Scholarly article on topic 'Spatial and Temporal Regulation of Receptor Endocytosis in Neuronal Dendrites Revealed by Imaging of Single Vesicle Formation'

Spatial and Temporal Regulation of Receptor Endocytosis in Neuronal Dendrites Revealed by Imaging of Single Vesicle Formation Academic research paper on "Biological sciences"

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{"clathrin-mediated endocytosis" / "long-term depression" / "transferrin receptor" / "Beta2 adrenergic receptor" / "AMPA receptor" / GluA1 / GluA2 / "post-synaptic density"}

Abstract of research paper on Biological sciences, author of scientific article — Morgane Rosendale, Damien Jullié, Daniel Choquet, David Perrais

Summary Endocytosis in neuronal dendrites is known to play a critical role in synaptic transmission and plasticity such as long-term depression (LTD). However, the inability to detect endocytosis directly in living neurons has hampered studies of its dynamics and regulation. Here, we visualized the formation of individual endocytic vesicles containing pHluorin-tagged receptors with high temporal resolution in the dendrites of cultured hippocampal neurons. We show that transferrin receptors (TfRs) are constitutively internalized at optically static clathrin-coated structures. These structures are slightly enriched near synapses that represent preferential sites for the endocytosis of postsynaptic AMPA-type receptors (AMPARs), but not for non-synaptic TfRs. Moreover, the frequency of AMPAR endocytosis events increases after the induction of NMDAR-dependent chemical LTD, but the activity of perisynaptic endocytic zones is not differentially regulated. We conclude that endocytosis is a highly dynamic and stereotyped process that internalizes receptors in precisely localized endocytic zones.

Academic research paper on topic "Spatial and Temporal Regulation of Receptor Endocytosis in Neuronal Dendrites Revealed by Imaging of Single Vesicle Formation"

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Spatial and Temporal Regulation of Receptor Endocytosis in Neuronal Dendrites Revealed by Imaging of Single Vesicle Formation

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Authors

Morgane Rosendale, Damien Jullie, Daniel Choquet, David Perrais

Correspondence

david.perrais@u-bordeaux.fr

In Brief

Rosendale et al. visualize single endocytic vesicle formation in mature, plasticity-competent neurons using the pulsed pH assay. They found that perisynaptic, clathrin-coated endocytic zones are not subject to specific regulation. Their localization alone is enough to explain preferential internalization of AMPA receptors at those sites.

Highlights

• Nascent endocytic vesicles can be visualized in hippocampal neurons with the ppH assay

• Vesicles form with no change in clathrin fluorescence but with dynamin recruitment

• Perisynaptic endocytic zones preferentially internalize synaptic receptors

• Perisynaptic endocytic zones are not specifically regulated by long-term depression

Rosendale et al., 2017, Cell Reports 18,1840-1847 ciossMark February 21, 2017 © 2017 The Author(s).

http://dx.d0i.0rg/l 0.1016/j.celrep.2017.01.081

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Cell Reports

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Spatial and Temporal Regulation of Receptor Endocytosis in Neuronal Dendrites Revealed by Imaging of Single Vesicle Formation

Morgane Rosendale,1'2'3 Damien Jullié,1'2'4 Daniel Choquet,12 and David Perrais1'2'5'*

University of Bordeaux, 33000 Bordeaux, France

2Centre National de la Recherche Scientifique, Interdisciplinary Institute for Neuroscience, UMR 5297, 33000 Bordeaux, France 3Present address: Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan 4Present address: University of California, San Francisco, San Francisco, CA 94143, USA 5Lead Contact

'Correspondence: david.perrais@u-bordeaux.fr http://dx.doi.org/10.10167j.celrep.2017.01.081

SUMMARY

Endocytosis in neuronal dendrites is known to play a critical role in synaptic transmission and plasticity such as long-term depression (LTD). However, the inability to detect endocytosis directly in living neurons has hampered studies of its dynamics and regulation. Here, we visualized the formation of individual endocytic vesicles containing pHluorin-tagged receptors with high temporal resolution in the dendrites of cultured hippocampal neurons. We show that transferrin receptors (TfRs) are constitutively internalized at optically static clathrin-coated structures. These structures are slightly enriched near synapses that represent preferential sites for the endocytosis of postsynaptic AMPA-type receptors (AMPARs), but not for non-synaptic TfRs. Moreover, the frequency of AMPAR endocytosis events increases after the induction of NMDAR-dependent chemical LTD, but the activity of perisynaptic endo-cytic zones is not differentially regulated. We conclude that endocytosis is a highly dynamic and stereotyped process that internalizes receptors in precisely localized endocytic zones.

