A mechanism underlying AMPA receptor trafficking during ... - PNAS

A mechanism underlying AMPA receptor trafficking during cerebellar long-term potentiation Wataru Kakegawa and Michisuke Yuzaki* Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Communicated by Roger Y. Tsien, University of California at San Diego, La Jolla, CA, October 11, 2005 (received for review June 30, 2005)

Long-term potentiation (LTP) is mediated by the activity-driven delivery of GluR1 glutamate receptors via Ca2ⴙ兾calmodulin-dependent protein kinase II activity in various brain regions. Recently, postsynaptic LTP was shown to be induced at parallel fiber–Purkinje cell synapses by stimulating the parallel fibers at 1 Hz or applying a NO donor. Here, we demonstrate that NO-evoked postsynaptic LTP in mice cerebellum was blocked by botulinum toxin and enhanced by prior treatment with phorbol ester, which is known to induce GluR2 endocytosis. Interestingly, such LTP was not affected by a Ca2ⴙ兾calmodulin-dependent protein kinase II inhibitor or a peptide binding to a protein interacting with C kinase 1, but was blocked by a peptide binding to N-ethylmaleimidesensitive factor, which specifically binds to GluR2. Therefore, although the synaptic incorporation of GluR2 has been reported to be a constitutive pathway, NO-induced postsynaptic LTP in Purkinje cells is likely mediated by a pathway involving N-ethylmaleimide-sensitive factor-dependent GluR2 trafficking. cerebellum 兩 glutamate receptor 兩 long-term depression 兩 nitric oxide

he ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidtype glutamate receptors (AMPARs), which exist as heteromers consisting of GluR1 through GluR4, are crucial for longlasting changes in synaptic strength, such as long-term potentiation (LTP) and long-term depression (LTD). Several lines of evidence indicate that activity-dependent AMPAR trafficking, which determines the number of postsynaptic AMPARs, is one of the major mechanisms underlying LTP and LTD (1–3). LTD at parallel fiber (PF)–Purkinje cell synapses (PF-LTD) is thought to be the basis of certain forms of motor learning in the cerebellum (4). Like N-methyl-D-aspartate-type glutamate receptor (NMDAR)-dependent LTD in hippocampal neurons (5), GluR2-containing AMPARs are specifically removed from synapses via regulated endocytosis during cerebellar LTD (6, 7). Studies using brain-slice and cell-culture preparations have indicated that increases in intracellular Ca2⫹ levels and PKC activation in Purkinje cells are necessary for PF-LTD induction (8–10). In addition, although its role in cell-culture preparations is contentious, NO released from PF terminals (11) is essential for PF-LTD induction in slice preparations (12, 13). Indeed, genetic ablation of neuronal NO synthase impaired PF-LTD (14) and cerebellum-dependent motor learning in mice (15). Furthermore, subdural applications of NO blockers to the cerebellum abrogated motor learning related to smooth pursuit eye movements in monkeys (16). Unlike PF-LTD, which occurs exclusively at postsynaptic sites, LTP at PF–Purkinje cell synapses (PF-LTP), typically induced by PF stimuli at 4–8 Hz, occurs at presynaptic sites by an increase in glutamate release from presynaptic PF terminals (17–19). However, theoretical studies on motor learning in the cerebellum have indicated the necessity of postsynaptic PF-LTP (20). Recently, the stimulation of PFs alone at 1 Hz was found to induce a postsynaptic form of PF-LTP (1-Hz LTP) (21), which could reverse PF-LTD (22, 23). Interestingly, postsynaptic PFLTP could also be elicited by applying a NO donor without PF stimulation (21). In addition, 1-Hz LTP was completely blocked by an NO synthase inhibitor or NO scavenger (21), and NO-

