapses in the observed time frame of a fewhundred seconds13,26. As expected, FRAP of NR1/NR2A-pHGFP was

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OverlayFigure 4 ECM-based surface compartments restrict AMPAR diffusion.

(a) Images of the cumulative surface explored on GluR1-pHGFP–expressing

dendrites (green) by quantum dots bound to GluR1-pHGFP–containing

AMPARs in 1 min of observation before (blue) and 10 min after (red)

hyaluronidase treatment. Magnification of the boxed area is shown below the

image. Receptors moved in restricted areas of variable size (control, blue

trajectory). After a 10-min digestion with hyaluronidase, individual receptors

explored a larger surface than before the treatment (middle, red trajectory).The overlay (right) illustrates a clear increase in mobility after ECM removal.

(b) Summary plot of the evolution of the explored surface between the control

recording period and 10 min after hyaluronidase treatment (n ¼ 7, paired

t test, P o 0.001). (c) Plot of the msd versus time before (ntrajectories ¼ 184)

and after hyaluronidase treatment (ntrajectories ¼ 196). (d) The fraction of

receptors with high Dinst increased after hyaluronidase treatment (Mann-

Whitney test, P o 0.001). (e–g) FLIP experiments on cells transfected with

GluR1-pHGFP. (e) A spot on the neuronal surface (0 mm) was continuously

bleached for 300 s and the loss of fluorescence at various distances (for

example, 3 mm, red circle) was measured. (f) The gradual loss of fluorescence

at the primary bleaching point (0 mm) and at 3-mm distance was plotted from

0–300 s for control cells and hyaluronidase-treated cells. Note the faster and

stronger bleach in hyaluronidase-treated cells at 3 mm. (g) Graph showing the

bleaching time to reach a 50% fluorescence loss (t50%) at 3 mm for control

and hyaluronidase-treated cells. For hyaluronidase-treated cells, t50% was

significantly shorter than for control cells (hyaluronidase, 62.60 ± 10.48%,

n ¼ 10; control, 197.6 ± 56.41%, n ¼ 10; P o 0.05, Student’s t test). Also

note the larger variance of control values (F test, P o 0.0005). * P o 0.05,

*** P o 0.001.

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substantially smaller than that of AMPARs. Moreover, hyaluronidasetreatment did not change NR2A mobility (control, 24 ± 4% recovery after300 s; hyaluronidase, 24 ± 3%; n ¼ 9; Fig. 5c). These data indicate thatremoval of the ECM facilitates the exchange of synaptic AMPARs, but hasno effect on NMDA receptors in the given time frame.

To analyze synaptic receptor exchange in more detail, we performedSPT experiments using antibodies to GluR1–quantum dots to trackendogenous receptors. Synapses were marked by Homer1c-dsRedtransfection and ECM by HABP-568 live staining (Fig. 5d,e). Plottingmsd versus time revealed no difference in the confinement for synapticAMPARs after ECM removal (Fig. 5f). Furthermore, neither theinstantaneous diffusion (median control: 0.014, IQR 0.006/0.025 mm2

s�1; median hyaluronidase: 0.015, interquartile range (IQR) 0.006/0.03mm2 s�1; Fig. 5g) nor the immobile fraction of synaptic receptors(control, 14.9 ± 5%; hyaluronidase, 9 ± 4%; P 4 0.05) were changedsignificantly. These results are consistent with the finding that synapticcontacts are devoid of hyaluronan-based ECM (Fig. 1c), and intra-synaptic receptor diffusion may not be affected by hyaluronidasetreatment. However, comparing the fraction of receptors that exchangebetween extrasynaptic and synaptic compartments we found an B40%increase after hyaluronidase treatment (17 ± 1.9% versus 24 ± 2.6%,Student’s t test, P o 0.05) and the synaptic dwell time of GluR1decreased after ECM removal (control, 4.2 ± 0.2 s; hyaluronidase,3.0 ± 0.1 s; Student’s t test, P o 0.05). Thus, ECM removal leads toan increased exchange of GluR1 between synaptic and extrasynapticcompartments without affecting their mobility in the synapse.