INTRODUCTION

Endocytosis is fundamental for neuronal function. Constitutive endocytosis regulates the number of AMPA receptors (AMPARs) at the postsynaptic level, and blocking this process blocks NMDA-receptor (NMDAR)- dependent long-term depression (LTD) at hippocampal excitatory synapses (Luscher et al., 1999; Man et al., 2000). In cultured neurons, application of NMDA greatly enhances the internalization of AMPARs via expression mechanisms apparently shared with synaptic LTD (Beattie et al., 2000; Lee et al., 2002; Ehlers, 2000). Molecular and cellular mechanisms regulating postsynaptic AMPAR trafficking have thus been studied in great detail (Anggono and Huganir, 2012), but key questions remain unanswered, such as

where and when receptors are being internalized following NMDAR stimulation. This issue is particularly relevant, considering the synaptic specificity of LTD. Two extreme possibilities can be envisaged. On the one hand, receptor endocytosis could be locally regulated around stimulated synapses and contribute to such specificity. Accordingly, endocytic zones, i.e., clathrin-coated structures (CCSs), have been visualized in dendritic spines around the post-synaptic density (PSD) of excitatory synapses (Lu et al., 2007; Racz et al., 2004; Blanpied et al., 2002). On the other hand, receptors could diffuse rapidly in the neuronal plasma membrane (Choquet and Triller, 2013) and be internalized away from the depressed synapses. Effectively, ultrastructural studies seldom identify clathrin-coated invaginations within spine heads but, rather, at the base of spine necks or in dendritic shafts (Cooney et al., 2002; Tao-Cheng et al., 2011). To distinguish between these two non-exclusive models, it would be necessary to detect the activity of endocytic zones and monitor their modulation during synaptic plasticity in living neurons.

So far, AMPAR internalization has been mostly studied through antibody feeding experiments (Beattie et al., 2000; Ehlers, 2000; Lee et al., 2002; Rocca et al., 2008). Although sensitive and quantitative, this method has a poor temporal resolution and does not provide accurate information on the location of receptor internalization. To provide a dynamic view of the endocytic process, other studies have used pH-sensitive super-ecliptic pHluorin (SEP)-tagged receptors to monitor endosome acidification following NMDA application (Ashby et al., 2004; Lin and Huganir, 2007). However, measuring overall cellularfluo-rescence does not pinpoint the moment and localization of endocytic vesicle formation. In addition, a large fraction of SEP-tagged receptors is found in intracellular weakly acidic compartments that also acidify during NMDA application, influencing the overall SEP signal (Rathje et al., 2013). Therefore, a more direct and resolutive method is needed to map the activity of endocytic zones in neuronal dendrites.

Clathrin-mediated endocytosis (CME) has been studied in great depth in immortalized cell lines, mostly by observing the disappearance of fluorescent clathrin from CCSs, which signals the departure of a coated vesicle from the plasma membrane (McMahon and Boucrot, 2011). However, such analysis is not applicable to mature, plasticity-competent cells because

1840 Cell Reports 18, 1840-1847, February 21, 2017 © 2017 The Author(s).

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

CrossMark

Figure 1. Formation of Endocytic Vesicles in Dendrites Visualized with the ppH Assay

(A) Portion of a dendrite visualized with TIRF microscopy of a neuron transfected with TfR-SEP at pH 7.4 and pH 5.5 (5x contrast). Spots visible at pH 5.5 are intracellular receptors.

(B) Example endocytic event detected with the ppH at time 0. Right: merged TfR (pH 7.4) and Clc with the location of the event (cross).

(C) Average fluorescence (fluo.) of 1,694 events in four cells in the green and red channels, aligned to the time of vesicle detection. There is no detectable variation of Clc fluorescence. Gray areas represent the 95% confidence interval of randomized data indicating a clear clathrin enrichment at the site of vesicle formation.

(D) Portion of a dendrite transfected with TfR-SEP and Dyn-mCh at pH 7.4.

(E) Same as in (B).

(F) Average fluorescence of Dyn-mCherry for 1,409 events in four cells. The fluorescence is maximal at —4 s (time of scission: —2 s).

(G) Kymograph of a portion of dendrite showing stable CCSs as visualized with Clc-mCh (bottom) or TfR-SEP at pH 7.4 (top). Sudden bursts of SEP fluorescence are due to exocytic events at pH 7.4 (yellow arrowheads) and to endocytic events at pH 5.5 (green arrowheads).