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evoked LTP and 1-Hz LTP were mutually occlusive (22). Thus, the intracellular Ca2⫹ levels were thought to determine the direction of changes in synaptic plasticity; PF-LTD would occur in Purkinje cells with high Ca2⫹ levels, whereas PF-LTP would be induced under low intracellular Ca2⫹ conditions (23). However, the molecular mechanisms underlying such bidirectional control of postsynaptic plasticity are unclear. A general scheme that has emerged from various studies proposes that GluR1 is the key subunit responsible for driving AMPARs to the synapses in response to NMDAR stimulation and the activation of Ca2⫹兾 calmodulin-dependent protein kinase II (CaMKII) during postsynaptic LTP (24–26). In contrast, the GluR2 subunit is thought to be constitutively delivered to synapses, replacing existing synaptic AMPARs, independently of neuronal activity (25, 26). Although recent researches have revealed several intracellular molecules involved in AMPAR trafficking, it is still unclear whether such subunit-specific principles for AMPAR trafficking could account for the expression of different forms of synaptic plasticity that occur in various regions of the brain. In this study, we showed that postsynaptic LTP at PF–Purkinje cell synapses was caused by a mechanism, the Ca2⫹-dependent insertion of GluR2-containig AMPARs via a mechanism that depends on N-ethylmaleimide-sensitive factor (NSF), but not on protein interacting with C kinase 1 (PICK1) or CaMKII. Materials and Methods Electrophysiology. Coronal cerebellar slices (250 ␮m) were prepared from ICR young mice (postnatal day 14–21) by using a tissue slicer (DSK, Kyoto) in accordance with our institution’s guidelines. Whole-cell voltage-clamp recordings from visually identified Purkinje cells were performed with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), and the PCLAMP system (Version 9.2, Axon Instruments) was used for data acquisition and analysis. Patch pipettes were pulled from borosilicate glass capillaries to achieve a resistance of 3–5 M⍀ when filled with a solution containing 130 mM K-gluconate, 10 mM KCl, 10 mM Hepes, 1 mM MgCl2, 4 mM Na2ATP, 1 mM Na2GTP, and 16 mM sucrose. In most experiments, 5 mM 1,2-bis-(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid (BAPTA; Sigma) was also added to the intracellular solution to detect PF-LTP easily by inhibiting PF-LTD induction (21). In the experiments shown in Fig. 4A, 30 mM BAPTA was added, and the amount of K-gluconate was reduced accordingly to maintain the proper osmolarity. The external Ringer’s solution contained 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 26 mM Conflict of interest statement: No conflicts declared. Abbreviations: AC, adenylate cyclase; AIP, autocamtide-2 related inhibitory peptide; AMPAR, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor; BAPTA, 1,2-bis-(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid; BoTx, botulinum toxin; CaMKII, Ca2⫹兾calmodulin-dependent protein kinase II; CARP, Ca2⫹-permeable AMPAR plasticity; DEANO, diethylamine NO sodium salt; EPSC, excitatory postsynaptic current; LTD, long-term depression; LTP, long-term potentiation; NMDAR, N-methyl-Daspartate-type glutamate receptor; NSF, N-ethylmaleimide-sensitive factor; PF, parallel fiber; PICK1, protein interacting with C kinase 1; PPF, paired-pulse facilitation; SNARE, soluble NSF attachment protein receptor; TPA, 12-O-tetradecanoyl-phorbol-13-acetate. *To whom correspondence should be addressed. E-mail: [email protected]. © 2005 by The National Academy of Sciences of the USA

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NaHCO3, 1.25 mM NaH2PO4, 10 mM D-glucose, and 0.1 mM picrotoxin (to inhibit GABAergic synapses), bubbled with 95% O2 and 5% CO2 at room temperature. To evoke the excitatory postsynaptic current (EPSC) at PF–Purkinje cell synapses (PFEPSC), a stimulating electrode was placed in the molecular layer (pulse width, 10–30 ␮s; 40–120 ␮A), and the selective stimulation of PFs was confirmed by the paired-pulse facilitation (PPF) of PF-EPSCs with a 50-ms stimulation interval. For the quantal PF-EPSC recordings, Sr2⫹ was substituted for Ca2⫹ in the external solution to elicit the asynchronous release of neurotransmitter from presynaptic terminals (27), and the evoked asynchronous PF-EPSCs were analyzed by using a MINI ANALYSIS program (Synaptosoft, Decatur, GA). Each measurement was performed independently, and observed events ⬍10 pA were discarded. For the synaptic plasticity experiments, PF-EPSCs were evoked successively at a frequency of 0.1 Hz from Purkinje cells clamped at ⫺80 mV. After stable PF-EPSCs had been observed for at least 10 min, the external solution containing each drug that elicits chemical-induced plasticity was perfused. Series resistances were monitored every 10 s by measuring the peak currents in response to 2-mV, 50-ms hyperpolarizing steps throughout the experiments; the measurements were discarded if the resistance changed by ⬎20% of its original value. The normalized EPSC amplitude on the ordinate represents the EPSC amplitude for the average of six traces for 1 min divided by that of the average of six traces for 1 min immediately before drug application. The current traces were filtered at 1 kHz and digitized at 4 kHz for the evoked EPSCs and 10 kHz for the quantal EPSCs. Data Analysis and Statistics. Results are reported as the mean ⫾ SEM, and the statistical significance was defined at P ⬍ 0.05, as determined with the Mann–Whitney U test unless stated otherwise.

