Extracellular Proteolysis by Matrix Metalloproteinase-9 Drives Dendritic Spine Enlargement and Long-Term Potentiation Coordinately

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Extracellular proteolysis by matrix metalloproteinase-9 drives dendritic spine enlargement and long-term potentiation coordinately

Xiao-bin Wanga, Ozlem Bozdagib, Jessica S. Nikitczukb, Zu Wei Zhaib, Qiang Zhoua,1, and George W. Huntleyb,1

Departments of aNeurology and bNeuroscience, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029

Edited by Edward G. Jones, University of California, Davis, CA, and approved October 8, 2008 (received for review July 25, 2008) Persistent dendritic spine enlargement is associated with stable remodeling, which has both beneficial and maladaptive conse- long-term potentiation (LTP), and the latter is thought to underlie quences. In brain, they are secreted by neurons and glia in an long-lasting memories. Extracellular proteolytic remodeling of the inactive (pro) form, and they become proteolytically active when synaptic microenvironment could be important for such plasticity, several regulatory steps that result in removal of the propeptide are but whether or how proteolytic remodeling contributes to persis- triggered in response to specific stimuli (9). For example, studies tent modifications in synapse structure and function is unknown. have shown that in response to LTP induction, MMP-9 rapidly Matrix metalloproteinase-9 (MMP-9) is an extracellular protease becomes proteolytically active at perisynaptic sites and essential for that is activated perisynaptically after LTP induction and required maintenance of LTP (10–13). Thus, perisynaptic MMP-9 proteol- for LTP maintenance. Here, by monitoring spine size and excitatory ysis in response to LTP induction may be critical for local remod- postsynaptic potentials (EPSPs) simultaneously with combined eling of dendritic spine structure and function necessary to support 2-photon time-lapse imaging and whole-cell recordings from hip- long-term synaptic plasticity. pocampal neurons, we find that MMP-9 is both necessary and Here, we test this idea by combining 2-photon time-lapse imaging sufficient to drive spine enlargement and synaptic potentiation with whole-cell patch clamp recording from area CA1 neurons to concomitantly. Both structural and functional MMP-driven forms investigate directly the role of MMP-9 in LTP-associated structural ␤ of plasticity are mediated through 1-containing integrin recep- and functional plasticity. The results identify an instructive role for tors, are associated with integrin-dependent cofilin inactivation MMP-9 in coordinating synaptic structural and functional plasticity within spines, and require actin polymerization. In contrast, during LTP. postsynaptic exocytosis and protein synthesis are both required for MMP-9-induced potentiation, but not for initial MMP-9-induced Results spine expansion. However, spine expansion becomes unstable Stable Spine Expansion and LTP Both Require MMP Proteolysis. We when postsynaptic exocytosis or protein synthesis is blocked, blocked endogenous MMP proteolysis with bath-applied MMP indicating that the 2 forms of plasticity are expressed indepen- inhibitors while applying a theta-burst pairing (TBP) protocol dently but require interactions between them for persistence. that induces rapid and persistent spine enlargement and LTP (5). When MMP activity is eliminated during theta-stimulation-induced Spine size and synaptic responses [excitatory postsynaptic po- LTP, both spine enlargement and synaptic potentiation are tran- tentials (EPSPs)] were monitored simultaneously in CA1 neu- sient. Thus, MMP-mediated extracellular remodeling during LTP rons by 2-photon time-lapse imaging and whole-cell patch clamp has an instructive role in establishing persistent modifications in recording (Fig. 1A). Spines were visualized by intracellular both synapse structure and function of the kind critical for learning labeling with an inert fluorescent dye, calcein, contained in the and memory. recording patch pipette. A stimulating glass electrode was po- sitioned Ϸ20 ␮m from the imaged spines to elicit EPSPs and actin ͉ cofilin ͉ integrin ͉ synaptic plasticity ͉ protein synthesis TBP, a configuration that maximizes the likelihood that synapses on the imaged spines are activated (5, 14, 15). In the absence of ong-lasting memory is based on long-term modifications of MMP inhibitors, TBP induced a rapid and persistent increase in Lsynapse structure and function. In hippocampal area CA1, spine volume (Fig. 1B) and an immediate but small increase in naturalistic patterns of theta-stimulation readily induce long-term EPSP slope, which gradually increased further until reaching a potentiation (LTP) of the excitatory, glutamatergic Schaffer col- plateau by Ϸ30 min (Fig. 1C) as expected (5). In contrast, in the lateral afferent inputs that target dendritic spines (1), which are presence of an MMP-9 inhibitor (Inhibitor II), TBP produced a small, actin-rich dendritic protrusions that harbor the majority of spine enlargement and synaptic potentiation that were transient the excitatory synapses (2). Studies show that dendritic spines (Fig. 1 A–C). Spine volume increased immediately to values undergo significant morphological remodeling in association with comparable with those in untreated slices, but then returned long-lasting plasticity (3). Spine growth, for example, is associated gradually to baseline values (Fig. 1B). In parallel, EPSP slope with the induction of LTP and is thought to be important for also increased immediately to levels comparable with those seen supporting persistent changes in synaptic strength (4–6). However, in untreated slices, but then fell to baseline levels (Fig. 1C). We little is known about signals that instruct and coordinate persistent modifications in synapse structure and function during LTP.