(H) Distribution of time intervals separating consecutive events occurring at single CCSs. Dashed line represents median (168 s).

Error bars represent SEM.

clathrin shows very little turnover in neurons after 1 week in culture, thus making CCSs appear optically static (Blanpied et al., 2002). Another method, called the pulsed pH (ppH) assay, is used in cell lines to visualize endocytic vesicle formation. It is based on the reversible extinction of SEP fluorescence by periodic application of acidic solution to isolate the fluorescence of internalized receptors protected from the transient pH change (Merrifield et al., 2005). It can thus reveal, with a temporal resolution of 2 s, the formation of endocytic vesicles at both disappearing and apparently stable CCSs.

Here, we adapted the ppH assay to make it usable on cultured hippocampal neurons. We show that endocytic vesicles form repetitively at optically static CCSs. All receptors observed in this study can be internalized throughout the dendrites, but AMPARs can additionally and preferentially be endocytosed in perisynaptic CCSs, a spatially defined set of endocytic zones located adjacent to PSDs. However, our results show that AMPAR removal from synapses during NMDAapplication cannot be explained by a specific upregulation of the activity of this population of CCSs. Rather, vesicle formation appears to be a stereotyped event occurring at precisely located endocytic zones.

RESULTS

The ppH Assay Reveals the Activity of CCSs in Neurons

We applied the ppH assay to hippocampal neurons in culture transfected with the transferrin receptor fused to SEP (TfR-SEP), a receptor constitutively internalized through CME. As in cell lines, TfR-SEP receptors visible at pH 7.4 were colocalized

with clathrin light chain fused to mCherry (Clc-mCh), and most of the signal disappeared at pH 5.5, revealing intracellular structures (Figure 1A). Therefore, the appearance of a spot in an image taken during a pH-5.5 interval indicates the formation of a clathrin-coated vesicle (CCV) that pinched off from the plasma membrane during the preceding 2-s interval, when the extracellular medium was at pH 7.4 (Figure 1B). Such endocytic events could be readily detected throughout the dendritic tree at a stable frequency of 35 ± 10 10—3 events min—1.mm—2 (n = 9). However, as opposed to what had previously been observed in other cell types (Merrifield et al., 2005), the fluorescence at pH 7.4 and that of clathrin hardly changed during these events (Figure 1C). To confirm that the detected events are, indeed, newly formed endocytic vesicles, we imaged cells cotransfected with TfR-SEP and dynamin fused to mCherry (Dyn-mCh), a protein essential for membrane scission (Ferguson et al., 2009). Similarto nonneuronal cells (Taylor et al., 2011) Dyn-mCh is partly colocalized with TfR-SEP in dendrites (Figure 1D) and is recruited maximally at the time of endocytic vesicle formation (Figures 1E and 1F).

We further assessed the innocuousness of rapid pH changes on neuronal physiology. First we co-applied fluorescent transferrin (Tfn-A568, 5 mg/mL) during the ppH assay. Tfn-A568 colocalized with TfR-SEP at CCSs (Figures S1A and S1B) and internalized with similar kinetics, whether neurons were subjected to the ppH assay or not (Figures S1C and S1D). Second, because the pH-5.5 solution generates currents due to the opening of acid-sensing ion channels, we blocked the ppH-induced currents using amiloride (Xiong et al., 2004) (Figures S1E and S1F). Amiloride did not change the frequency of endocytic

events (Figures S1G and S1H), indicating that acid-induced depolarization does not affect TfR internalization. Therefore, we concluded that the ppH assay does not affect constitutive endocytosis in neurons. Nevertheless, amiloride was added in all subsequent experiments where low-pH solution was used to avoid potential artifacts affecting the regulation of other processes by repeated depolarizations. Finally, we looked for an alternative to low-pH solution to quench SEP fluorescence reversibly. We found that trypan purple almost completely quenched extracellular TfR-SEP fluorescence in a reversible manner (Jullie et al., 2014) (Figure S2A). Moreover, it did not induce currents or affect neuronal excitability (Figure S2B). Consequently, we used it to reveal endocytic events in a similar way as the ppH assay, simply by quenching SEP with trypan instead of low pH (Figure S2C). This protocol enabled the detection of endocytic events with similar characteristics as the ones detected with the ppH assay (Figure S2D), albeit at lower frequencies (13.7 ± 2.3 10~3 events mirT2.min~1) (n = 8). We propose that this is due to incomplete quenching of surface fluorescence and/or to accumulation of trypan at the cell surface. Indeed, unlike for the ppH assay, whole-cell fluorescence steadily decreased (Figure S2E), and the frequency of detected events started to drop after 4 min of recording (Figure S2F). We concluded that the pulsed-trypan assay (pTry) reveals a similar endocytic process as that of the ppH assay but can only be used quantitatively on short recordings.