chased from Molecular Probes, 12-O-tetradecanoyl-phorbol-13acetate (TPA) and the synthesized peptides (pep-R845A [KAMKVAKNPQ], pep-K844A [ARMKVAKNPQ], pep-EVKI [KKEGYNVYGIEEVKI], and pep-SGKA [KKEGYNVYGIESGKA]) were purchased from Sigma, and botulinum toxin (BoTx) and autocamtide-2 related inhibitory peptide (AIP) were purchased from Calbiochem. Results Postsynaptic LTP at PF–Purkinje Cell Synapses in Mice. To charac-

terize the molecular mechanisms underlying postsynaptic PFLTP, we performed whole-cell patch-clamp recordings from Purkinje cells in cerebellar slices. We adopted a chemical protocol developed for rat cerebellar slices to induce postsynaptic PF-LTP (21, 22). Application of a potent NO donor DEANO (10 ␮M) to the mouse cerebellar slices induced significant LTP (136 ⫾ 6%, t ⫽ 25–30 min, n ⫽ 6; Fig. 1 A and B), with little changes in the kinetics (see Fig. 8, which is published as supporting information on the PNAS web site) and the PPF ratio (P ⫽ 0.705 by paired t test; Fig. 1 C and D) of the PF-EPSCs, a short-term plasticity that reflects changes in the probability of presynaptic transmitter release. These results indicate that the NO-induced PF-LTP was not presynaptically expressed. As reported for rat cerebellar slice preparations (22), prior potentiation by NO occluded 1-Hz LTP (data not shown), indicating a common signaling mechanism in mice cerebellum. When intracellular Ca2⫹ was chelated by the inclusion of 5-mM BAPTA in the internal saline, DEANO induced a significantly larger PF-LTP (170 ⫾ 12%, t ⫽ 25–30 min, n ⫽ 9; Fig. 1 A and B) than it did without BAPTA (P ⫽ 0.034), suggesting that NO may have elicited LTD at synapses where the postsynaptic Ca2⫹ levels were high, possibly reducing the level of the overall LTP Kakegawa and Yuzaki

Fig. 1. Cerebellar LTP is postsynaptically induced by NO in mice. (A) NOinduced PF-LTP recorded from Purkinje cells in the absence (E, n ⫽ 6) and the presence (F, n ⫽ 9) of 5-mM BAPTA in the patch pipette. EPSC amplitudes were normalized with those at time 0. (B) Histogram showing NO-induced potentiation under each condition. Potentiation was estimated as the percentage of normalized EPSC amplitudes averaged 25–30 min after DEANO application (solid bar in A). (C) Representative EPSC responses to the paired-pulse stimulation (50-ms interval) of PFs before (Pre) and 30 min after (Post) NO-induced LTP in the absence (Left) and presence (Right) of 5-mM BAPTA in the internal saline. (D) Plots showing the change in the PPF ratio before (Pre) and after (Post) LTP. The PPF ratio was defined as the ratio of the amplitude of the second EPSC to that of the first EPSC. The horizontal lines connect the pre and post data points for individual cells. (E) PF-evoked quantal PF-EPSC responses before (Pre; Upper) and after (Post; Lower) LTP. (Insets) Magnifications of the areas surrounded by the squares. (F) Cumulative probability plot showing the distribution of quantal PF-EPSC amplitude before (Pre; shaded line, n ⫽ 8) and after (Post; solid line, n ⫽ 9) LTP induction. Events with an amplitude of ⬎10 pA were counted during the time range of 700 ms between 70 and 770 ms after the synchronous PF-EPSC peak time, accumulated for 10 sweeps at 0.1 Hz.

