Contribution of cytoskeleton to the internalization ofAMPA receptorsQiang Zhou, Min-Yi Xiao, and Roger A. Nicoll*

Departments of Cellular and Molecular Pharmacology and Physiology, University of California, San Francisco, CA 94143

Contributed by Roger A. Nicoll, December 4, 2000

Trafficking of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid (AMPA) receptors (AMPARs) at synapses has been suggestedto play an important role in the expression of synaptic plasticity.Both the regulated and the constitutive trafficking of synapticAMPARs are thought to involve the insertion and removal ofreceptors by means of an exocytotic and endocytotic process,respectively. In contrast, N-methyl-D-aspartate (NMDA) receptors(NMDARs), which are colocalized with AMPARs at excitatory syn-apses, appear to be much less dynamic. Here, we present evidencesupporting the idea that synaptic AMPARs turn over through aconstitutive endocytotic process and that glutamate applicationgreatly enhances this turnover of AMPARs. The glutamate-inducedinternalization of AMPARs requires a rise in postsynaptic Ca21. TheAMPAR internalization is mimicked by latrunculin A, a drug thatselectively depolymerizes actin and is blocked by jasplakinolide, adrug which stabilizes actin filaments. The rate of endocytosis is notaltered by glutamate application, whereas a clear enhancement isobserved with insulin application. We propose a model in whichthe glutamate-induced dissociation of AMPARs from their anchoron the postsynaptic membrane involves actin depolymerization,which allows the released AMPARs to segregate from the NMDARsand diffuse to a presumably perisynaptic site, where they be-come available to an endocytotic machinery and are selectivelyinternalized.

Recent evidence suggests that trafficking of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) re-

ceptors (AMPARs) at synapses plays an important role in theexpression of synaptic plasticity (1–5). Furthermore, it has beenproposed that AMPARs are constitutively cycling in and out ofthe synapse on a rapid time scale (2, 4, 6–8). This constitutivecycling appears to play a role in activity-dependent trafficking ofAMPARs, because disruption of the cycling occludes expressionof long-term depression in hippocampal slices (2, 6). Based onresults with inhibitors of exocytosis and endocytosis, it appearsthat both the regulated (6, 9–12) and the constitutive (6, 7, 13)trafficking of synaptic AMPARs involve the insertion andremoval of receptors by an exocytotic and endocytotic process,respectively.

Glutamate induces disappearance of AMPARs from synapticsites on a rapid time scale (14). This change is selective toAMPARs, in that synaptic N-methyl-D-aspartate (NMDA) re-ceptors (NMDARs) remain intact, and is accompanied by areduction in the frequency of AMPAR miniature excitatorypostsynaptic currents (mEPSCs) (14). Recently, it has beenshown that glutamate induces internalization of AMPARs by aclathrin-dependent pathway (10).

The sequence of events required for internalization is stillunclear. Because AMPARs are clustered in the postsynapticdensity (PSD) and are intermingled with NMDARs (15–17),presumably the selective internalization of AMPARs requiresthe release of AMPARs from their anchors at the PSD, so thatthey can be segregated before their internalization. In thecurrent study, we used glutamate as the stimulus to study themechanism of AMPAR endocytosis. We sought to answer twoquestions: (i) Can glutamate disassociate AMPARs from theiranchoring to the postsynaptic membrane? (ii) Does glutamate

enhance the rate of endocytosis? We found that the Ca21 influxassociated with glutamate application is required for internal-ization, and we present evidence that depolymerization of actinfilaments caused by this rise in intracellular Ca21 releasessynaptic AMPARs to the endocytotic machinery. Finally, wewere unable to detect any gross change in the rate of endocytosisfollowing the application of glutamate. These results suggest amodel in which glutamate receptor activation increases theconcentration of Ca21 in the dendritic spine, causing actindepolymerization. This releases AMPARs from their anchoringto the PSD and allows access of the receptors to a presumablyperisynaptic endocytotic pathway.