Remarkably, as noted earlier, despite their intense endocytic activity, CCSs marked by Clc-mCh were very stable (Figures 1B, 1C, and 1G). Therefore, we wondered whether single CCSs could undergo several rounds of endocytosis. In 10-min recordings, 52% of all segmented CCSs (404 out of 776 CCSs in four cells) produced at least one detectable vesicle (644 events). Among these productive CCSs, 55% yielded two to six vesicles. The interval between two consecutive events in the same CCS varied broadly, with a median of 168 s (Figure 1H). In conclusion, despite their optical stability, CCSs are highly active, and the produced vesicles can be readily detected by the ppH assay.

We finished validating the ppH assay as a quantitative measure of endocytosis by examining the stimulated internalization of the p2 adrenergic receptor (P2AR), a G-protein-coupled receptor robustly endocytosed in neurons following agonist binding (Jullie et al., 2014; Yudowski et al., 2006). Under basal conditions, SEP-P2AR was homogenously distributed on the plasma membrane, and we detected 6.8 ± 1.2 10~3 events min^^mr2. Oppositely, in the presence of its agonist isoproterenol (10 mM), SEP-P2AR clustered at preexisting CCSs (Figures S3Aand S3B), and its frequency of internalization increased by 152% ± 58% (p = 0.01) (Figure S3C). Interestingly, surface receptor accumulation at CCSs during stimulation did not translate into a higher number of receptors inside vesicles, as determined by a quantification of SEP fluorescence at the time of vesicle formation (Figure S3D). Overall, this analysis confirms that the dynamics of agonist-dependent receptor internalization can be monitored using the ppH assay.

Synaptic Receptors Are Preferentially Internalized Close to Synapses

To investigate whether synapses could represent preferential sites of endocytosis regulation, we compared the localization

of internalization of TfR and AMPAR in neurons cotransfected with Homer1c-RFP, a marker of PSDs. First, we observed that TfR-SEP endocytic events were detected in all parts of the den-drites, both in shafts and spines (n = 6; Figures 2A and 2B). To assess whether TfR-containing vesicles formed at random locations with regard to synapses, we measured the distance between each event and its closest PSDs. We then simulated chance distributions using randomly scattered events. From this analysis, we found no observable difference between real and randomized distributions (Figure 2C; Kolmogorov-Smirnov [KS] test, p = 0.34), indicating that constitutive endocytosis occurs in all parts of dendrites. We next monitored the internaliza-tion of AMPARs. These events occurred at CCSs, as shown by the enrichment of clathrin at the time of vesicle formation (Figure S4). The basal frequency of SEP-GluA1-containing events was 2.1 ± 1.1 10~3 events mm_2.min (n = 8), while that of SEP-GluA2-containing events was 2.5 ± 0.4 10~3 events mm_2.min_1 (n = 14). Surprisingly, AMPAR-containing vesicles were also detected throughout the dendrites and not only close to PSDs where receptors are enriched (Figures 2D-2I). For GluA1 events, although measured and randomized distributions were not statistically different (KS test, p = 0.89), they did not overlap completely either. Notably, we measured a 12% enrichment of GluA1 events being detected within 300 nm of a synapse, as compared to chance (Figure 2F, top, first two bins). More strikingly, we found a 20% enrichment of GluA2 events occurring close to synapses so that the random and real distribution of events were statistically different (Figure 2I, top, first two bins; KS test, p = 0.039). These data are consistent with GluA2 being more concentrated and less mobile at synapses than GluA1 (Makino and Malinow, 2009). Therefore, AMPARs, as opposed to TfRs, are preferentially internalized in the vicinity of synapses.