(12, 13). Similarly, concentrations of Ca2⫹ chelater in a recording pipette are shown to determine the direction of changes in synaptic plasticity at PF–Purkinje cell synapses (23). In contrast to postsynaptic PF-LTD, relatively little data supporting the postsynaptic origin of PF-LTP are available, other than the PPF analysis mentioned above. Recently, the low-affinity competitive AMPAR antagonist ␥-D-glutamylglyPNAS 兩 December 6, 2005 兩 vol. 102 兩 no. 49 兩 17847

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Materials. Diethylamine NO sodium salt (DEANO) was pur-

Fig. 2. Suppression of PF-LTP by postsynaptic perfusion of BoTx. (A) Averaged data of normalized EPSC amplitude showing the effect of intact (E, n ⫽ 8) and heat-inactivated (F, n ⫽ 10) BoTx (100 nM) in the internal solution. EPSC amplitudes were normalized with those at time 0. (B) Histogram showing potentiation averaged at 25–30 min after DEANO application in the presence of intact (⫹) and heat-inactivated (inact.) BoTx.

cine was shown to inhibit PF-EPSC after PF-LTP as effectively as it did before PF-LTP (23), indicating that the amount of glutamate released from PFs did not change after the induction of PF-LTP. However, whether the PF terminals could release multiple synaptic vesicles per action potential, a prerequisite of analyses with low-affinity antagonists (28), was uncertain. Thus, to further confirm that NO-induced PF-LTP was postsynaptic in origin, we replaced extracellular Ca2⫹ with Sr2⫹, leading to asynchronous transmitter release (27), and analyzed PF-induced quantal EPSCs (Fig. 1E). After NO-induced PF-LTP, the amplitude of the quantal EPSCs from the stimulated PF terminals was significantly increased (P ⫽ 0.025; Fig. 1F), providing complementary evidence that the NO-induced PF-LTP was postsynaptic in origin. NO-Induced Cerebellar LTP Is Mediated by Soluble NSF Attachment Protein Receptor (SNARE)-Dependent Exocytosis. To fulfill its role as

an efficient mechanism for reversing postsynaptically induced PF-LTD, which is induced by AMPAR endocytosis, postsynaptic PF-LTP should be accompanied by an increase in the number of postsynaptic AMPARs. To examine this hypothesis, we added 100 nM BoTx (light chain), which digests the SNARE complex necessary for exocytosis, to the internal saline. BoTx induced a slight, but not statistically significant, reduction in the PF-EPSC amplitude (87 ⫾ 4% at 40 min, n ⫽ 8; P ⫽ 0.131), consistent with earlier reports that SNARE-dependent exocytosis is involved in maintaining the synaptic content of AMPARs (29–32). In addition, after stabilization of the PF-EPSC amplitudes, the application of DEANO to Purkinje cells loaded with BoTx significantly reduced PF-LTP (113 ⫾ 7%, t ⫽ 25–30 min, n ⫽ 8, P ⫽ 0.010; Fig. 2), whereas normal PF-LTP occurred in Purkinje cells loaded with heat-inactivated BoTx (157 ⫾ 9%, t ⫽ 25–30 min, n ⫽ 10; Fig. 2). These results indicated that the SNAREdependent exocytosis of AMPARs is necessary not only for the maintenance of constitutive neurotransmission, but also for NO-induced LTP at PF–Purkinje cell synapses. In addition, the effect of BoTx further supports the postsynaptic origin of NO-induced PF-LTP. PKC activation by phorbol ester has been shown to induce the endocytosis of AMPARs and PF-LTD in Purkinje cells (6, 9, 10). Thus, if NO-induced PF-LTP was caused by the exocytosis of AMPARs, the prior induction of PF-LTD by phorbol ester should enhance subsequent NO-induced PF-LTP. Because it was technically very difficult to perform whole-cell recordings long enough to saturate one form of plasticity and then evoke its counterpart (22), we preincubated cerebellar slices with a phorbol ester TPA (1 ␮M) before the recordings. As expected, DEANO induced significantly larger LTP in the cerebellar slices 17848 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0508910102