Materials and MethodsNeuronal Culture and Immunocytochemistry. Hippocampal cultureswere prepared as described (14) and were used for experimen-tation 2–3 wk after plating. Surface AMPARs were labeled withan antibody against an extracellular epitope (amino acid 271–285) of the rat GluR1 subunit (Oncogene Research, Boston,MA) for 15 min at 37°C, at a concentration of 5 mgyml. After aquick wash, neurons were incubated with 100 mM glutamate for15 min, unless otherwise noted. Cells were then washed and fixedwith 4% paraformaldehyde and 4% sucrose in PBS for 20 minat room temperature. AMPARs on the surface were labeled withanti-rabbit Cy3-conjugate for 1 h at room temperature. Then,cells were permeabilized with 0.25% Triton for 5 min, followedby incubation with anti-rabbit Cy2-conjugate to visualizeAMPARs inside the cells.

To measure uptake of transferrin (Tf), cells were incubatedwith Texas Red-conjugated Tf (50 mgyml; Molecular Probes) for5 or 10 min. After thorough washing, cells were imaged imme-diately. The intensity of fluorescent puncta was measured andaveraged for each cell. To visualize the synaptic spines, cells werepermeabilized and incubated in PBS containing 10% BSA for 1 hto block nonspecific binding. They were then incubated withrhodamine-conjugated phalloidin (Rd-phalloidin; 1:10,000 dilu-tion; Molecular Probes) for 1 h at room temperature.

Fluorescence Microscopy. An MRC1024 laser-scanning confocalmicroscope (Bio-Rad) attached to a Nikon upright microscopewas used to image the distribution of AMPARs. A thin opticalsection was obtained by using a half-open confocal aperture. Theimage plane was selected so that it focused on the middle of acell (as evidenced by the presence of nucleus), and excludedsignals from above and below the focal plane (which couldrepresent signal from the surface). Laser power of 1–30% wasused, depending on the intensity of the labeling. The 488-nm and

Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; AMPAR,AMPA receptor; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; mEPSC, miniatureexcitatory postsynaptic currents; Tf, transferrin; lat A, latrunculin A; jas, jasplakinolide.

*To whom reprint requests should be addressed. E-mail: nicoll@phy.ucsf.edu.

The publication costs of this article were defrayed in part by page charge payment. Thisarticle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.§1734 solely to indicate this fact.

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073ypnas.031573798.Article and publication date are at www.pnas.orgycgiydoiy10.1073ypnas.031573798

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594-nm lines of the argon-krypton laser were used for excitation,and band-pass filters were used for emission. A 603 oil-immersion objective (NA 1.4) was used. For time-lapse exper-iments involving FM1–43, cells were first incubated in externalsolution containing 1 mM FM1–43 at room temperature for 5min so that the edge of the cell body could be clearly resolved anda proper image plane through the middle of a cell could beselected. Then, FM1–43 was perfused at 37°C and images weretaken every 60 s. A charge-coupled device camera (Hamamatsu,Middlesex, NJ) with a 603 oil-immersion objective (NA 1.4)affixed to a Zeiss inverted microscope was used for other imageacquisition. When comparisons between groups of culturesyfields were made, the exposure time was kept constant.

Analysis. METAMORPH imaging software (Universal Imaging,Media, PA) was used for all image analysis. To quantify recep-tors on the cell surface, pixel intensity of the fluorescent punctaon the surface was measured and averaged for each cell. Thepixel intensity inside the entire cell body, except the nuclearregion, was measured to quantify the internalized receptors.

Electrophysiology. mEPSCs were recorded at room temperaturefrom 10- to 14-day-old cultured neurons by using anAxopatch-1D amplifier (Axon Instruments, Foster City, CA)with low-resistance patch pipettes (3–7 MV). Pipette solutionscontained 145 mM Kzgluconate, 8 mM NaCl, 10 mM Hepes, 0.2mM EGTA, 2 mM MgATP, and 3 mM NaGTP, adjusted to pH7.3 with 1 M KOH. For recording pure AMPAR-mediatedmEPSCs, the control extracellular perfusion solution contained140 mM NaCl, 3.5 mM KCl, 10 mM Hepes, 20 mM glucose, 2mM CaCl2, 2 mM MgCl2, 100 mM picrotoxin, and 1 mMtetrodotoxin, and was perfused at a speed of 0.2–0.3 mlymin. Forrecording dual component mEPSCs mediated by both AMPARsand NMDARs, the perfusion solution contained no MgCl2 andinstead 20 mM glycine was included. Cells were held at 270 mV,and currents were low-pass filtered at 2 kHz and digitallysampled at 5 kHz. Series and input resistances were monitoredthroughout each experiment by using IGOR PRO software (Wave-Metrics, Lake Oswego, OR). mEPSCs were recorded and storedon videotape and analyzed offline with MINI ANALYSIS PROGRAMsoftware (Synaptosoft, Leonia, NJ). Threshold mEPSC ampli-tude was set at 5 pA for AMPAR mEPSCs and 10 pA for dualcomponent mEPSCs. To examine the effect of latrunculin A (latA) on AMPAR mEPSCs, a single large coverslip was broken intotwo: one was used for control recordings and the other wasincubated with 20 mm lat A at 37°C for 20 min before recording.mEPSCs were recorded from both groups of cells. The averageamplitude and frequency of mEPSCs were calculated from10-min records. To examine the effect of lat A on dual compo-nent mEPSCs, a 10-min baseline was recorded and then 20 mMlat A was perfused for 20 min. mEPSCs recorded during thebaseline period and 10 min after wash out of lat A were averaged.The AMPAR component was measured at the peak of the dualcomponent, whereas the NMDAR component was measured asthe average amplitude between 100 and 110 ms after the onsetof mEPSCs. Application of 100 mM D(-)-2-Amino-5-phospho-novalerate (D-APV) at the end of the recording verified that thismeasurement accurately reflects pure NMDAR responses (seeFig. 4D).