We wondered whether this discrepancy could be explained by a precise spatial organization of endocytic zones along den-drites. Previous studies have shown that many CCSs reside close to excitatory synapses (Blanpied et al., 2002; Lu et al., 2007; Petrini et al., 2009), but whether they are actually enriched there has not been documented. To address this point quantitatively, we imaged neurons cotransfected with Homer1c-RFP and Clc-GFP to measure the distance between CCSs and PSDs from segmented images (Figures 2J-2L). We confirmed, as in Lu et al. (2007), that 87% ± 2% of PSDs have an adjacent CCS (i.e., less than 300 nm, Figure 2K, bottom). However, simulations show that 75% of PSDs would also have an adjacent CCS if the latter were randomly scattered throughout the cell (Figure 2K, bottom, black line). Nevertheless, these distributions are different (KS test, p < 0.001), and there is a 20% excess of PSDs with an attached CCS as compared to chance (Figure 2K, top, first bin). Therefore, we conclude that clathrin is actively enriched around PSDs. Accordingly, using the same dataset, we found that, while CCSs were, for the most part, located no further from their closest PSD than if randomly scattered (Figure 2L, bottom), we could detect an ~6% enrichment of CCSs lying closer to a PSD than by chance (Figure 2L, top; KS test, p < 0.001). We named this population of PSD-associated CCSs perisynaptic endocytic zones. We propose that this spatial arrangement of CCSs combined with the enrichment of AMPARs at the PSD

Figure 2. Localization of Receptor Endocytic Sites Relative to PSDs

(A) Example TfR-SEP event occurring at time 0 In a spine. Right: merged TfR (pH 7.4) and Homer1c with the location of the event (cross).

(B) Map of TfR-SEP endocytic events detected in an 8-min recording overlaid on the corresponding Homer1c-RFP image. Blue and pink crosses locate events detected more or less than 300 nm away from a PSD, respectively.

(C) Distribution of distances between an event and their nearest PSD (360 events in six cells, green curve). The corresponding randomized dataset (gray) and its median (black) are represented. Top: difference between the real and the median of the randomized distributions.

(D-F) Same as in (A)-(C) for cells transfected with SEP-GluA1 and Homer-RFP (40 events in six cells).

(G-I) Same as in (A)-(C) for cells transfected with SEP-GluA2, unlabeled GluA1, and Homer1c-RFP (70 events in 14 cells).

(J) Portion of a dendrite transfected with Homer1c-RFP and Clc-GFP. Right: top = merged image, bottom = segmented clusters of Homer1c and Clc displayed in red and green, respectively.

(K) Same as in (C), but measuring the distance between each PSD and the nearest CCS (2468 PSDs in six cells, red curve). Blue line: 300-nm threshold. (L) Same as in (K), in the same neurons, but measuring distances between the center of CCSs and their nearest PSD (8,008 CCSs, green curve).

can explain their preferential internalization over TfRs in the vicinity of synapses.

Regulation of Perisynaptic Endocytic Zones during Stimulated Receptor Internalization

Even though shaft and perisynaptic CCSs both appear able to internalize AMPARs and TfRs under basal conditions, we investigated whether perisynaptic endocytosis could be differentially regulated upon stimulation. First, we stimulated overall endocytosis using 5 mM insulin while monitoring TfR or GluA1 internalization (Man et al., 2000; Zhou et al., 2001). This treatment increased the frequency of TfR-SEP endocytic events within minutes by 54% ± 20% (p = 0.03; Figure 3A) and that of SEP-GluA1 events by up to 138% ± 119% (p = 0.05; Figure 3D). However, this treatment did not affect receptor loading inside the forming vesicles for both types of receptor (Figures 3B and 3E). Also, most importantly, it did not specifically affect the perisynaptic CCS population, leaving the localization of endocytic events unchanged (Figures 3C and 3F).

We thus investigated whether stimulating AMPAR endocytosis more specifically could reveal a differential regulation of perisynaptic CCSs. Specific internalization of GluA2 can be achieved by application of NMDA with signaling mechanisms mimicking synaptic LTD (Anggono and Huganir, 2012; Beattie et al., 2000; Lee et al., 2002) that differ from insulin treatment. In our conditions, NMDAR-mediated currents, known to be sensitive to extracellular pH (Traynelis et al., 1995), were inhibited by ~80% every pH-5.5 interval of the ppH (Figures S4A and S4C). However, this inhibition was totally reversible and did not prevent the stimulation of GluA2 endocytosis by the so-called chemical LTD (chemLTD) treatment (3 min NMDA [20 mM] and glycine [10 mM] in 0.3 mM Mg2+). Indeed, the frequency of GluA2-containing endocytic events increased significantly during (59% ± 29%, p = 0.03) and after (91% ± 35%, p = 0.08) stimulation, and this increase was blocked by the NMDA receptor antagonist APV (50 mM) (Figure 4A). To rule out any potential effect of the application of acidic solution, we repeated the stimulation with the pTry assay. We allowed for sufficient clearance of accumulated

Figure 3. Stimulation of Endocytosis by Insulin Does Not Affect the Location of Endocytic Sites

(A) Normalized cumulative frequency (Norm cumul freq) of endocytic events recorded In the six neurons used for Figures 2A-2C transfected with TfR-SEP and Homer1c-RFP before and during application of insulin (5 mM). Dotted line represents the extrapolation of the linear regression of the control period.