Fig. 3. Effect of phorbol ester treatment on NO-induced PF-LTP. After treatment with TPA (1 ␮M) for 10 min or DEANO (10 ␮M) for 5 min, each slice was incubated in the normal external solution for 1 h, and then the recording was performed. (A) DEANO-induced PF-LTP without (E; see Fig. 1 A) and with (F, n ⫽ 8) TPA pretreatment. (B) Histogram showing potentiation averaged at 25–30 min after DEANO application. (C) TPA-induced PF-LTD in the absence (E, n ⫽ 8) and the presence (F, n ⫽ 10) of DEANO pretreatment. (D) Histogram showing depression averaged at 25–30 min after TPA application. For the recordings of TPA-induced PF-LTD, 5-mM BAPTA was removed from the internal saline.

pretreated with TPA (211 ⫾ 15%, t ⫽ 25–30 min, n ⫽ 8, P ⫽ 0.048; Fig. 3 A and B). Conversely, treatment with DEANO enhanced subsequent TPA-induced PF-LTD (65 ⫾ 4%, t ⫽ 25–30 min, n ⫽ 10; Fig. 3 C and D), although the effect did not reach the level of statistical significance (without DEANO preincubation: 74 ⫾ 3%, t ⫽ 25–30 min, n ⫽ 8; P ⫽ 0.183). This lack of significance was probably because DEANO’s effect was much smaller when the intracellular Ca2⫹ was not chelated (Fig. 1B). These results support the view that NO-induced PF-LTP is caused by the exocytosis of AMPARs, which could reverse PF-LTD mediated by AMPAR endocytosis at PF–Purkinje cell postsynapses. Cerebellar LTP Depends on Postsynaptic Ca2ⴙ but Not CaMKII Activation. NMDAR-dependent LTP in the adult hippocampus is

mediated by the insertion of GluR1-dominant receptors into activated synapses, a process triggered by an increase in postsynaptic Ca2⫹ and the subsequent activation of CaMKII (24, 25). Therefore, we next examined whether AMPAR insertion during cerebellar LTP also depended on postsynaptic Ca2⫹ and CaMKII. Although adding 5-mM BAPTA to the internal saline enhanced PF-LTP (Fig. 1B), 30-mM BAPTA completely blocked NO-induced PF-LTP (100 ⫾ 3%, t ⫽ 25–30 min, n ⫽ 7; Fig. 4A). Similarly, high BAPTA concentrations ranging from 30 to 40 mM were necessary to block a Ca2⫹-dependent form of short-term plasticity (33) and 1-Hz LTP in rat Purkinje cells (23). These results support the hypothesis that the induction cascades of both PF-LTD and PF-LTP depend on an increase in postsynaptic Ca2⫹ but that the PF-LTP induction threshold is lower than that for PF-LTD (23). The inclusion of 5 mM BAPTA without the addition of Ca2⫹ in the internal saline was expected to result in a subnanomolar Ca2⫹ concentration far below the range (0.7–4 ␮M) necessary for CaMKII activation (34). Thus, the NO-induced PF-LTP Kakegawa and Yuzaki

probably did not involve the activity of CaMKII. However, because the dependency of CaMKII activity on Ca2⫹ could be modulated by its autophosphorylation levels, we further examined the contribution of CaMKII to NO-induced PF-LTP by adding a potent CaMKII blocker AIP to the internal saline. We confirmed the effectiveness of 1-␮M AIP by examining its blocking effect on the rebound potentiation of miniature inhibitory postsynaptic current in Purkinje cells (see Fig. 9, which is published as supporting information on the PNAS web site), a phenomenon that depends on CaMKII activation (35, 36). AIP (1 ␮M) did not affect the basal PF-EPSC amplitude (96 ⫾ 9%, t ⫽ 40 min, n ⫽ 5; Fig. 4B) or NO-induced PF-LTP (157 ⫾ 13%, t ⫽ 25–30 min, n ⫽ 5; Fig. 4B). Therefore, unlike LTP in the hippocampal CA1 regions, the postsynaptic LTP at the PF– Purkinje cells was not mediated by CaMKII-dependent pathways. Cerebellar LTP Involves a Postsynaptic NSF–GluR2 Interaction. If