Dendritic spine morphology and synaptic potentiation can both Author contributions: X.-b.W., O.B., Q.Z., and G.W.H. designed research; X.-b.W., O.B., be dynamically modulated by proteins of the extracellular matrix J.S.N., and Z.W.Z. performed research; X.-b.W., O.B., J.S.N., Z.W.Z., Q.Z., and G.W.H. (ECM) and the cell-surface proteins with which they interact, which analyzed data; and Q.Z. and G.W.H. wrote the paper. has long fueled the idea that regulated ECM remodeling has an The authors declare no conﬂict of interest. important role in synaptic plasticity (7). How precisely such remod- This article is a PNAS Direct Submission. eling could occur is not understood. In other tissues, regulated 1To whom correspondence may be addressed. E-mail: [email protected] or proteolytic remodeling of the ECM by matrix metalloproteinases [email protected]. (MMPs) is important for driving changes in cell shape and move- This article contains supporting information online at www.pnas.org/cgi/content/full/ ment (8). MMPs are mostly secreted, extracellularly-acting pro- 0807248105/DCSupplemental. teases that function under various contexts in local pericellular © 2008 by The National Academy of Sciences of the USA

19520–19525 ͉ PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807248105 Downloaded by guest on September 24, 2021 Locally Delivered MMP-9 Causes Spine Expansion and Synaptic Po- 1 tentiation. The experiments above indicate that MMP activity is 1 necessary for persistence of TBP-induced spine enlargement, but is it sufficient for inducing persistent spine enlargement alone in the absence of TBP? We answered this by using the combined imaging/ recording configuration described above while briefly puffing MMP-9 through the stimulating microelectrode onto naive (unpo- 2 2 tentiated) spines. We found that local exposure of naive spines to MMP-9 (0.5 ␮g/mL; 30 min) produced a persistent expansion in the volume of the dendritic spine heads in a majority of the spines imaged (Fig. 2A, arrowheads) and contemporaneously produced a consistent increase in EPSP slope that reached a plateau in 30–40 3 min and persisted stably throughout the recording period (Fig. 2 A 3 and B). When all of the spines within the imaged regions near the stimulating pipette were examined as a group, the average spine volume increased significantly compared with baseline levels (Fig. 2C and Fig. S3A). In contrast, the inactive pro-MMP-9 did not affect either EPSPs or spine volume (Fig. 2 B and C and Fig. S3B), presumably because the endogenous mechanisms leading to removal of the prosequence, normally triggered by TBS, are not engaged in the absence of TBS. Further analyses showed substantial spine expansion in response to MMP-9 over the entire range of initial spine volumes (Fig. S3C), indicating that both small and large spines are capable of expansion in response to MMP-9, consistent with several recent studies (5, 16). Together, these results demonstrate that local MMP-9 proteolysis is both necessary and sufficient for persistent spine enlargement and potentiation.

TBP Occludes MMP-9-Induced Structural and Functional Plasticity. We next asked whether the MMP-9-induced plasticity is the same spine and synaptic modifications that are triggered by TBP. We addressed this by occlusion experiments, in which TBP was applied first followed 15 min later by local delivery of MMP-9 to the TBP- potentiated spines through the stimulation pipette. TBP produced an immediate spine expansion and potentiation as expected, but subsequent delivery of MMP-9 did not produce further spine NEUROSCIENCE expansion or potentiation (Fig. S4). These results suggest that TBP and MMP-9 engage common signaling pathways or expression mechanisms.