(B) Average TfR-SEP fluorescence (fluo.) at the indicated pH, aligned to the time of vesicle detection, before and during insulin application. SEP fluorescence measured at pH 5.5 at the time of vesicle formation was 1,590 ± 48 (n = 413) and 1,444 ± 31 (n = 941) during baseline and stimulation, respectively, and was not significantly different (p = 0.11, Kolmogorov-Smirnov test).

(C) Same analysis as in Figure 2C during insulin application. The distribution of distances during stimulation is not different from random.

(D-F) Same as (A)-(C) for the six neurons used for Figures 2D-2F transfected with SEP-GluA1 and Homer1c-RFP. In (E), SEP fluorescence measured at pH 5.5 at the time of vesicle formation was 1,506 ± 74 (n = 40) and 1,591 ± 133 (n = 93) during baseline and stimulation, respectively, and was not significantly different (p = 0.43, Kolmogorov-Smirnov test). In (F), the analysis of distances shows the same ~10% enrichment at perisynaptic CCSs as in control. Error bars represent SEM.

Trypan by resting 10 min between baseline, stimulation, and washout (Figure S5D). As for the ppH assay, despite an ~80% inhibition of NMDARs by trypan (Figures S5A and S5C), the frequency of GluA2-containing events increased during NMDA treatment (88% ± 64%, n = 5; p = 0.3) (Figures S5F and S5G). Interestingly, while a continuous monitoring using the ppH assay shows that event frequency keeps increasing in the 3 min directly following stimulation, the paradigm used here shows that it comes back to baseline levels 10 min after stimulation (2% ± 39%, compared to baseline; Figure S5F). We concluded from this dataset that the effect of NMDAR activation on AMPAR endocytic rates is transient, an observation that neither surface measurements of GluA2 fluorescence (Figures S5H and S5I) (Lin and Huganir, 2007; Rathje et al., 2013) nor electrophysiological readouts measuring the long-lasting effects of LTD could detect.

We further analyzed our results obtained with the ppH assay with regard to the regulation of GluA2 internalization. Asforthe other receptors, increased frequency of vesicle formation was not accompanied by an increase in receptor loading in vesicles (Figure 4B),

highlighting the stereotypical behavior of vesicle formation. Interestingly, the location of endocytic events relative to PSDs, as compared to baseline, shifted with time so that the excess of vesicles forming in perisynaptic zones fell from ~20% before and during stimulation to ~12% after stimulation (Figures 4C and 4D). This finding goes against the possibility that chemLTD induction specifically upregulates the activity of perisynaptic CCSs. Oppositely, it is in accordance with a model in which GluA2-containing AMPARs are destabilized from synapses to become more homogenously distributed at the cell surface. As a result, they can be endocytosed in CCSs located at more and more randomly distributed locations by an otherwise unmodulated machinery.

DISCUSSION

Optically Static CCSs Are Endocytically Active in Mature Cultured Neurons

The ppH assay was originally designed in cell lines to detect the precise timing of vesicle formation. While reaching this goal

Figure 4. Internalization of AMPARs during NMDA Application

(A) Normalized cumulative frequency (Norm cumul freq) of endocytic events (green curve) for the six neurons used for Figures 2G-2I transfected with SEP-GluA2, unlabeled GluAl, and Homerlc-RFP before, during, and after application of NMDA; blue curve: same in the presence of APV (188 events in eight cells).

(B) Average fluorescence (fluo.) in the green channel at the indicated pH, aligned to the time of vesicle detection, before (left) and during (right) NMDA application. SEP fluorescence measured at pH 5.5 at the time of vesicle formation were 744 ± 107 (n = 70), 646 ± 74 (n = 90), and 599 ± 36 a.u. (n = 109) during baseline, stimulation, and wash, respectively, and was not significantly different (p = 0.18, Kruskal-Wallis test).