PF-LTP does not rely on the CaMKII–GluR1 pathway, how is AMPAR exocytosis mediated at PF–Purkinje cell synapses? Purkinje cells abundantly express the GluR2 and GluR3 subunits of AMPARs (37), and the specific interaction of GluR2 with NSF has been shown to be necessary for maintaining basal neurotransmission (29–32) and cerebellar PF-LTD (38). Thus, we examined whether NO-induced cerebellar LTP required NSF by using a decoy peptide (pep-R845A) that interfered with the GluR2–NSF interaction (32). The inclusion of pep-R845A in a patch pipette induced a gradient decrease in the PF-EPSC amplitude over 20 min (73 ⫾ 6% at 30 min; Fig. 5 A and B Left). In contrast, a control peptide pep-K844A did not induce a similar rundown of the EPSC amplitude (101 ⫾ 2% at 30 min, P ⫽ 0.007; Fig. 5 A and B Left), confirming pep-R845A’s specific effect on basal neurotransmission. Surprisingly, pep-R845A significantly reduced subsequent NO-induced PF-LTP (118 ⫾ 6%, t ⫽ 55–60 min, n ⫽ 6; Fig. 5 A and B Right), compared with the robust PF-LTP in Purkinje cells perfused with a control peptide (152 ⫾ 10%, t ⫽ 55–60 min, n ⫽ 6, P ⫽ 0.025; Fig. 5 A and B Right). These results suggest that, unlike LTP in other regions of the brain, PF-LTP was mediated by the NSFdependent insertion of GluR2-containing AMPARs into PF– Purkinje cell synapses. Recently, the activity-dependent lateral diffusion of GluR2containing AMPARs into synapses was reported to underlie Ca2⫹-permeable AMPAR plasticity (CARP) at PF-stellate cell postsynapses via a pathway involving both NSF and PICK1 (39, 40). Unlike LTP at PF–Purkinje cell synapses, the incorporation of GluR2 into PF-stellate cell synapses reduced the EPSC amplitudes; however, to further examine whether PF-LTP shared a common mechanism with this phenomenon, we used a decoy peptide (pep-EVKI) that specifically interfered with the Kakegawa and Yuzaki

Fig. 5. Suppression of NO-induced PF-LTP by the NSF-binding peptide. (A) The data of NO-induced PF-LTP recorded from Purkinje cells loaded with the NSF-binding peptide (pep-R845A; 100 ␮M; E, n ⫽ 6) and the control peptide (pep-K844A; 100 ␮M; F, n ⫽ 6) in the patch pipette. Because the rundown of PF-EPSC continued for up to 20 min, DEANO was applied at 30 min once the EPSC amplitudes became stable. (B) Histograms showing the PF-EPSC rundown at 30 min after the recording (Left) and the potentiation averaged at 25–30 min after DEANO treatment (Right) under each condition. To measure potentiation, the baseline was set to the averaged value of six traces immediately before drug application.

GluR2–PICK1 interaction (7). As previously reported (7), loading Purkinje cells with pep-EVKI specifically blocked the induction of PF-LTD (data not shown); however, it failed to block NO-induced PF-LTP (176 ⫾ 21%, t ⫽ 25–30 min, n ⫽ 5; Fig. 6 vs. 166 ⫾ 11%, t ⫽ 25–30 min, n ⫽ 5 in control peptide pep-SGKA; Fig. 6; P ⫽ 0.602). Therefore, the molecular mechanisms responsible for NO-induced PF-LTP are likely distinct from those underlying CARP at PF-stellate cell synapses. Discussion Postsynaptic LTP Mediated by NSF-Dependent GluR2 Exocytosis. A postsynaptic form of LTP at PF–Purkinje cell synapses has been postulated for a long time because it would endow these synapses with a true bidirectional computational capability relevant to the extinction phase of motor learning. Such postsynaptic PF-LTP, induced by either 1-Hz PF stimulation or NO donor application, has been recently discovered (22, 23); however, its underlying molecular mechanisms remain uncharacterized. In this study, we have provided complementary evidence that NO-induced PF-LTP is indeed postsynaptically expressed by demonstrating that the amplitudes of PF-evoked quantal EPSCs are increased after NO application (Fig. 1). Because NO-induced PF-LTP was blocked by BoTx (Fig. 2) and enhanced by prior treatment with phorbol ester, which induced PF-LTD associated with AMPAR endocytosis (Fig. 3), NO-induced PF-LTP likely involves the exocytosis of AMPARs. Thus, we propose that this form of LTP is a reverse