Integrin Receptors Mediate MMP-9-Induced Spine Expansion and Synaptic Potentiation. We next investigated the signaling cascade Fig. 1. Persistence of TBP-triggered spine expansion and LTP requires MMP proteolysis. (Ai). A representative CA1 neuron showing the position of the leading from MMP-9 activity to spine enlargement. We focused on local stimulating (Stim) and whole-cell recording (Rec) electrodes. (Scale bar: integrins because they can regulate cell shape on binding MMP- 50 ␮m.) (Aii and Aiii) Representative images (Aii) and EPSP traces (Aiii) showing exposed matrix proteins (8). We preincubated slices with a function- effects of the MMP inhibitor (Inhibitor II) on TBP-induced spine expansion and neutralizing ␤1-integrin antibody before exposure to MMP-9, be- potentiation. The spines and EPSP traces are shown before (1), 5 min after (2), cause the majority of hippocampal integrin heterodimers contain a and 45 min after (3) TBP, and correspond to times (in minutes) indicated in the ␤1 subunit (17, 18), and such heterodimers are required for population data shown in B and C. In this and subsequent figures, expanded consolidation of theta-LTP (19, 20). In the presence of the blocking and stable spines are indicated by arrowheads and arrow, respectively. (Scale antibody, MMP-9-induced spine expansion (Fig. 3A) and synaptic ␮ bars: 1 m, 5 mV, and 50 ms.) (B) Population data showing that immediate potentiation (Fig. 3B) were both abrogated. Control experiments TBP-triggered spine expansion is unaffected by MMP inhibitors (143.5 Ϯ 7.4% vs. 145.7 Ϯ 6.0% at 5 min post-TBP, P Ͼ 0.1), but persistence of expansion is confirmed that the neutralizing antibody alone had no effects on blocked (filled triangles) in comparison with control neurons (open triangles). baseline spine morphology or synaptic physiology (Fig. S5) (10). (C) Population data showing initial TBP-triggered potentiation is also unaf- Integrins drive changes in cell shape by affecting the actin cytoskel- fected by MMP inhibitors (filled circles, 144.9 Ϯ 17.3% inhibitor-treated vs. eton (21). Accordingly, we found that internal perfusion of the actin 153.3 Ϯ 19.7% untreated at 5 min post-TBP, P Ͼ 0.4), but persistence of polymerizing inhibitor latrunculin A (latA, 0.1 ␮M) delivered potentiation is blocked in comparison with long-lasting potentiation after through the patch pipette eliminated MMP-9-induced spine expan- TBP in control neurons (open circles). All statistical tests are Mann–Whitney U sion and synaptic potentiation (Fig. S6). tests. Gray lines in B and C indicate duration of bath-applied Inhibitor II. MMP-9 Triggers Integrin-Dependent Cofilin Phosphorylation in Spines. The results above suggest an important requirement for actin obtained identical results by using a different MMP inhibitor, remodeling in persistent spine enlargement and synaptic poten- GM6001 [supporting information (SI) Fig. S1]. Control exper- tiation. What regulates MMP-9-driven actin remodeling? Actin iments verified that the MMP inhibitors had no effects on either depolymerizing factor (ADF)/cofilin, among others, has F-actin basal synaptic transmission, as expected (10, 11), or spine volume severing and depolymerizing activity (22) and is negatively (Fig. S2). These data demonstrate that blocking endogenous regulated by phosphorylation at a single site (23). Previous MMP activity during LTP induction impairs stable spine expan- studies show that LTP causes increased dendritic spine content sion and prevents the full expression of synaptic potentiation. of phosphorylated cofilin (24), whereas inhibiting cofilin phos-

Wang et al. PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 ͉ 19521 Downloaded by guest on September 24, 2021 123

Fig. 3. MMP-9-induced structural and functional plasticity is mediated by integrins. (A) Preincubation with a neutralizing ␤1-integrin antibody prevents MMP-9-induced spine expansion. (B) Preincubation with ␤1-integrin antibody also prevents MMP-9-induced synaptic potentiation.