(C and D) Same as in Figures 2G-2I on the 90 events recorded during NMDA application (C) and the 109 events recorded after NMDA removal (D). Error bars represent SEM.

efficiently, it unexpectedly revealed the existence of endocytic events occurring in the absence of a concomitant disappearance of clathrin, which was taken as a hallmark of vesicle formation (Merrifield et al., 2005). These forming vesicles were shown to recruit, with similar kinetics, all the major classes of proteins associated with CME (Taylor etal., 2011). The analogy between those events and the long-standing dilemma observed in mature neurons, where CME is known to play a major role despite the lack of significant observable clathrin dynamics, led us to apply the ppH assay in neurons. Using this method, we demonstrate that constitutive endocytosis occurs throughout dendrites and soma and that single, long-lived CCSs can undergo several rounds of endocytosis while remaining optically stable. The events detected with the ppH assay are, indeed, bona fide newly formed endocytic vesicles, as confirmed by the recruitment of dynamin at the time of scission (Taylor et al., 2011), as well as by increased detections induced by insulin, a known activator of endocytosis (Man et al., 2000). By analogy, we suggest the possibility that clathrin stability may be the rule in many cell types with highly organized compartments such as focal adhesion sites (Batchelder and Yarar, 2010) and microvilli (Boulant et al., 2011) in epithelial cells. The ppH assay may, therefore, become an important tool to visualize the activity of optically stable endocytic zones in primary cells.

Definition of CCS Subpopulations in Neuronal Dendrites

We investigated whether subsets of CCSs could be defined by their capacity to concentrate and internalize specific receptors. We were able to define two subsets of CCSs by their location relative to PSDs. The first subset consists of the majority of CCSs, which appear to be independent of a PSD. The second consists of a smaller fraction (26%) found enriched in the vicinity of a PSD, which we define as perisynaptic CCSs. These are found near 87% of PSDs. Such close agreement between the present study and Lu et al. (2007) shows that both PSDs and

perisynaptic CCSs can be seen on a TIRF (total internal reflection fluorescence) microscope, introducing no bias toward missing synaptic and perisynaptic events. Vesicles labeled with TfR-SEP, a marker for constitutive endocytosis, were detected throughout the dendritic tree, with no preferential internalization around PSDs despite the enrichment of clathrin at these sites. This suggests that this receptor is depleted, but not excluded, from perisynaptic CCSs. Conversely, vesicles containing SEP-GluA1 or -GluA2 formed preferentially close to PSDs. The spatial arrangement of CCSs alone could explain the apparent preferential targeting of AMPARs to these sites. Indeed, CCSs located near PSDs are ideally positioned to capture diffusing receptors (Petrini etal., 2009). However, the detection of a large proportion of events away from PSDs shows that non-perisynaptic CCSs are also capable of AMPAR internalization. Our data, thus, do not argue for the definition of molecularly or functionally distinct subpopulations of CCSs. Rather, our data are consistent with a primary—but not exclusive—role of perisynaptic CCSs in internalizing AMPARs.

Modulation of AMPAR Internalization during chemLTD

NMDAR-mediated internalization of AMPARs has been proposed to be one of the mechanisms leading to synaptic LTD. In cultured neurons, previous studies report that the fraction of internalized receptors increases by about 100% after application of the chemLTD protocol for 5 to 10 min (Lee et al., 2002; Rocca et al., 2008). However, it remained undefined which fraction was due to increased endocytic rates or to a delay in fast receptor recycling (Citri et al., 2010). Here, we measured endocytic activity directly using the ppH and pTry assays. Knowing that NMDARs are sensitive to extracellular pH changes, we cannot exclude that the rate of stimulation could be even greater if NMDARs remained activated continuously for 3 min, as is commonly done, rather than every 2 s. Nonetheless, under our experimental conditions, we found that the frequency of

endocytosis events increased by 59% (in ppH) or 88% (in pTry) during NMDA application and by 91% in the 3 following minutes before returning back to baseline 10 min later. Notably, this increase occurred within seconds after the start of stimulation. Therefore, we directly show that the greater fraction of internalized receptors reportedly observed after chemLTD induction is mainly due to an increase in endocytic rates of AMPARs in the first minutes after stimulation. While this effect is only transient, we propose that the following downstream trafficking steps, such as targeting of AMPARs to late endosomes, can account for the long-lasting effects of LTD (Fernandez-Monreal et al., 2012).