Lateral diffusion modulates paired-pulse characteristics

The ability of a synapse to respond to high-frequency stimuli isimpaired when lateral mobility is blocked experimentally, suggesting

that the exchange of desensitized AMPARs for naive ones via lateraldiffusion at synapses affects fast synaptic performance9. The peri-synaptic ECM would be an ideal natural factor to control AMPARtrafficking into and out of synapses. We therefore hypothesized thatthe increased AMPAR mobility after ECM removal may affect fastsynaptic transmission. In a first set of experiments, we compared mini-ature excitatory postsynaptic currents (mEPSCs) in the presence of1 mM tetrodotoxin (TTX), 10 mM D(�)-2-amino-5-phosphonovalericacid (AP5) and 50 mM picrotoxin and evoked excitatory postsynapticcurrents (eEPSCs) in the absence of TTX from hyaluronidase-treatedand untreated primary hippocampal neurons. Application of hyalu-ronidase did not change mEPSC frequency, kinetics or amplitude(Supplementary Fig. 6 online), indicating that no major changes insynaptic properties occurred on enzyme treatment. We then shifted theextracellular Mg2+/Ca2+ ratio from 2:2 to 1:7 (mM), which increasesmEPSC frequency as a result of a marked change in presynaptic releaseprobability. Neither mEPSC kinetics nor amplitude differed betweenhyaluronidase-treated and control cells under these conditions. Toexplore a potential effect of hyaluronidase treatment on the kineticproperties of AMPARs, we measured glutamate-evoked currents inoutside-out patches. There was no difference in the kinetics ofdeactivation and desensitization, as determined by short (1 ms) andlong pulses (100 ms) of glutamate (10 mM). In addition, the recoveryfrom desensitization was unchanged (Supplementary Fig. 7 online).Thus, removal of the ECM and the subsequent higher degree offreedom for diffusive membrane proteins did not have an overt effecton spontaneous and evoked synaptic activity or kinetic properties ofAMPAR. The membrane resting potential and the action potentialamplitude and width were essentially unchanged, suggesting that theexcitability of the neuron was unaffected by hyaluronidase treatment(Supplementary Fig. 7).

To test for the ability of the neurons to respond to high-frequencystimuli, we measured paired-pulse ratios (PPRs) using dual whole-cellrecordings from monosynaptically connected neurons (Supplemen-tary Fig. 8 online). The amplitudes and kinetic properties of the firstEPSCs were unaffected by hyaluronidase treatment, which is consistentwith mEPSCs measurements (Supplementary Fig. 8). For a paired-pulse interstimulus interval (ISI) of 50 ms, the vast majority of control

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220 300

0 s 300 s

32 28 16 17 8 9

Figure 5 ECM removal enhances exchange between synaptic and

extrasynaptic AMPARs without altering intrasynaptic mobility. (a) Example

of a FRAP experiment at a synapse of a GluR1-pHGFP–transfected neuron

bleached by a single laser pulse (dotted yellow region) and fluorescence

recovery 300 s post-bleach. (b) Fluorescence recovery of GluR1-pHGFP at a

synapse on a hyaluronidase-treated cell (red) and control cell (black). The

solid line represents the fitted curve. (c) Synaptic fluorescence recovery was

significantly increased for GluR1-pHGFP (control, 45.8 ± 2%; hyaluronidase,56.6 ± 2%; Student’s t test, P o 0.001) and GluR2-pHGFP (control,

38.9 ± 3%; hyaluronidase, 49.2 ± 2%; Student’s t test, P o 0.001), but

not for NR1- and NR2A-pHGFP–transfected cells (control, 24.5 ± 4%;

hyaluronidase, 24.3 ± 3%). *** P o 0.001. (d) GluR1 was detected using

quantum dot–coupled antibody to GluR1 on Homer1c-mDsRed–transfected

neurons and subjected to SPT. Trajectories were superimposed with synapses

(Homer1c-DsRed, green) and HABP (blue). The red circle marks the beginning

and the red triangle marks the end of the trajectory. (e) Diffusion coefficient of

the GluR1-quantum dot particle depicted in d, plotted against time. Note the

decrease in Dinst as the GluR1–quantum dot particle entered the synapse (green

bar) or the ECM (blue bars). (f) Plot of the msd against time showed no major

difference in the confinement of synaptic GluR1–quantum dot on hyaluronidase-

treated (ntrajectories ¼ 47) compared with control cells (ntrajectories ¼ 82). (g) In

the synapse, the distribution of the Dinst was not changed. The numbers of cells

for FRAP experiments are given in the bar graphs; for each cell, four regions

were photobleached and four different hippocampal neuron cultures were used

for each construct. Error bars indicate s.e.