increase was blocked by the ␤1-integrin-neutralizing antibody, confirming that MMP-9-mediated effects on actin remodeling are mediated through integrins. We verified the increased levels of phosphocofilin in MMP-9-potentiated slices anatomically by using immunocytochemistry (Fig. 4 B and C). We found significantly greater numbers of phosphocofilin-labeled puncta within CA1 stratum radiatum, the site of the potentiated synapses, in comparison with control slices; such labeled puncta were significantly larger in comparison with those from control slices, which could not be attributed to differences in fluorescence intensity (data not shown). Coimmunolabeling with the excitatory postsynaptic density marker PSD-95 confirmed that the majority of such phosphocofilin-labeled puncta were associated with dendritic spines (Fig. 4C), as expected (see further discussion in SI Text, Note 1) (24, 26). Fig. 2. MMP-9 drives persistent spine expansion and LTP at naive spines. (A) A representative experiment from a single CA1 neuron showing persistent Postsynaptic Exocytosis or Protein Synthesis Is Required for MMP-9 spine expansion and synaptic potentiation after brief puffing of MMP-9 onto Potentiation but Not Initial Spine Enlargement. We next asked naive spines. Numbers indicate corresponding times on the recording. (Scale ␮ whether the MMP-9-induced spine enlargement was mechanis- bars: 1 m, 5 mV, and 50 ms.) (B) Population data showing that MMP-9 induces tically dissociable from MMP-9-induced potentiation. We inves- a persistent potentiation (open circles, 213.1 Ϯ 38.9% at 75 min post-MMP-9 exposure; P Ͻ 0.001, n ϭ 10). Neurons exposed to inactive pro-MMP-9 show no tigated this first by inhibiting postsynaptic exocytosis with inter- changes in EPSPs (filled circles). (C) Population data showing that MMP-9 nal loading of the light chain of botulinum toxin type B (BoTox, induces persistent spine expansion (open triangles, 143.9 Ϯ 6.1% of the 0.5 ␮M) while exposing imaged spines to MMP-9 (Fig. 5 A–C). baseline level at 75 min post-MMP-9 exposure; n ϭ 95 spines/10 cells; P Ͻ Postsynaptic exocytosis is required for LTP (27), and studies 0.001). Such effects on spine size are not seen in response to inactive pro- suggest that LTP-associated spine expansion is unstable (5) or MMP-9 (filled triangles). All statistical tests are paired t tests. In this and absent (28) when exocytosis is blocked. Under conditions where subsequent figures, black bar indicates duration of local MMP-9 puffing. exocytosis in the postsynaptic neuron was blocked, MMP-9- induced potentiation was mostly eliminated (Fig. 5 A and B). In contrast, MMP-9-induced spine expansion occurred initially and phorylation impairs LTP maintenance (25). We tested whether appeared identical to that for control neurons exposed to MMP-9-induced spine enlargement was associated with in- MMP-9 alone (Fig. 5 A and C). However, such initial expansion creased levels of phosphorylated cofilin. Slices were bath- was transient, and it decayed significantly from initial peak exposed briefly to MMP-9, which produces persistent potenti- values (Fig. 5 A and C). ation of CA3–CA1 synapses by 30 min posttreatment (10). We then tested the requirement for protein synthesis in MMP-9 Lysates were then probed with antisera specific to cofilin phos- structural and functional plasticity, because previous studies have phorylated at Ser-3 and examined by Western blotting. MMP-9 indicated that in the presence of protein synthesis inhibitors, LTP induced a significant increase in levels of phosphorylated cofilin, is largely blocked and spine expansion becomes unstable (5, 29). We without effects on total cofilin protein levels (Fig. 4A). This found that in the presence of cycloheximide, MMP-9 potentiation

19522 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807248105 Wang et al. Downloaded by guest on September 24, 2021 Fig. 4. MMP-9 drives integrin-dependent cofilin phosphorylation in dendritic spines. (A) Representative West- ern blotting showing levels of phosphocofilin (p-cof) and total cofilin (cof) in untreated control sections, ones treated with MMP-9, ones treated with the ␤1-integrin neutralizing antibody alone, or ones treated with MMP-9 in the presence of the ␤1-integrin-neutralizing antibody. Quantitative analysis shows significant elevation in levels NEUROSCIENCE of phosphocofilin after MMP-9 exposure (*, P Ͻ 0.001 in comparison with other conditions, n ϭ 5 rats, ANOVA) that is blocked by the ␤1-integrin-neutralizing antibody. (B) Confocal microscope images taken from area CA1 stratum radiatum showing phosphocofilin immunofluo- rescent puncta in untreated control slices or ones exposed to MMP-9. (Scale bar: 5 ␮m.) The graphs indicate significantly greater numbers and sizes of puncta in the MMP-9 stimulated slices in comparison with control slices (*, P Ͻ 0.01, unpaired t tests, n ϭ 5 rats). (C) High-power confocal images from an MMP-9-stimulated slice showing that labeling for the postsynaptic density marker PSD-95 (green), which is concentrated in spines, codis- tributes with that for phosphocofilin (red), as indicated by the yellow pixels shown in the merge and Insets (ar- rows). (Scale bar: 5 ␮m.)