ResultsGlutamate Causes Internalization of AMPA Receptors. Two ap-proaches were used to ensure that signals inside the cell actuallyreflect internalized AMPARs in the cytoplasm: saturation la-beling of the surface receptors under nonpermeabilizing condi-tions and the use of thin confocal optical sections. In cells treatedwith glutamate, there is less AMPAR fluorescent staining on thecell surface, and more internal staining, compared with un-

treated cells (Fig. 1A). This agrees with previous results (10),indicating that glutamate causes AMPARs to move from thesurface to the interior of the cells. We quantified this change bycalculating the ratio of fluorescence intensity inside the cellsversus those of puncta on the surface (RinyRout) and used thisratio as an index for the degree of internalization, so thatcomparisons could be made across cells and experiments. Thisratio is significantly higher for cells treated with glutamate thanfor cells not treated with glutamate (Fig. 1B). A similar effect isseen when AMPA is applied (Fig. 1C). The above result isunlikely to be the result of antibody binding directly triggeringAMPAR internalization, because this would require that gluta-mate selectively enhances antibody-driven internalization ofAMPARs.

We next examined whether glutamate application causes areduction in the number of surface synaptic AMPARs. Wefocused on the staining of GluR1 puncta along the dendrites withthe shape resembling that of dendritic spines, because thesepuncta are highly colocalized with a presynaptic marker (eithersynaptophysin or SV2; data not shown) and hence representsynaptic AMPARs. In nontreated cultures (15- to 21-day-old),f luorescent staining reveals large, round, and high-intensity

Fig. 1. Glutamate and AMPA enhanced internalization of AMPARs. (A)Detection of internalization of AMPARs. In untreated cultures, clear stainingof GluR1 is seen on the surface, along the edge of the cell body and dendrites(red), whereas the staining inside the cell body is more homogeneous andweak (green). This distribution is altered by incubation with glutamate:staining becomes weaker on the surface, but stronger inside the cell body thatoccupies the perinuclear region. (Scale bar: 10 mm.) (B) Quantification ofinternalization of AMPARs. Ratio of the fluorescence intensity inside the cellbody vs. on the surface was calculated and used as an index for internalization.The results were normalized to the control group. In the groups treated withglutamate, a higher ratio is obtained (for control, n 5 19 cells from 6 exper-iments; for glutamate, n 5 16 cells from 6 experiments). (C) A similar changeis observed when AMPA is applied (for control, n 5 18 cells from 5 experi-ments; for AMPA, n 5 19 cells from 5 experiments).

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dendritic puncta (Fig. 2A). In contrast, the staining in glutamate-treated cultures is weaker and occupied smaller areas, consistentwith a decrease in synaptic AMPARs. This apparent internal-ization of synaptic AMPARs is also seen with NMDA applica-tion (Fig. 2B), suggesting that Ca21 influx through NMDARsmay trigger internalization. Ca21 influx also appears to berequired for the AMPA-induced internalization of AMPARs.Blockade of voltage-gated Ca21 channels with 100 mM Cd21

blocks the internalization of AMPARs, whereas Cd21 alone haslittle effect (Fig. 2C). These results suggest that Ca21 influxthrough either NMDARs or voltage-gated Ca21 channels is thetrigger for AMPAR internalization.