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cells (17 out of 20) showed paired-pulse depression (PPD). However,the PPR increased after treatment with hyaluronidase (SupplementaryFig. 8). This increase was a result of both a decreased PPD and a highernumber of neurons that showed paired-pulse facilitation (5 out of18 cell pairs). To rule out presynaptic effects, we used local iontophor-etic glutamate application, as has been recently reported9,27. Applica-tion of glutamate for 1 ms with iontophoretic currents of B100–200nA evoked inward currents of 168.8 ± 17 pA (n¼ 16) with fast rise anddecay times, as present in synaptic transmission. Paired glutamateapplication at various ISIs resulted in PPD in control cells (Fig. 6a,b).Under these experimental conditions, hyaluronidase treatment stronglyreduced PPD of short ISI between 10–100 ms and the addition of thedesensitization-blocking agent cyclothiazide (50 mM) caused an evengreater reduction in PPD (Fig. 6b). Artificial immobilization ofreceptors by antibody cross-linking decreased the PPR below controlvalues (Fig. 6b), indicating a modulation of the PPR by the mobility ofAMPAR in the dendritic membrane. No PPD was observed undereither condition for ISIs above 200 ms.

DISCUSSION

Although the brain’s ECM was first described a century ago by thepioneers of cellular neuroanatomy, including Golgi and Cajal, itsfunction has remained enigmatic1. The fully differentiated ECMassembles late during brain development after the major period ofsynaptogenesis. We found that the net-like ECM acts as a local diffusionbarrier for AMPA receptors on dendrites, controlling the accessibility oflocal receptor populations to the synapse. At the functional level, theperisynaptic ECM affects synaptic depression during high-frequencystimulation, presumably by restricting the exchange of desensitizedreceptors for naive functional ones from extrasynaptic sites.

The ECM forms compartments on the neuronal surface

In the SPT experiments, we found that individual GluR1 receptors areconfined to small surface compartments of variable sizes, whichresemble ECM-free areas on mature neurons1. We therefore proposethat the net-like ECM of the brain forms surface compartments thatvary in their individual size and act as lateral diffusion constraints forsurface molecules. The restricted exchange of surface molecules

between neighboring compartments could allow a neuron to locallycontrol the composition of surface molecules. For example, activesynapses may regulate their repertoire of surface molecules throughlocal recruitment in their surface compartment20,28. Thus, surfacecompartmentalization would enable a neuron to modify a particularsynaptic response without affecting neighboring synapses. Besides localtrafficking29,30, somatic endo- and exocytosis of AMPARs and lateraldiffusion along the dendrite has been proposed to contribute to theturnover of surface AMPARs31. The presence of surface compartmentsin mature neurons makes the somatic supply of receptors unlikely to besufficient to provide receptors at distant synapses and favors thehypothesis of a local delivery of AMPARs32, which is further strength-ened by the recent finding of dendritic synthesis of AMPARs33.

However, the ECM seems to not form a rigid and impervious barrier,as long-distance diffusion, although diminished, is still present onECM-bearing cells. More likely, the hyaluronic acid–based ECM is aviscous diffusion constraint that slows and confines the mobility ofmolecules that enter ECM-covered areas. FRAP data obtained withNrCAMDcytDfn-GFP shows that the ECM-derived change in lateralmobility depends substantially on the extracellular domain. Even FRAPof GPI-GFP, which has no known interaction partner in the ECMand has a very fast fluorescence recovery (half maximum recovery, 4.1 ±0.3 s), was increased after ECM removal. Furthermore, we did notobserve increased FRAP of NMDAR, whose mobilization stronglydepends on PKC activation13, arguing against a nonspecific activationof intracellular signaling cascades by ECM removal. Altogether, thesedata indicate that a general function of the ECM is to act as a passivediffusion barrier. Whether active interactions between transmembranereceptors and ECM components also contribute to the regulation ofsurface trafficking will be addressed by future studies. Certainly, itwould be an interesting, but complex, scenario to have passive andactive mobility regulating factors in the ECM. Clearly, after ECMremoval, receptor mobility is still restricted to distinct dendriticsegments, suggesting additional diffusion constraints at the dendrite.These may arise from specialized structures such as endo- or exocytoticsites outside synapses in conjunction with intracellular scaffoldsorganized by the actin cytoskeleton30,32.