was greatly reduced and did not persist (Fig. 5D). In contrast, enlargement and potentiation are transient. Our results suggest that MMP-9-induced spine expansion occurred initially and reached MMP-9 functions during LTP as a local, instructive extracellular values comparable with controls, but thereafter decayed steadily to signal that consolidates synaptic structural and functional modifi- baseline (Fig. 5E). Together, these results demonstrate that MMP- cations coordinately, ensuring persistent modifications critical for 9-induced spine expansion occurs initially even when MMP-9 learning and memory (30, 31). potentiation is absent or greatly diminished. This result suggests After TBP, there is an immediate increase in both spine size and that the 2 forms of plasticity are expressed independently, but their EPSPs. The former is presumably attributable to rapid actin persistence requires mechanistic interactions between them. remodeling, at least partially mediated by cofilin inactivation, occurring seconds to minutes after LTP induction (4, 5, 20, 24, Discussion 32–34), and the latter likely to rapid phosphorylation of AMPA Induction of LTP leads rapidly to perisynaptic MMP-9 proteolysis receptors (AMPARs) (35). The MMP blockers had no effect on the (10, 11). We show here that such MMP-9 proteolysis is both immediate, TBP-triggered changes in spine size and potentiation, necessary and sufficient to drive spine enlargement and synaptic indicating that such rapid plasticity is mechanistically independent potentiation concomitantly. Both forms of MMP-9-driven plasticity of MMP proteolysis. Proteolysis is probably maximally active later require ␤1-integrin-dependent actin remodeling and protein syn- than the immediate forms of TBP-triggered plasticity, because thesis. When MMP activity is eliminated during TBP, both spine increases in MMP-9 levels and proteolysis are detectable by 15–30

Wang et al. PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 ͉ 19523 Downloaded by guest on September 24, 2021 min after LTP induction (10, 11). NMDA receptors are required for such activation, but the downstream steps that then lead to MMP 123 activation remain unknown and presumably account for some delay. It is conceivable that the onset of MMP-9 proteolysis could occur sooner, because pro-MMP-9 is present within the neuropil (10, 36) and could in theory be quickly activated by signaling molecules such as nitric oxide (37) or BDNF (38) that are released by LTP induction (39). The immediate TBP-triggered increase in spine size and potentiation is followed by a consolidation process through which both structural and functional forms of plasticity are stabilized over the first Ϸ30 min (5, 40, 41). Studies suggest that although initial TBP-triggered spine expansion and LTP occur independently of each other, the 2 forms of plasticity then interact to enable consolidation of both (5, 6, 16). Our results are consistent with this observation and suggest that MMP-9 is an active component of the consolidation process, thereby having an instructive role in both spine expansion and synaptic potentiation. First, onset of LTP- triggered MMP-9 proteolysis matches closely the timeframe of consolidation; the fact that MMP inhibitors blocked persistence of TBP-triggered spine enlargement and potentiation is consistent with impairment in consolidation. Second, the effects of MMP inhibitors on stability of LTP are time-dependent, becoming inef- fective after the period of consolidation (10). Third, MMP-9-driven spine enlargement and potentiation were both mediated exclusively by ␤1-integrins and require protein synthesis. It has been estab- lished that ␤1-integrins are critical for consolidation of theta-LTP and actin polymerization (18, 20, 42). Although it remains unknown precisely how MMP-9 engages integrin signaling, proteolysis of perisynaptic ECM could expose latent recognition sequences that then activate cell-surface integrins (43). We found that MMP-9/integrin-dependent cytoskeletal reorganization, presumably mediated in part by MMP-9-triggered inactivation of cofilin, is a critical and common effector of both MMP- 9-induced structural and functional plasticity. Potentiation associated with stable LTP involves insertion and synaptic incor- poration of new AMPARs, a process that occurs on the order of 15–20 min post-LTP induction and is thought to require actin remodeling (5, 6, 28, 44, 45). The absence of MMP-9-induced potentiation in the presence of latA may reflect disruption of MMP-9/integrin-dependent trafficking of AMPARs. Although this possibility remains to be tested, ␤3-integrins regulate homeostatic synaptic plasticity through AMPA receptor trafficking (46). In addition to the expression of stable potentiation, synaptic incorpo- ration of glutamate receptor 1 also appears necessary for LTP- associated initial spine expansion to persist (6, 16). Our finding that blocking postsynaptic exocytosis prevented MMP-9-induced potentiation, but affected only the stability of the MMP-9-induced spine expansion, suggests that MMP-9/integrin-dependent pathway controls AMPAR insertion, which confers subsequent stability on