In addition to this activity-dependent internalization ofAMPARs, there is physiological evidence for a constitutive

turnover of AMPARs (2, 6, 7, 13). Consistent with these results,AMPARs labeled with Fab fragments of the GluR1 antibodyshifted from the surface to the interior of the cell over time(Fig. 2D). The use of Fab fragments makes it unlikely thatthe internalization is triggered by the crosslinking of boundantibodies.

Depolymerization of Actin Filaments Causes Internalization ofAMPARs. Next, we addressed possible mechanisms underlying theinternalization of AMPARs. One possibility is that Ca21 influxtriggers dissociation of AMPARs from their anchoring at thepostsynaptic membrane. In particular, elevated intracellular[Ca21] ([Ca21]i), is known to depolymerize actin (18), and, inaddition, drugs that selectively depolymerize actin filamentscause the disappearance of AMPARs from synapses (19) and areduction in mEPSC amplitude (20). These results suggest thatAMPARs may be anchored to the postsynaptic membrane byactin filaments. To examine this notion in more detail, weapplied jasplakinolide (jas), a drug which stabilizes actin fila-ments (21), to see whether it affected the internalization ofAMPARs. Incubation with jas alone did not cause any signifi-cant change in internalization of AMPARs compared with thecontrol, but it blocked the effect of glutamate (Fig. 3A1). Inaddition to preventing the dissociation of AMPARs from the

Fig. 2. Further characterization of glutamate-induced internalization ofAMPARs. (A) Glutamate reduces AMPARs at the synapse. Large and strongpunctate staining of GluR1 is seen in the untreated culture (arrowheads),whereas small and weaker puncta are observed in cultures treated withglutamate (arrows). The staining of GluR1 in untreated cultures is mainlylocated at extrusions from the dendrites, resembling dendritic spines. Similarresults were obtained in five other fields in three experiments. (Scale bar: 10mm.) (B) NMDA causes internalization of AMPARs (n 5 20 cells and 19 cells forcontrol and 50 mM NMDA, respectively, from 6 experiments). (C) AMPA-induced internalization of AMPARs requires Ca21 influx through voltage-gated Ca21 channels. Internalization of AMPARs by AMPA is blocked bytreating the cultures with 100 mM Cd21. Cd21 alone does not affect internal-ization (n 5 18 cells, 18 cells, 10 cells, and 11 cells for control, Cd21, AMPA(1Cd21), and AMPA respectively, from three experiments). (D) Constitutiveturnover of AMPARs. Cultures were incubated with Fab fragments of GluR1antibody for the periods indicated, then quickly washed, fixed, and labeled inthe same way to visualize the distribution of AMPARs. More internalization isobserved after longer incubation, indicating a gradual internalization ofsurface AMPARs (n 5 20 cells, 20 cells, 20 cells, and 17 cells for 0 min, 10 min,30 min, and 60 min, respectively, from five experiments).

Fig. 3. Depolymerization of actin filaments causes internalization ofAMPARs. (A) Internalization of AMPAR is blocked by prior incubation withjasplakinolide (jas). Cultures were first treated with 2 mM jas for 2 h beforethey were labeled with GluR1 antibody, and treated with control or gluta-mate-containing solutions. Glutamate-induced internalization is blocked byjas, whereas jas by itself has no effect (A1, n 5 12, 15, 16, and 15 cells forcontrol, jas, jas 1 Glu, and Glu, respectively, from four experiments). Thiseffect may be due, at least in part, to an inhibition of endocytosis by jas as theuptake of Tf is also reduced (A2, n 5 45 and 42 cells for control and jas,respectively, from five experiments). (B) Latrunculin A (lat A) mimics the effectof glutamate on AMPAR internalization. Cultures were incubated with lat Afor 1 h at the concentration indicated, and the distribution of AMPAR wasevaluated as above. Lat A causes internalization of AMPAR in a dose-dependent manner (n 5 19, 19, 21, 15, 14, and 16 cells for control, 5 mM, 10 mM,20 mM, 50 mM, and glutamate, respectively, from four experiments).