We observed a higher mobility of GluR1- and GluR2-containingreceptors on immature compared with mature neurons (Fig. 2). This isconsistent with a previous report, where a decrease in Dinst of GluR2-containing AMPARs during maturation has been observed34. Removalof the ECM restored a large degree of juvenile receptor mobility in ourFRAP experiments, suggesting that the formation of the ECM accountsfor a certain extent of the decrease in lateral mobility in mature neurons.

ECM-controlled receptor exchange and synaptic plasticity

Removal of the ECM leads to an increase in extrasynaptic Dinst and to ahigher rate of AMPAR exchange between extrasynaptic and synapticsites, which is accompanied by an increased PPR. This observation isconsistent with our recent finding that experimental immobilization ofAMPARs, which restricts receptor exchange, leads to a decreased PPR9.There, we had concluded that lateral diffusion accounts for the recoveryfrom PPD by exchanging desensitized receptors for naive ones9.However, it is not fully understood whether synaptic, or ratherextrasynaptic, mobile receptors contribute to this exchange. Synapticmobility was unchanged in quantum dot experiments and our datatherefore suggests that AMPARs that exchange with extrasynaptic sitescontribute, to a large extent, to the more robust PPR at short pulseintervals. Thus, extrasynaptic receptors may constitute a reservoir thatpermanently exchanges with synaptic receptors in a stochastic mannerand thereby replaces rapidly emerging desensitized synaptic receptors

Controla b

50 pA

Hy1.0

0.8n.s.

200

100I 1 (p

A)

0

Con Hy

Hy + C

yclo

Hy + X

-link

PP

R (

I 2/I 1)

0.6

0.4

0.2

010 100

Interval (ms)

50 ms

Figure 6 Synaptic short-term plasticity is altered after ECM removal.

(a) Example traces of glutamate-evoked AMPAR-mediated currents by local

iontophoresis of glutamate; note the decrease in PPD after hyaluronidase

treatment. (b) Plot of the recovery rate as a function of interstimulus interval

(10, 20, 50, 100, 200 and 500 ms); control (open black circles, n ¼ 9–14),

hyaluronidase-treated cells (red circles, n ¼ 8–14), hyaluronidase-treated

cells with 50 mM cyclotiazide (open black squares, n ¼ 4–5) and

hyaluronidase-treated cells with antibody cross-link (see Online Methods,

red squares, n ¼ 5–15). Recovery rates were significantly different (one-way

ANOVA significant until time interval of 100 ms, P o 0.001). The insert

shows the amplitude of the first glutamate pulse for each condition, which

was unchanged for all treatments. Error bars represent s.e.m.

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(Supplementary Fig. 8). In this model, the size of the reservoir wouldbe one important determinant for synaptic fidelity to high-frequencytransmitter release. The larger the mobile receptor reservoir, the morerobustly a synapse resists PPD. The heterogeneous appearance of theECM suggests that differences in compartment size, and thereforedifferences in the pool of mobile receptors at synapses, may account forsubtle variations in synaptic properties.

Alternatively, the ECM may modulate synaptic properties through itsnegative charge, which may provide a polyanionic rim aroundsynapses35. It has been proposed that the charge of the synapticmembrane shapes the AMPAR-mediated current by influencing theclearance of the negatively charged glutamate from the synapse36. Ananionic environment could therefore alter the duration of the glutamatesignals, which may influence the number of desensitized receptors.Model calculations proposed that glutamate may support the clusteringof AMPARs at release sites37. Both the nature of the glutamate signalsand the diffusion rates of AMPARs are critical parameters in this model.Thus, the size and the location of ECM-derived compartments withrespect to the synapse can influence synaptic function.