(C) Initial spine expansion in response to MMP-9 occurred normally in neurons loaded with BoTox (142.8 Ϯ 7.0% at 15 min post-MMP-9 exposure, P Ͼ 0.3, Mann–Whitney U test), but was transient. At 75 min postpuffing, when the experiments were terminated for technical reasons, spine volume had decayed significantly from initial peak values (115.2 Ϯ 1.8% at 75 min post-MMP-9 puffing, P Ͻ 0.01, paired t test, n ϭ 108 spines/13 cells). At this time, the average spine volume remained significantly larger than baseline (P Ͻ 0.05, paired t test). We speculate that spine volume would continue to decay to baseline over time. Fig. 5. Blocking postsynaptic exocytosis or protein synthesis prevents MMP- (D) MMP-9-induced potentiation is significantly reduced by protein synthesis 9-induced potentiation and destabilizes MMP-9-induced spine expansion. (A) inhibition (black circles). The ghostline (gray circles) indicates comparative control Sample images and EPSP traces showing the transient spine expansion (ar- values (MMP-9 alone) shown in Fig. 2B.(E) MMP-9-induced spine expansion in the rowheads) and small increase in EPSP size in a representative neuron internally presence of cycloheximide (black triangles) is transient, reaching control values loaded with light chain of BoTox (postsynaptic). (Scale bars: 1 ␮m, 5 mV, and initially (129.2 Ϯ 4.3% MMP-9 alone vs. 138.7 Ϯ 8.6% MMP-9 plus cycloheximide 50 ms.) Numbers indicate corresponding times in A–C.(B) Population data show- immediately after MMP-9 exposure, P Ͼ 0.2, Mann–Whitney U test), but then ing that synaptic potentiation is largely absent in neurons loaded with BoTox decays to baseline values (at 60 min, 95.4 Ϯ7.6% of baseline; nϭ72 spines/8 cells). The after exposure to MMP-9 (119.1 Ϯ 11.5% of baseline at 75 min after MMP-9 ghostline (gray triangles) indicates comparative control values (MMP-9 alone) shown puffing vs. 99.2 Ϯ 5.1% at 0 min before MMP-9 puffing, P Ͼ 0.1, paired t test). in Fig. 2C. Gray bars indicate duration of bath-applied cycloheximide.

19524 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807248105 Wang et al. Downloaded by guest on September 24, 2021 spine enlargement, and is consistent with previous studies showing pulses. Imaging was performed on a 2-photon laser scanning system. We verified that postsynaptic exocytosis is required for stable spine expansion that the various experimental conditions did not cause a substantial shift in the and potentiation (5). Protein synthesis is also required for consol- distribution of spine volume by calculating the percentage of imaged spines that idating spine expansion and LTP (5, 29), and our data are consistent show significant increases in volume (Fig. S7). with this requirement for MMP-9-induced structural and functional Western Blotting of MMP-9-Treated Sections. Methods are detailed in SI Text, plasticity. Our data do not distinguish whether such protein syn- Note 3. Slices were bath-exposed to MMP-9 (30 min) in the presence or thesis is downstream of integrin activation, or whether it is down- absence of ␤1-integrin-blocking antibody; control sections were untreated. stream of an additional, unidentified MMP-9-triggered pathway. In After washout (30 min), whole-hippocampal lysates were prepared. Protein any event, MMP-9 is activated in hippocampus by inhibitory was separated by SDS/14% PAGE and probed with anti-phosphocofilin and avoidance learning and is required for consolidating memory (47, anti-cofilin antibodies. 48). Thus, the cellular plasticity mechanisms we describe here likely underlie the contribution of MMP-9 to cognitive function. Immunolabeling and Analysis. Methods are detailed in SI Text, Note 4. Slices were exposed to MMP-9 or left untreated as described above. After fixation, they were Materials and Methods processed immunofluorescently for codistribution of phosphocofilin and PSD-95 and analyzed with Metamorph. Animals. Acute hippocampal slices (350-␮m thick) were from 14- to 21-day-old Sprague–Dawley rats. Use of the animals conformed to institutional and National Statistical Analysis. All data are expressed as mean Ϯ SEM. Statistical analyses are Institutes of Health guidelines. listed in the text and individual figure legends.