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postsynaptic membrane, jas might also affect endocytosis assuggested by Lamaze et al. (22). We tested this possibility byexamining the effect of jas on the uptake of Tf. Jas does causea reduction in the uptake of Tf (Fig. 3A2); however, it is unlikelythat this modest reduction in endocytosis can fully account forthe blockade of internalization of AMPARs caused by gluta-mate. We next examined the effect of lat A, a drug thatdepolymerizes actin, on the internalization of AMPARs. Incu-bation with lat A caused a clear internalization of AMPARs, ina dose-dependent manner (Fig. 3B). This result supports previ-ous findings suggesting that actin depolymerization, which istriggered by a rise in [Ca21]i, may be the underlying initiatingmechanism for the observed internalization of AMPARs.

The above results with lat A were obtained under conditionsthat would be expected to cause substantial depolymerization ofactin filaments that, in turn, would be expected to result in thedisappearance of dendritic spines (19). To ensure that disruptionof spines could not account for the effect of lat A, we incubatedcultures with lower concentrations of lat A and for shorter timeperiods. Under these conditions, spines can still be observed(Fig. 4A, Lower, arrows), and the percentage of spines withGluR1 staining decreases when compared with untreated cells(Fig. 4B). This loss of AMPARs from spines is accompanied bya reduction in the amplitude and frequency of AMPAR mEPSCs(Fig. 4C). Importantly, this change in mEPSC amplitude isselective for the AMPAR component, because when dual com-ponent mEPSCs (AMPAR and NMDAR) were recorded, onlythe AMPAR component is reduced in the presence of lat A(Fig. 4D).

Glutamate Does Not Alter the Rate of Endocytosis. In addition toreleasing AMPARs from their anchoring to the postsynapticmembrane, glutamate might also alter the rate of endocytosis byenhancing the efficiency of the endocytotic pathway. Two ap-proaches were used to address this issue. First, we examined theeffect of glutamate on the uptake of FM1–43. FM1–43 is a styryldye that labels the endocytotic compartment if it is present in theextracellular fluid when endocytosis occurs. FM1–43 has beenused extensively to study endocytosis of synaptic vesicles (23), aswell as internalization in nonneuronal cells (24). Uptake ofFM1–43 into the postsynaptic cell can occur in the absence ofglutamate, and this uptake is not affected by a subsequentapplication of high K1, indicating that the labeled compartmentsare not synaptic vesicles (Fig. 5A1). This uptake is blocked byprior treatment of cells with a hypertonic sucrose solution,consistent with involvement of a clathrin-mediated pathway(Fig. 5A2; refs. 25 and 26). Glutamate has no apparent effect onthe uptake rate of FM1–43, as monitored by time-lapse exper-iments (Fig. 5B). Second, we examined whether uptake of Tf isaltered by glutamate application. For the two incubation periodstested, no difference was observed in the presence or absence ofglutamate (Fig. 5C). To test the sensitivity of our assay forendocytosis, we next examined the effects of insulin on FM1–43and Tf uptake. Insulin has been shown to accelerate bothendocytosis and exocytosis in nonneuronal cells (27–29) andinternalization of AMPARs in neurons (12). Insulin did enhancethe uptake of FM1–43 (Fig. 6A) and Tf (Fig. 6B), which wasaccompanied by an enhanced internalization of AMPARs (Fig.6C). These results indicate that glutamate, unlike insulin, doesnot alter the bulk endocytosis in neurons. However, it remainsa possibility that the internalization of AMPARs represents asmall subset of the overall endocytosis measured with eitherFM1–43 or Tf, in which case an effect of glutamate on thisprocess would have been missed.

DiscussionWe have presented evidence supporting the idea that synapticAMPARs turn over through a constitutive endocytotic process

and that glutamate application greatly enhances this turnover ofAMPARs. The glutamate-induced internalization of AMPARsis apparently triggered by Ca21 influx, because it is also causedby application of NMDA, and is blocked by Cd21 when AMPAis used. The AMPAR internalization is mimicked by lat A, a drugthat selectively depolymerizes actin and is blocked by jasplakino-lide, a drug which stabilizes actin filaments, suggesting that theglutamate-induced dissociation of AMPARs from their anchoron the postsynaptic membrane may be because of actin depo-lymerization. We further show that the efficiency of the endo-cytotic pathway is not altered by glutamate application, whereasa clear enhancement is observed with insulin application.