Fig. 6. Insensitivity of NO-induced PF-LTP to the PICK1 binding peptide. (A) Summarized data showing NO-induced PF-LTP from Purkinje cells loaded with the PICK1-binding peptide (pep-EVKI; 100 ␮M; F, n ⫽ 5) and the control peptide (pep-SGKA; 100 ␮M; E, n ⫽ 5) in the pipette saline. (B) Histograms showing potentiation averaged at 25–30 min after DEANO treatment. PNAS 兩 December 6, 2005 兩 vol. 102 兩 no. 49 兩 17849

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Fig. 4. Cerebellar LTP depends on postsynaptic Ca2⫹ but not CaMKII activation. (A) Averaged data of NO-induced PF-LTP recordings in the presence of 30-mM BAPTA in the pipette saline (n ⫽ 7). (B) Averaged data (F, n ⫽ 5) of NO-induced PF-LTP recorded in the presence of a CaMKII blocker AIP (1 ␮M) in the internal saline. AIP had little effect on the basal PF-EPSC amplitude during the recordings (E, n ⫽ 5).

because PICK1–GluR2 complex blocks the lateral diffusion of GluR2 into synapses. Therefore, although the lateral diffusion of GluR2 could also be involved in NO-induced LTP at PF– Purkinje cell synapses, the molecular mechanisms responsible for this phenomenon are likely to be distinct from those for CARP at PF-stellate cell synapses.

Fig. 7. A model showing the mechanisms underlying bidirectional synaptic plasticity at PF–Purkinje cell synapses. NO released from stimulated PF terminals induces LTD via a pathway dependent on PICK1 and GRIP, when Purkinje cells are depolarized and intracellular Ca2⫹ levels are high, as reported (12, 13). When intracellular Ca2⫹ levels are low, NO induces LTP via a pathway involving a GluR2–NSF interaction and exocytosis sensitive to BoTx, but insensitive to PICK1 and CaMKII.

process of PF-LTD, not only at the functional level (22), but also at the molecular level, resetting the PF-LTD-induced endocytosis of AMPARs. NO-evoked PF-LTP was severely blocked by pep-R845A (Fig. 5), which specifically inhibits the interaction of GluR2 with NSF but not with AP2 clathrin adapter protein (32). NSF has been shown to play crucial roles in maintaining the synaptic content of GluR2-containing AMPARs, possibly by controlling the SNARE-dependent exocytosis of GluR2 (41) or promoting the lateral diffusion of GluR2 through the disruption of the PICK1– GluR2 complex (38, 42, 43). Indeed, pep-R845A reduced basal PF–Purkinje cell neurotransmission (Fig. 5). However, NSF activities are not required for activity-dependent AMPAR trafficking during LTP in the hippocampus (26). Therefore, we propose the existence of an NSF-dependent mechanism through which GluR2 is inserted into PF–Purkinje cell synapses during LTP in response to NO production or 1-Hz PF stimulation (Fig. 7). The synaptic incorporation of GluR2 has been reported to involve an unregulated, constitutive pool of receptors (26). However, the activity-induced and NSF-dependent lateral diffusion of GluR2 into PF-stellate cell synapses during CARP has been recently reported (39, 40). Similarly, it is possible that during NO-induced PF-LTP, GluR2 may be first inserted into extrasynaptic regions via BoTx-sensitive exocytosis, and then laterally diffused into the synapses via an NSF-dependent pathway. However, CARP occurs at synapses expressing Ca2⫹permeable, GluR2-lacking AMPARs, whereas PF–Purkinje cell synapses express Ca 2⫹ -impermeable, GluR2-containing AMPARs (44). In addition, unlike PF-LTP (Fig. 1), the induction of CARP requires the influx of Ca2⫹ into stellate cells (45). Furthermore, unlike LTP at PF–Purkinje cell synapses (Fig. 6), CARP depends on an interaction with PICK1 (39, 40), probably 17850 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0508910102