Whole-Cell Recordings, Image Acquisition, and Analysis. Methods and reagents ACKNOWLEDGMENTS. We thank Dr. Vanja Nagy for help and discussion at ␤ are described in SI Text, Note 2. MMP inhibitors, 1-integrin-blocking antibody, early stages of the project. G.W.H. was supported by National Institute of and cycloheximide were bath-applied; and active- or inactive- (pro)-MMP-9 was Mental Health Grant MH-075783. Q.Z. was supported by grants from the delivered locally through the glass stimulating pipette by controlled pressure Whitehall Foundation and Ellison Medical Foundation.

1. Larson J, Wong D, Lynch G (1986) Patterned stimulation at the theta frequency is 26. Racz B, Weinberg RJ (2006) Spatial organization of cofilin in dendritic spines. Neuro- optimal for the induction of hippocampal long-term potentiation. Brain Res 368:347– science 138:447–456. 350. 27. Lledo PM, Zhang X, Sudhof TC, Malenka RC, Nicoll RA (1998) Postsynaptic membrane 2. Matus A, Ackermann M, Pehling G, Byers HR, Fujiwara K (1982) High actin concentra- fusion and long-term potentiation. Science 279:399–403. tions in brain dendritic spines and postsynaptic densities. Proc Natl Acad Sci USA 28. Park M, Penick EC, Edwards JG, Kauer JA, Ehlers MD (2004) Recycling endosomes supply 79:7590–7594. AMPA receptors for LTP. Science 305:1972–1975. 3. Yuste R, Bonhoeffer T (2001) Morphological changes in dendritic spines associated 29. Tanaka J, et al. (2008) Protein synthesis and neurotrophin-dependent structural plas- with long-term synaptic plasticity. Annu Rev Neurosci 24:1071–1089. ticity of single dendritic spines. Science 319:1683–1687. 4. Matsuzaki M, Honkura N, Ellis-Davies GC, Kasai H (2004) Structural basis of long-term 30. Fedulov V, et al. (2007) Evidence that long-term potentiation occurs within individual potentiation in single dendritic spines. Nature 429:761–766. hippocampal synapses during learning. J Neurosci 27:8031–8039. 5. Yang Y, Wang XB, Frerking M, Zhou Q (2008) Spine expansion and stabilization 31. Whitlock JR, Heynen AJ, Shuler MG, Bear MF (2006) Learning induces long-term associated with long-term potentiation. J Neurosci 28:5740–5751. 6. Kopec CD, Real E, Kessels HW, Malinow R (2007) GluR1 links structural and functional potentiation in the hippocampus. Science 313:1093–1097. plasticity at excitatory synapses. J Neurosci 27:13706–13718. 32. Honkura N, Matsuzaki M, Noguchi J, Ellis-Davies GC, Kasai H (2008) The subspine 7. Dityatev A, Schachner M (2003) Extracellular matrix molecules and synaptic plasticity. organization of actin fibers regulates the structure and plasticity of dendritic spines. Nature Rev 4:456–468. Neuron 57:719–729. 8. Sternlicht MD, Werb Z (2001) How matrix metalloproteinases regulate cell behavior. 33. Okamoto K, Nagai T, Miyawaki A, Hayashi Y (2004) Rapid and persistent modulation Annu Rev Cell Dev Biol 17:463–516. of actin dynamics regulates postsynaptic reorganization underlying bidirectional plas- NEUROSCIENCE 9. Ethell IM, Ethell DW (2007) Matrix metalloproteinases in brain development and ticity. Nat Neurosci 7:1104–1112. remodeling: Synaptic functions and targets. J Neurosci Res 85:2813–2823. 34. Lin B, et al. (2005) Theta stimulation polymerizes actin in dendritic spines of hippocam- 10. Nagy V, et al. (2006) Matrix metalloproteinase-9 is required for hippocampal late- pus. J Neurosci 25:2062–2069. phase long-term potentiation and memory. J Neurosci 26:1923–1934. 35. Lee HK, Barbarosie M, Kameyama K, Bear MF, Huganir RL (2000) Regulation of distinct 11. Bozdagi O, Nagy V, Kwei KT, Huntley GW (2007) In vivo roles for matrix metal- AMPA receptor phosphorylation sites during bidirectional synaptic plasticity. Nature loproteinase-9 in mature hippocampal synaptic physiology and plasticity. J Neuro- 405:955–959. physiol 98:334–344. 36. Szklarczyk A, Lapinska J, Rylski M, McKay RD, Kaczmarek L (2002) Matrix metal- 12. Okulski P, et al. (2007) TIMP-1 abolishes MMP-9-dependent long-lasting long-term loproteinase-9 undergoes expression and activation during dendritic remodeling in potentiation in the prefrontal cortex. Biol Psychol 62:359–362. adult hippocampus. J Neurosci 22:920–930. 