To monitor the internalization of AMPARs, we used sequen-tial labeling with secondary antibodies under nonpermeabilizing

Fig. 4. Lat A causes loss of AMPARs from synapses. (A) After cultures weretreated with 20 mM lat A for 20 min, dendritic spines colocalized with GluR1staining can still be seen (arrowheads, red for spines, green for GluR1), butthere are also spines without GluR1 staining (arrows). (Scale bar: 10 mm.) (B)The percentage of spines with colocalized GluR1 staining decreases aftertreatment with lat A, indicating that AMPARs are dissociated from theiranchorings in the postsynaptic density (n 5 10 and 9 for control and lat A,respectively, from three experiments). (C) A significant reduction in both theamplitude and frequency of AMPAR mEPSCs is observed in cultures treatedwith lat A. Sample AMPAR mEPSCs are shown in Left for untreated and latA-treated cultures, whereas population data from 11 cells are shown in theRight. (D) Reduction in AMPAR mEPSCs occurs in the absence of changes inNMDAR mEPSCs. Dual component mEPSCs were collected, and averagedresponses from the same cell are shown in the Left. After perfusion with lat A,a significant reduction in the peak amplitude is observed, but there is nochange in the slow component (n 5 5 cells). Pharmacological isolation ofAMPAR mEPSC with APV shows that the peak is mainly due to AMPARactivation, whereas the slow component is exclusively mediated by NMDARs.

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and permeabilizing conditions and confocal microscopy to vi-sualize the distribution of AMPARs. Judging from the size anddistribution of the fluorescent puncta inside the cell body, it isvery likely that this staining represents internalized AMPARsinside endosomes (30, 31). It is unclear from the present resultswhether the visualized internalized AMPARs are being storedtemporarily in the cytoplasm before being recycled back onto the

surface, or are on their way to being degraded. Because onlythose AMPARs on the surface before treatment were labeledwith antibody, we can only trace the fate of these receptorsfollowing stimulationymanipulation. Therefore, we can onlyconclude that there is a higher internalization rate of AMPARson stimulation with glutamate. The above results demonstratinga glutamate-induced translocation of AMPARs to the interior ofthe cell are in agreement with previous results (10).

A number of studies have shown that the cytoskeleton isinvolved in anchoring neurotransmitter receptors to the postsyn-aptic membrane. At the neuromuscular junction rapsyny43-kDaappears to anchor acetylcholine receptors to a subsynapticcytoskeletal scaffold (32). Actin, in particular, is also importantin the stability of surface neuronal nicotinic acetylcholine re-ceptors (nAChRs) on chick ciliary ganglion neurons. A gradualdispersal of nAChR clusters and eventual loss of surface recep-tors is observed after cell dissociation (33). This loss of surfacenAChR is accelerated by incubation with lat A, whereas treat-ment with jas prevents this loss and maintains the clusters (33).Furthermore, this loss parallels the rundown of nAChR currentduring whole cell recording, which is postulated to be the resultof Ca21 influx through activated nAChR and subsequent actindepolymerization (34). In cerebellar Purkinje cells, the numberof d glutamate receptor clusters (which are predominantlylocated on the dendritic spines) is dramatically reduced byincubation with the actin-depolymerizing drugs, cytochalasin Dor lat A, suggesting that these receptors are anchored to the actincytoskeleton (35).

In hippocampal slices, Kim and Lisman (20) observed areduction in the amplitude of AMPAR EPSCs, but no change inNMDAR EPSCs when cells were perfused with lat B, anactin-depolymerizing drug. In hippocampal culture, incubationwith lat A causes a significant reduction in the number ofAMPAR-containing spines on pyramidal neurons, suggestingthat actin filaments are required for synaptic localization ofAMPARs and the maintenance of their clustering (19). Al-though Allison et al. (19) reported that lat A also caused adecrease in synaptic NMDARs, and a rundown of NMDA

Fig. 5. Glutamate does not alter the rate of endocytosis. (A) FM1–43 labelsparticles endocytosed through a clathrin-dependent pathway. Incubation ofcultures with 1 mM FM1–43 causes distinct labeling around the perinuclearregion of the cell body (A1). The particles labeled are not synaptic vesiclesbecause they cannot be released on subsequent depolarization with high K1