The assembly of secreted proteins to form the net-like ECM impliesthe presence of surface receptors as coordinators. Proteins of the Igsuperfamily are good candidates to act as ECM receptors because oftheir ability to bind components of the ECM, such as brevican,tenascin-R and neurocan16,18,38. Also hyaluronic acid–binding proteinssuch as CD44 or hyaluronan synthase may define the location anddimension of ECM surface compartments39. Many of these potentialECM receptors are transmembrane proteins that can be anchored tothe cytoskeleton or intracellular scaffolds. Thus, the extent of the ECM-derived surface compartments may be controlled by the intracellularscaffold or, alternatively, may be shaped by proteolysis of ECMcomponents, for example, after strong synaptic stimuli40–42.

It has previously been suggested that the ECM contributes to themaintenance of neuronal networks by inhibiting structural rearrange-ments at synapses5,43,44. Our results indicate that the ECM furtherstabilizes mature synaptic properties by defining the mobile receptorreservoir that is available for individual synapses. The size of thereservoir seems to be an important determinant for high-frequencyfiring, which typically occurs in a mature neuronal network. Thus, theadult ECM may inhibit juvenile plasticity, but might be essential for thefunctionality of mature synapses.

METHODS

Methods and any associated references are available in the onlineversion of the paper at http://www.nature.com/natureneuroscience/.

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSWe thank C. Poujol and P. Legros for help with video microscopy, C. Breillat,B. Tessier and D. Bouchet for expert technical assistence, J. Falk for NrCAM andGPI-GFP constructs, M. Carta and P. Opazo for support with the slice cultures,O. Kobler for help with three-dimensional image processing, and A. Triller andA. Fejtova for very helpful discussions. This work was supported by grants fromthe Centre National de la Recherche Scientifique, the Conseil Regional d’Aquitaine,the Ministere de la Recherche, the Fondation pour la Recherche Medicale, theEuropean Commission (CT-2005-005320), the Deutsche Forschungsgemeinschaft(GU230/5-2/5-3), a Max Planck award from the A.v. Humboldt Foundationand the Max Planck Society. During part of the work, R.F. was supported bya fellowship from the Swiss National Fonds.

AUTHOR CONTRIBUTIONSR.F. and M.H. designed and performed all experiments and assembled a firstdraft of the manuscript. D.P. performed outside-out patch experiments. C.I.S.,D.C. and E.D.G. formulated the working hypothesis and wrote the corresponding

grant applications. All authors contributed steadily to the discussion of the actualexperiments and to the writing of the manuscript.

Published online at http://www.nature.com/natureneuroscience/

Reprints and permissions information is available online at http://www.nature.com/

reprintsandpermissions/

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ONLINE METHODSPrimary neuronal cultures and transfection, hyaluronidase treatment and

receptor cross-link. Cultures of hippocampal neurons were prepared from E18

Sprague-Dawley rats following a previously described method45. Briefly, cells

were plated at a density of 100–200 � 103 cells per ml on poly-lysine pre-coated

cover slips. Cultures were maintained in serum-free neurobasal medium

(Invitrogen) and kept at 37 1C in 7.4% CO2 for 21–28 DIV. During this

period, half of the medium was exchanged weekly.

Neurons were co-transfected at 5–7 DIV with Homer1c-dsRed and either

pHGFP-GluR1, pHGFP-GluR2 or pHGFPNR1/2 using Effectene (Quiagen).

We mixed 2 mg of DNA with 50 ml of Effectene and 16 ml of Enhancer in 300 ml

of reaction buffer, and then added the mixture to cultured neurons, which were

transferred to serum-free neurobasal medium 10 min beforehand. After an

incubation period of 60 min, neurons were placed in the old medium again.

Transfected neurons were kept for another 15–23 d after transfection.

The ECM was either removed by overnight incubation with 100 U ml�1 or

by 10-min incubation with 1,000 U ml�1 hyaluronidase or 10 min 0.01 U ml�1

chondroitinase ABC (Sigma). Spine morphology was analyzed on hyaluronidase-

treated and hyaluronidase-untreated Homer1c-DsRed–transfected neurons. For

cross-linking with GluR2-containing AMPARs, neurons were pre-incubated

with the primary antibody for 10 min followed by incubation with the

secondary antibody for 10 min. After washing, neurons were used to examine

synaptic currents9.