Remarkably, none of the stimulation paradigms tested here led to a specific upregulation of the activity of perisynaptic CCSs. Therefore, we propose that the increased internalization rate of AMPARs upon chemLTD induction may be due to modifications of the receptor itself, such as phosphorylation (Ang-gono and Huganir, 2012) or unbinding from protein interactors that would destabilize it from its scaffolds in the PSD (Constals et al., 2015;Tomitaet al., 2004). However, in the more physiological context of synaptically induced LTD (as opposed to a bath-applied chemLTD paradigm), only synaptic NMDARs are activated, and a small fraction of synapses are being modified. The receptors associated to these depressed synapses could be targeted solely to their closest CCS, thus spatially limiting their internalization. The spatial organization of CCSs may, therefore, be crucial in this context and contribute to the fast kinetics of AMPAR internalization and LTD expression as predicted by modeling (Czondor et al., 2012). More precise protocols associating synaptic stimulation and endocytic vesicle formation will be needed to address this issue.

In conclusion, our data support a model in which an efficient and tightly organized network of endocytic machinery proteins coupled to a precise regulation of AMPAR diffusion and trapping properties could be key to the fast kinetics and synaptic specificity of LTD induction.

EXPERIMENTAL PROCEDURES

Primary Neuronal Cultures and Transfection

Neurons were prepared and cultured as in Jullie et al., 2014 (see the Supplemental Information for details).

TfR-SEP, SEP-P2AR, SEP-GluA1, Clc-mCh, and Homer1c-RFP(tdTomato) plasmids have been described previously (Jullie et al., 2014; Merrifield et al., 2005; Petrini et al., 2009; Taylor et al., 2011). SEP-GluA2 was cloned in an inducible pBI-Tet-on vector (Clontech Laboratories). AP-GluA1 was expressed under the human Synapsin promoter (see the Supplemental Information for details).

Neurons were transfected at 7-10 days in vitro (DIV), except when expressing Dyn1-mCh (transfected at 13 DIV). Transfection was performed using Effectene (QIAGEN), following the company protocol, except for SEP-GluA2-expressing neurons used for chemLTD experiments under the ppH assay and neurons cotransfected with SEP-GluA1/2 and clc-mCherry, which were transfected using the calcium phosphate technique (see the Supplemental Information for details).

Fluorescence Imaging

Imaging was performed at 35°C on a TIRF microscope as previously described (Shen et al., 2014) on neurons at 14-22 DIV. The ppH assay was performed as in Shen et al. (2014) (see the Supplemental Information for details of solutions and other stimulation protocols).

Patch-Clamp Recordings

Recording pipettes and solutions were prepared as in Jullie et al. (2014) (see the Supplemental Information for details). Cells were recorded in the same conditions as for fluorescence imaging.

Image Analysis

Semi-automatic detection of endocytic events and fluorescence quantification were performed as described previously for cell lines (Shen et al., 2014; Taylor et al., 2011). See the Supplemental Information for details on neuron-specific notes about this analysis.

Data Representation

Values are given as mean ± SEM. Statistical p values were obtained using two-tailed paired Student's t tests unless otherwise stated. Cumulative frequency plots were normalized to 100 for each cell at the end of a baseline recording when applicable. Seethe Supplemental Information for the construction of cumulative distance plots, randomization, and statistical tests for differences.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures and five figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.celrep.2017.01.081.

AUTHOR CONTRIBUTIONS

M.R. and D.P. conceived the study and wrote the manuscript. M.R. performed most experiments and analyses. D.P. performed part of the experiments for Figures 1, 4, and S4 and wrote most of the MATLAB scripts for analysis. D.J. performed part of the experiments for Figures S1 and S3. All authors helped in formulating the models and contributed to the preparation of the manuscript.

ACKNOWLEDGMENTS

We thank William Abdou and Abinaya Ravisankarfor preliminary experiments, the cell culture core facility of the IINS for neuronal cultures, the molecular biology technical assistance for help with the plasmids, and Olivier Thoumine for critically reading the manuscript. This work was supported by the Centre National de la Recherche Scientifique (Interface program), the Fondation Recherche Medicale (FRM; grant ING20101221208 to D.P. and grant FDT20140921063 to M.R.), the Agence Nationale pour la Recherche (CaPeBlE ANR-12-BSV5-005) (to D.P.), the ERC (ADOS grant) (to D.C.), a pre-doctoral fellowship from the University of Bordeaux, and a Labex BRAIN fellowship (to M.R.).

Received: August 31, 2016 Revised: December 16, 2016 Accepted: January 30, 2017 Published: February 21, 2017

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