13. Meighan PC, Meighan SE, Davis CJ, Wright JW, Harding JW (2007) Effects of matrix 37. Gu Z, et al. (2002) S-nitrosylation of matrix metalloproteinases: Signaling pathway to metalloproteinase inhibition on short- and long-term plasticity of Schaffer collateral/ neuronal cell death. Science 297:1186–1190. CA1 synapses. J Neurochem 102:2085–2096. 38. Dagnell C, et al. (2007) Effects of neurotrophins on human bronchial smooth muscle 14. Mainen ZF, Malinow R, Svoboda K (1999) Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature 399:151–155. cell migration and matrix metalloproteinase-9 secretion. Transl Res 150:303–310. 15. Zhou Q, Homma KJ, Poo MM (2004) Shrinkage of dendritic spines associated with 39. Schuman EM, Madison DV (1991) A requirement for the intercellular messenger nitric long-term depression of hippocampal synapses. Neuron 44:749–757. oxide in long-term potentiation. Science 254:1503–1506. 16. Kopec CD, Li B, Wei W, Boehm J, Malinow R (2006) Glutamate receptor exocytosis and 40. Barrionuevo G, Schottler F, Lynch G (1980) The effects of repetitive low frequency spine enlargement during chemically induced long-term potentiation. J Neurosci stimulation on control and ‘‘potentiated’’ synaptic responses in the hippocampus. Life 26:2000–2009. Sci 27:2385–2391. 17. Pinkstaff JK, Detterich J, Lynch G, Gall C (1999) Integrin subunit gene expression is 41. Staubli U, Chun D (1996) Factors regulating the reversibility of long-term potentiation. regionally differentiated in adult brain. J Neurosci 19:1541–1556. J Neurosci 16:853–860. 18. Chan CS, et al. (2006) Beta1-integrins are required for hippocampal AMPA receptor- 42. Staubli U, Chun D, Lynch G (1998) Time-dependent reversal of long-term potentiation dependent synaptic transmission, synaptic plasticity, and working memory. J Neurosci by an integrin antagonist. J Neurosci 18:3460–3469. 26:223–232. 43. Giannelli G, Falk-Marzillier J, Schiraldi O, Stetler-Stevenson WG, Quaranta V (1997) 19. Chan CS, Weeber EJ, Kurup S, Sweatt JD, Davis RL (2003) Integrin requirement for Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science hippocampal synaptic plasticity and spatial memory. J Neurosci 23:7107–7116. 277:225–228. 20. Kramar EA, Lin B, Rex CS, Gall CM, Lynch G (2006) Integrin-driven actin polymerization 44. Shi S, Hayashi Y, Esteban JA, Malinow R (2001) Subunit-specific rules governing AMPA consolidates long-term potentiation. Proc Natl Acad Sci USA 103:5579–5584. receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 105:331–343. 21. Wiesner S, Legate KR, Fassler R (2005) Integrin-actin interactions. Cell Mol Life Sci 45. Krucker T, Siggins GR, Halpain S (2000) Dynamic actin filaments are required for stable 62:1081–1099. long-term potentiation (LTP) in area CA1 of the hippocampus. Proc Natl Acad Sci USA 22. Carlier MF, Pantaloni D (1997) Control of actin dynamics in cell motility. J Mol Biol 97:6856–6861. 269:459–467. 23. Agnew BJ, Minamide LS, Bamburg JR (1995) Reactivation of phosphorylated actin 46. Cingolani LA, et al. (2008) Activity-dependent regulation of synaptic AMPA receptor depolymerizing factor and identification of the regulatory site. J Biol Chem 270:17582– composition and abundance by beta3 integrins. Neuron 58:749–762. 17587. 47. Nagy V, Bozdagi O, Huntley GW (2007) The extracellular protease matrix metal- 24. Chen LY, Rex CS, Casale MS, Gall CM, Lynch G (2007) Changes in synaptic morphology loproteinase-9 is activated by inhibitory avoidance learning and required for long- accompany actin signaling during LTP. J Neurosci 27:5363–5372. term memory. Learn Mem 14:655–664. 25. Fukazawa Y, et al. (2003) Hippocampal LTP is accompanied by enhanced F-actin 48. Meighan SE, et al. (2006) Effects of extracellular matrix-degrading proteases matrix content within the dendritic spine that is essential for late LTP maintenance in vivo. metalloproteinases 3 and 9 on spatial learning and synaptic plasticity. J Neurochem Neuron 38:447–460. 96:1227–1241.

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