Antibodies and drugs. The antibody to the N-terminal epitope of the GluR1

subunit was described previously46. We used a commercial antibody to an

N-terminal epitope of the GluR2 subunit to detect GluR2 (BD Pharmigen).

Rabbit brevican antibodies were custom-made and used as described3. Com-

mercial monoclonal antibodies were used against Tenascin-R (Zymed), Homer

(Synaptic Systems) and GFP (Roche). Fluorescent secondary antibodies to

rabbit or mouse (Alexa 488/568) and rhodamin phalloidin were purchased

from Invitrogen (Molecular Probes). Hyaluronidase was obtained from

Calbiochem, HABP from Seikagaku, and TTX, picrotoxin and AP5 from

Tocris. All other chemicals and drugs were purchased from Sigma-Aldrich

(USA).

AMPA receptor labeling and synaptic live staining. Quantum dot 655 goat

F(ab¢)2 antibody to rabbit IgG conjugate (H+L) highly cross-absorbed and

quantum dot 655 goat F(ab¢)2 antibody to mouse IgG conjugate (H+L) highly

cross-absorbed were obtained from Quantum Dot (Invitrogen). Receptors were

stained using quantum dots pre-coated with antibody to GluR1 or monoclonal

antibody to GFP. Quantum dots (0.1 mM) were incubated with 1 mg of

antibody in 10 ml of phosphate-buffered saline (PBS) for 15–30 min. Unspecific

binding was blocked by adding casein (Vector Laboratories) to the pre-coated

quantum dot 15 min before use. Neurons were incubated 5–10 min at 37 1C in

culture medium with pre-coated quantum dots (final dilution of 0.1–0.01 nM).

The incubation was followed by four washing steps of 30 s each. All incubations

and washes were performed in pre-warmed extracellular HEPES-buffered

solution (see below).

Live staining followed by cell fixation. Living neurons were incubated with

antibody to GluR1 (1:200), brevican (1:2,000) or Tenascin-R (1:1,000) in culture

medium at 37 1C for 10 min, fixed in 4% paraformaldehyde (wt/vol) in PBS for

15 min and permeabilized with 0.3% Triton X-100 (wt/vol) for 1 min. After

three washes in PBS with 2% BSA (wt/vol) to block unspecific binding, cells

were incubated for 60 min at 21–25 1C with antibody to rabbit (Alexa 568/488/

647, 1:1,000). Cells were washed and mounted in Moviol. Preparations were kept

at 4 1C until examination. Images were acquired using a confocal microscope

(Leica) and processed for qualitative and quantitative analyses with Metamorph-

Software (Universal Imaging) or ImageJ (US National Institutes of Health).

For quantification of GluR1 surface expression, control and hyaluronidase-

treated hippocampal neurons at 24 DIV were simultaneously live stained with

antibody to GluR1 and images were taken on a Zeiss axioplan fluorescence

microscope. After background subtraction using Metamorph-Software, the

average intensity of GluR1 fluorescence was measured.

Dissociated hippocampal neurons were treated overnight with hyaluron-

idase and were subsequently biotinylated using a biotinylation kit from Pierce.

After cells were lysed using Hank’s buffered salt solution with Ca2+ and Mg2+

containing 1% Triton X-100 and biotinylated proteins were extracted using

excess of avidin sepharose (Pierce), equal amounts of eluates were subjected to

SDS-PAGE and western blotting. Western blots were probed with antibody to

GluR1 (Synaptic Systems) and then scanned and quantified using Odyssey

imaging system (Licor). Spine size, density and length were measured on

Homer1c-dsRed–overexpressing neurons. In addition to clearly labeling the

postsynaptic density, overexpressed Homer1c-dsRed was found in dendrites

and spines. Images were analyzed using ImageJ.

Single molecule optical microscopy. Cells were imaged at 35–37 1C in an open

chamber mounted onto an inverted microscope (IX70 Olympus) equipped

with a 60� (NA ¼ 1.35, Olympus) or 100� objective (NA ¼ 1.3, Olympus).

