Activation of extrasynaptic NMDA receptors induces LTD in rat hippocampal CA1 neurons

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Review

Activation of extrasynaptic NMDA receptors induces LTD in rat hippocampal CA1 neurons Dan-dan Liu a , Qian Yang a,b , Sheng-tian Li a,∗ a b

Bio-X Institute, Shanghai Jiao Tong University, 800 Dongchuan Road, 200240 Shanghai, PR China Department of Cell Biology and Anatomy, the LSU Health Science Center, New Orleans, LA 70112, USA

a r t i c l e

i n f o

Article history: Received 8 September 2012 Received in revised form 12 December 2012 Accepted 13 December 2012 Available online xxx Keywords: Extrasynaptic NMDA receptor Long-term depression Theta burst MK-801 Hippocampus

a b s t r a c t In the adult rat hippocampus, activation of N-methyl-d-aspartate receptors (NMDARs) is required for the induction of certain forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Several studies have indicated the opposing role of synaptic NMDARS (S-NMDARs) versus extrasynaptic NMDARs (ES-NMDARs) in CREB-dependent gene regulation and neuronal survival/death. The contribution of ES-NMDARs in synaptic plasticity, however, remains unclear. Here we investigated the contribution of ES-NMDARs on LTD induction in CA1 neurons of rat hippocampal slices. ES-NMDARs were selectively activated by theta burst stimulation (TBS) after selective blockade of SNMDARs with pairing of 5 Hz stimulation and MK-801, an irreversible use-dependent antagonist of NMDARs. Application of TBS in naïve slices evoked a transient potentiation. In contrast, the activation of ES-NMDARs evoked a robust LTD. These results suggest the involvement of ES-NMDARs in LTD induction. © 2012 Elsevier Inc. All rights reserved.

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Slice preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Electrophysiological recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. ES-NMDAR-evoked induction of LTD in hippocampal CA1 neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Presynaptic mechanisms were not involved in ES-NMDAR-evoked LTD induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Long-term depression (LTD), as well as long-term potentiation (LTP), is a persistent activity-dependent change in synaptic efficacy which is considered to be an important mechanism for information storage in the brain. In hippocampal CA1 pyramidal neurons, Nmethyl-d-aspartate receptor (NMDAR)-mediated Ca2+ influx plays

∗ Corresponding author. Tel.: +86 021 34207976 801; fax: +86 021 34207976 801. E-mail address: [email protected] (S.-t. Li).

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a key role in the induction of both LTP (Herron et al., 1986; Morris et al., 1986) and LTD (Dudek and Bear, 1992; Mulkey and Malenka, 1992). On the other hand, the distribution of NMDARs in different neuronal dendritic locations, including the synaptic area (S-NMDARs) and extrasynaptic sites (ES-NMDARs) have been found and extensively studied through blockage of S-NMDARs by using MK-801, an irreversible use-dependent antagonist of NMDARs, and low frequency stimulation during the last decade (Hessler et al., 1993; Weisskopf and Nicoll, 1995; Huang and Stevens, 1997; Chavis and Westbrook, 2001; Hardingham et al., 2002; Tovar and Westbrook, 2002; Harris and Pettit, 2008; Groc et al.,

0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2012.12.003

Please cite this article in press as: Liu, D.-d., et al., Activation of extrasynaptic NMDA receptors induces LTD in rat hippocampal CA1 neurons. Brain Res. Bull. (2013), http://dx.doi.org/10.1016/j.brainresbull.2012.12.003

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2009; Speed and Dobrunz, 2009; Wang et al., 2011). Studies have emphasized the distinct functions of S-NMDARs and ES-NMDARs. Although it has been reported that either activation of S- or ESNMDARs was equally capable of excitotoxicity (Sattler et al., 2000; Wroge et al., 2012), the accumulating evidence suggests that ESNMDARs play an important role in triggering excitotoxic neuronal damage. Hardingham et al. showed in cultured neurons that activation of ES-NMDARs triggers cAMP-responsive element-binding protein (CREB) shut-off and the cell death pathway, while calcium influx through S-NMDARs induces CREB activity-dependent brain derived neurotrophic factor (BDNF) gene expression. Studies of GeneChip DNA microarray analyses showed that activation of NMDARs regulates different transcriptional responses dependent on their synaptic or extrasynaptic location (Medina, 2007). In addition, studies have implied that ES-NMDARs contribute to the induction of LTD rather than LTP: bath application of NMDA, which causes simultaneous activation of both S- and ES-NMDARs, can either attenuate LTP (Kato et al., 1999) or promote LTD induction (Lee et al., 1998; Kamal et al., 1999; Massey et al., 2004; Yang et al., 2005; Kollen et al., 2008) at different concentrations. The role of ES-NMDARs in induction of LTD by synaptic released glutamate, however, has not yet been established directly. In the current study, we examined whether selective activation of ES-NMDARs induces LTD in juvenile rat hippocampal CA1 pyramidal neurons.