CaMKII-Independent AMPAR Trafficking. NO-evoked PF-LTP was enhanced in the presence of 5-mM BAPTA (Fig. 1) and was independent of CaMKII activities (Fig. 4). Thus, this process likely does not involve a CaMKII-dependent GluR1 trafficking mechanism, which was originally identified in the adult hippocampal CA1 synapses and accounts for synaptic plasticity in various regions of the brain, including the visual cortex (46), somatosensory cortex (47), and spinal cord (48). Recently, several forms of synaptic plasticity mediated by CaMKIIindependent AMPAR trafficking have been reported. For example, in the hippocampal CA1 neurons or the barrel cortex of developing animals, LTP is independent of CaMKII activities (49, 50); instead, protein kinase A (PKA), which is activated by a Ca2⫹兾calmodulin-dependent adenylate cyclase (AC), is thought to control the trafficking of AMPARs that contain a long C terminus, such as GluR1 and long splicing variants of GluR2 and GluR4 (50 –52). In contrast to LTP mediated by the AC–PKA pathway, postsynaptic LTP at PF–Purkinje cell synapses was induced in the presence of 5-mM (Fig. 1) or 20-mM BAPTA (23). In addition, AMPARs containing a long C terminus would not be inhibited by pep-R845A because they do not possess an NSF-binding motif (2, 3). Furthermore, 1-Hz LTP was induced in an AC-null cerebellum (21). Therefore, postsynaptic LTP at PF–Purkinje cell synapses is unlikely to be mediated by the AC–PKA pathway. The AC-dependent AMPAR trafficking mechanism seems to be operational in immature synapses where the expression levels of the key enzyme ␣CaMKII are low. In contrast, ␣CaMKII is highly expressed in Purkinje cells from the early postnatal days and throughout adulthood (53). In addition, although the absolute expression levels of GluR1 proteins are controversial, GluR1 is expressed at least at certain levels in Purkinje cells (54). Thus, it is unclear why the CaMKIImediated GluR1 trafficking mechanism was not operational at PF–Purkinje cell synapses. According to the ‘‘slot’’ theory (55), NMDARs are essential for recruiting and binding to CaMKII in activated synapses and provide a slot necessary for GluR1 insertion. Because mature Purkinje cells do not express functional NMDARs (33, 56 – 60), this fact may explain why the CaMKII–GluR1 trafficking mechanism was not operational at PF–Purkinje cell synapses. Indeed, in hippocampal CA3 pyramidal neurons, CaMKII-dependent GluR1 insertion does not occur at mossy fiber synapses that express low levels of functional NMDARs, whereas it does occurs at associational fiber synapses that express NMDARs (61). Similarly, most motor neurons in the brainstem and spinal cord lose the expression of NR2 subunits during postnatal development and thus express low levels of functional NMDAR in adults (62). Therefore, although the CaMKII–GluR1 trafficking mechanism is generally used at most synapses, it is possible that other mechanisms, including the CaMKII-independent and NSFdependent GluR2 trafficking mechanism described here, may be present in certain brain regions. Functions of NO at Postsynaptic Sites. The molecular mechanisms

underlying NO-induced PF-LTP remain to be explored. NO is involved in a variety of learning-related forms of synaptic plasticity, including structural changes at synapses. The most common signaling pathway for NO is retrograde signaling by NO, which activates guanylate cyclase and cGMP-dependent Kakegawa and Yuzaki

protein kinase at the presynaptic terminals and modifies neurotransmitter release (63, 64). In addition, the NO-cGMP pathway has been suggested to control the postsynaptic clustering of GluR1 in cultured hippocampal neurons (65). Unlike these phenomena, 1-Hz or NO-induced postsynaptic LTP in Purkinje cells is completely independent of the NO-cGMP pathway (21). Recently, S-nitrosylation, a process that covalently attaches an NO moiety to cysteines on target proteins, has emerged as a prevalent signaling mechanism (66). For example, because the activation of small G protein Ras plays a crucial role in activity-dependent AMPAR trafficking in hippocampal CA1 neurons (67), and because Ras-Snitrosylation was confirmed to be induced by NO, small G proteins could be a downstream target of NO responsible for postsynaptic PF-LTP. Similarly, NSF was recently shown to be

We thank J. Motohashi and N. Kakiya for technical assistance. This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (to W.K. and M.Y.), the Takeda Science Foundation (W.K.), the Narishige Neuroscience Research Foundation (W.K.), a Toray Science and Technology Grant (to M.Y.), and a Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects (to M.Y.).

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directly S-nitrosylated and promoted the surface expression of GluR2 in cultured cortical neurons (68). Thus, although physiological functions of S-nitrosylated NSF during synaptic plasticity in various brain regions remain to be determined, this phenomenon could account for NO-induced postsynaptic LTP in the cerebellum. Further studies are warranted to understand the mechanisms underlying the various forms of plasticity controlled by NO at postsynaptic sites.