Quantum dots and Homer1C-DsRed were detected using a xenon lamp

(excitation filter HQ500/20X (Chroma), Mitotrack 560RDF55 (Omega)) and

appropriate emission filters (HQ560/80M (Chroma Technology), 655WB20

(Omega Optical)). Fluorescent images from quantum dots were acquired with

an integration time of 33 ms with up to 2,000 consecutive frames. Signals were

recorded with a back-illuminated thinned CCD camera (Cascade 512BFT,

Roper Scientific).

Quantum dot–labeled GluR1 receptors were monitored on randomly

selected dendritic regions for up to 20 min of total experimental time.

Recording of the synaptic marker over time revealed that the mobility of

synapses was much slower in comparison to the mobility of the receptors (data

not shown). Mobility of synapses themselves did not seem to affect our location

method. Acquisition of the synaptic labeling before and after quantum dot

recording as well as quantum dots fixed on the cover slip allowed us to

compensate mechanical drifts of the stage, which would have lead to a false

interpretation of receptor location.

Receptor tracking and analysis. The tracking of single quantum dots was

performed with homemade software based on Mathlab (Mathworks). Single

quantum dots were identified by their blinking fluorescent emission and their

diffraction-limited signals. Owning to the random blinking events of the

quantum dots, the trajectory of a quantum dot–tagged receptor could not be

tracked continuously. Subtrajectories of the same receptor were reconnected

when the positions before and after the dark period were compatible with

borders set for maximal position changes between consecutive frames and

blinking rates. The values were determined empirically: 1–2 pixels for maximal

position change between two frames and maximal dark periods of 25 frames.

Mean square displacement curves were calculated for reconnected trajec-

tories of at least 100 frames. Diffusion coefficients were calculated by a linear fit

of the first four points of the msd plots versus time. The resolution limit for

diffusion was 0.001 mm2 s�1, as determined by msd calculations of fixed

quantum dots. The resolution precision was B40 nm. Dwell times of

individual receptors given in the results were measured from trajectories in

which the entry and exit from the compartments could be identified.

Synaptic or ECM compartments were identified by homer1c-DsRed expres-

sion or HABP staining, respectively. Pixels assigned to synapses or ECM were

defined as a set of connected pixels obtained using two-dimensional object

segmentation by wavelet transformation47

Electrophysiology. The extra cellular medium contained 145 mM NaCl,

2.5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES and 10 mM

D-glucose (pH 7.4). To block GABAA receptors, we added 50 mM picrotoxin to

the extra cellular medium. The bath temperature was kept at 33–35 1C.

Borosilicate pipettes were used to produce patch electrodes with resistances

of 3–5 MO. A standard pipette solution was used to characterize neuronal

properties in voltage and current-clamp conditions during development

(Supplementary Fig. 7) and contained 140 mM potassium gluconate, 2 mM

MgCl2, 4 mM NaATP, 0.1 mM EGTA, 10 mM HEPES, 10 mM phosphocrea-

tine, 0.4 mM GTP (pH 7.25). To record mEPSC and to decrease space-clamp

difficulties, we used another recording solution 125 mM CH3CsSO3, 2 mM

MgCl2, 1 mM CaCl2, 4 mM NaATP, 10 mM EGTA, 10 mM HEPES and 0.4 mM

GTP (pH 7.25).

Recordings in voltage and current clamp mode were performed

with an EPC10 double patch-clamp amplifier (HEKA Electronics).

Data were acquired and stored using Pulse-Pulse fit software

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version 8.62 (HEKA Electronics, Lambrecht, Germany) and analyzed

with IGOR (WaveMetrics) and GraphPad Prism software.

Spontaneous events were analyzed by Minianalysis (Synaptosoft). Local

activation of receptors was performed by iontophoresis of glutamate using

an amplifier from NPI Electronics. Pipettes for iontophoretic stimulation had

resistances between 40–60 MO when filled with 150 mM sodium glutamate

(pH 7.4). A small retaining current was needed to keep glutamate inside the

pipette (usually between 10–50 nA). Current pulses between 30 and 600 nA and

1–2 ms duration were required to evoke AMPAR-mediated currents between

amplitudes of 30–600 pA under control conditions.