solution (A1, 60 mM K1 solution for 1 min, which normally releases all FM1–43contained in synaptic vesicles, data not shown). Uptake of FM1–43 into the cellbody is blocked when cultures were preincubated with hypertonic sucrosesolution (A2, 450 mM sucrose solution, total osmolarity was 750 mOsm,treated for 20 min before FM1–43 loading). [Scale bars: 10 mm (A1), 5 mm (A2).](B) Glutamate does not alter the rate of uptake of FM1–43. The fluorescenceintensity of FM1–43 inside the cell body increases roughly linearly with time,and this rate does not differ whether glutamate was present or not. To ensurethat the optical section was selected correctly, the fluorescence intensityinside the nucleus was also monitored, which does not change significantlyduring the same time course (n 5 15 cells from 15 experiments). (C) Glutamatedoes not alter the uptake of Tf. Uptake of Tf was examined with two incu-bation periods: 5 min or 10 min. In neither case does glutamate alter theuptake significantly. Uptake of Tf depends on a clathrin-mediated pathwaybecause it is severely reduced by prior incubation with hypertonic sucrosesolution (n 5 26, 24, 29, 29, 14, 16 cells for Tf 5 min, AMPA 1 Tf 5 min, Tf 10min, AMPA 1 Tf 10min, Tf 10 min, and sucrose 1 Tf 10 min, respectively, fromfive experiments).

Fig. 6. Insulin increases the rate of endocytosis. FM1–43 and Tf labeling wereused to measure the effects of insulin (5 mM) on the rate of endocytosis. Anincreased uptake of FM1–43 (A: n 5 12 cells from 11 experiments) and Tf (B:n 5 34 and 25 cells for control and insulin from 5 experiments) is detected,which is accompanied by an enhanced internalization of AMPARs (C: n 5 16and 14 cells for control and insulin, from 3 experiments).

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whole-cell current has been observed following actin depoly-merization (36), our physiological experiments found littlechange in the NMDAR component, suggesting that, with ourtreatment, there is a preferential loss of AMPARs, consistentwith previous physiological results (20). Furthermore, stainingfor the NR1 subunit of the NMDAR remains intact followingthe NMDA-induced loss of actin staining at synapses (21).Importantly, we were able to demonstrate the loss of synapticAMPARs with short lat A incubation times when spines werestill largely intact. This is consistent with previous studiesshowing that lat A or related drugs do not strongly affect spinemorphology on the time scale of a few hours (19, 20, 37). We alsofound that stabilizing actin filaments with jasplakinolide blockedAMPAR internalization, consistent with the proposal that glu-tamate induced internalization involves actin depolymerization.However, this result must be treated with caution, becausejasplakinolide also inhibited Tf internalization, albeit to a lesserextent.

We also examined the effect of glutamate on the rate ofendocytosis, by comparing the uptake rate of FM1–43 and Tf inthe absence and presence of glutamate. FM1–43 labels virtuallyall endocytotic compartments, including the Tf pathway. Wehave observed consistently that more compartments are labeledby FM1–43 than by Tf (data not shown). Our failure to detecta significant change in the rate of uptake during glutamateapplication could mean that the endocytotic pathway involved inthe internalization of AMPARs represents a small subset of theentire endocytotic pathway labeled with FM1–43. Thus, we

tested a more selective endocytotic pathway, the one used by Tf,which has been used widely to assess effects on receptor-mediated endocytosis. Again, we failed to detect any change. Onthe other hand, insulin, which enhances bulk endocytosis in othercell types, caused a clear enhancement of endocytosis andenhanced internalization of AMPARs, in accord with previouswork (12). Our results therefore argue against a glutamate-dependent increase in endocytosis, although it remains possiblethat the pathway used for the internalization of AMPARs isconsiderably smaller than that used by Tf.

In summary, our results are in general accord with previousfindings, indicating that AMPARs are anchored to the postsyn-aptic membrane by actin filaments. The precise mechanism forthis anchoring is unclear, because a direct interaction betweenAMPARs and actin has not been reported. We propose a modelin which Ca21 influx at synaptic sites during the application ofglutamate untethers synaptic AMPARs, but not NMDARs bydepolymerizing actin filaments. This would allow the freedAMPARs to diffuse away from the colocalized NMDARs to aperisynaptic site where they become available to the endocytoticmachinery and are selectively internalized. It will be of interestto determine whether long-term depression induced by synapticstimulation also involves a similar mechanism.

We thank Drs. M. Frerking, R. Edwards, and D. Bredt for theircomments on the manuscript. R.A.N. is supported by grants fromNational Institutes of Health and the Bristol–Myers Squibb Co. and is amember of the Keck Center for Integrative Neuroscience and the SilvioConte Center for Neuroscience Research.

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