Outside-out recordings. Outside-out patches were pulled from 14–21 DIV

neurons. Internal solution contained 130 mM CsCl, 2 mM MgCl2, 10 mM

EGTA, 10 mM HEPES and 4 mM Na2ATP. Pipette resistance was 3.5–4.5 MO.

After patch formation, the pipette was placed under the flow of a theta appli-

cation pipette containing HEPES-buffered solution in one line and HEPES-

buffered solution, 10 mM glutamate and 20 mM sucrose in the other line to

clearly visualize the interface between solutions. The application pipette was

immerged in the bath and heated to 37 1C for at least 1 cm. It is thus assumed

that solutions were close to that temperature. Fast application was achieved with

a piezo-electric manipulator (Burleigh). After the recording, the application was

controlled by measuring the junction current between the two solutions. To

measure recovery from desensitization, it is important to verify that 1-ms appli-

cations effectively saturate receptors. We verified this by measuring the amplitude

of the currents evoked by 1- or 100-ms applications (Supplementary Fig. 6). If the

former was less than 80% of the latter, the recording was discarded; on average,

current amplitudes were 497.2 ± 160 pA (n ¼ 10) for control and 334.3 ± 143

pA (n ¼ 10) for treated neurons, and the amplitude ratios (1 ms/100 ms) were

0.9 ± 0.03 and 0.91 ± 0.05 for control and treated neurons, respectively.

FRAP. Transfected neurons (21–30 DIV) were placed on the heated stage

(37 1C) of an inverted microscope (Leica CTR 6500) and continually perfused

with preheated (37 1C) extracellular solution (composition as described above).

For low-pH solution, HEPES was replaced by MOPS and adjusted to pH 5.5.

To test the population of surface pHGFP-GluR1–containing AMPARs of a

particular cell, we used a gravity-driven rapid solution exchange using a

theta-glass electrode containing low-pH solution in one channel and NH4Cl

(50 mM) in the other channel to determine the ratio between the fluorescent

intensities48. Fluorescence was excited using a monochromator (Cairn) con-

trolled by Metamorph software (Universal Imaging). To photobleach locally, we

used a sapphire laser 488-20 (Coherent) at 30% power to avoid photodamage.

The laser was coupled to the microscope via a galvometric mirror (Roper

Scientific), which allowed us to photobleach several regions in a short time

window. Recovery from photobleaching was monitored by consecutive acquisi-

tion at a 10-Hz acquisition rate. Recovery curves were corrected for continuous

photobleaching and background noise as described elsewere49.

FLIP. For FLIP experiments, the laser beam was parked at the dendritic shaft at

a power of 10% and a additional 75% intensity filter was used to avoid

photodamage in the continuous bleached region. Continuous laser illumina-

tion was interrupted during image acquisition at a frequency of 0.2 Hz. Control

of surface expression and experimental conditions were similar to those for the

FRAP experiments described above.

Statistics. Statistical values are given as mean ± s.e.m. or medians ± 25%/75%

interval, if not stated otherwise. Statistical significances were performed by

using GraphPad Prism (GraphPad Software). Normal distributed datasets were

tested by one-way ANOVA followed by a Newman-Keuls test to compare

individual pairs of data. Non-Gaussian distributed datasets were tested

by Kruskal-Wallis or Mann-Whitney tests for paired observations. Cumulative

distributions were tested using the Kolmogorov-Smirnov-test. * P o 0.05,

** P o 0.01 and *** P o 0.001.

45. Banker, G. & Goslin, K. Developments in neuronal cell culture. Nature 336, 185–186(1988).

46. Mammen, A.L., Huganir, R.L. & O’Brien, R.J. Redistribution and stabilization of cell sur-face glutamate receptors during synapse formation. J. Neurosci.17, 7351–7358 (1997).

47. Racine, V. et al. Visualization and quantification of vesicle trafficking on a three-dimensional cytoskeleton network in living cells. J. Microsc. 225, 214–228 (2007).

48. Ashby, M.C., Ibaraki, K. & Henley, J.M. It’s green outside: tracking cell surface proteinswith pH-sensitive GFP. Trends Neurosci. 27, 257–261 (2004).

49. Axelrod, D., Koppel, D.E., Schlessinger, J., Elson, E. & Webb, W.W. Mobility measure-ment by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16,1055–1069 (1976).

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