5 Hz control

All experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Experimentation Committee of Shanghai Jiaotong University. A total of 16 healthy juvenile male Sprague-Dawley rats were provided by the Shanghai Laboratory Animal Center, Chinese Academy Sciences (application no. SYXK (Hu) 2007-0005). 2.2. Slice preparation Hippocampal slices were prepared as described previously (Yang et al., 2010) from male Sprague-Dawley rats (4–5 week old). After being deeply anaesthetized with halothane, the brain was removed immediately and placed in an ice-cold artificial cerebrospinal fluid (ACSF) solution containing the following (mM): 119 NaCl, 2.5 KCl, 1.3 MgSO4 , 2.5 CaCl2 , 1.0 NaH2 PO4 , 26.2 NaHCO3 , and 11 glucose. ACSF was continuously bubbled with 95%O2 and 5%CO2 . Transverse hippocampal slices (400 ␮m) were cut at 0–4 ◦ C using a vibratome tissue slicer (Vibratome, St. Louis, MO). 2.3. Electrophysiological recording Field EPSP recordings were performed as described previously (Lin et al., 2011). In all experiments, picrotoxin (100 ␮M, GABAA receptor antagonist) (Sigma–Aldrich) was included in the ACSF, and a cut was made to separate the CA3 region from the CA1 region to avoid epileptiform activity. The amplitudes of the field excitatory postsynaptic potentials (EPSPs) were calculated as the initial slope of the EPSP. Similar to the previous reports, we applied a short train of LFS (5 Hz/16 s) in the presence of MK-801 (20 ␮M, pretreated for 20 min) (Tocris Cookson Bristol) to selectively block synaptic NMDARs (Chen and Diamond, 2002; Clark and Cull-Candy, 2002; Hardingham et al., 2002; Tovar and Westbrook, 2002; Lozovaya et al., 2004; Scimemi et al., 2004; Harris and Pettit, 2008). After complete wash out of MK-801 for 30 min (Harris and Pettit, 2007), ES-NMDARs were activated by stimulating the presynaptic inputs with a theta-burst (TBS): a total of 180 trains were delivered at 1 Hz, each train contained 5 pulses at 100 Hz LTD values were calculated as the ratio of averaged response 50–60 min after the induction and that 20 min before the induction. Paired-pulse stimulations were given with a 200 ms interstimulus interval (ISI) with equal strength. A Multiclamp 700B amplifier (Molecular Devices) was used in all experiments, and the data were stored on a personal computer and analyzed (filtered at 3 kHz, sampled at 10 kHz) using PClamp 10 (Molecular Devices). 2.4. Statistical analysis Data were expressed as mean ± SEM. The statistical significance was determined using the One-way ANOVAs for three groups’ comparison or ANOVAs for repeated measured data, or paired t-test for fiber volley and PPF measurements that tested before and after the induction of LTD. A P value <0.05 refers to the existing differences statistically.

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Fig. 1. Selective blockade of S-NMDARs during field potential recordings. Field EPSPs of pyramidal cells-mediated Schaffer collateral-commissural fiber activation were recorded. Upper, sample traces evoked by 5 Hz stimulation delivered for 16 s with or without MK-801. The amplitude of NMDA-EPSPs was calculated as the average amplitude of 30–32 ms from the onset of stimuli. Box ‘a’ is the part of the trace 25–35 ms from the onset of stimuli; while box ‘b’ is the enlarged box ‘a’. Lower, the amplitude of NMDA-EPSPs evoked by the first stimulation was compared with that of the 80th stimulation in control slices (left) and MK-801 treated slices (right). Note that the amplitude of NMDA-EPSPs was increased in control but not in MK-801 treated slices.

3. Results 3.1. ES-NMDAR-evoked induction of LTD in hippocampal CA1 neurons It has been demonstrated that ES-NMDARs could be activated selectively in cultured neurons by using MK-801 (Hardingham et al., 2002; Tovar and Westbrook, 2002). Since the short train of 5 Hz stimulation activates S- but few ES-NMDARs (Harris and Pettit, 2008), the co-application of MK-801 with the 5 Hz stimulation could irreversibly block S-NMDARs. Using acute slice preparations we firstly tested whether the combination of MK-801 with 5 Hz stimulation (for 16 s) could block synaptic NMDA-EPSPs measured by field potential recordings. Since the AMPA receptor-dependent component of field EPSPs almost decayed to the baseline level within 30 ms, the amplitude of the NMDAR-dependent component of field EPSPs (NMDA-EPSP) was measured as the mean amplitude of 30–32 ms after the onset of the field EPSP (Fig. 1). Our results showed that the amplitude of NMDA-EPSPs was not altered by applying the 5 Hz stimulation in the presence of MK801 (Fig. 1, right, the first NMDA-EPSP, 40.6 ± 9.5 ␮V; the last (80th) NMDA-EPSP, 45.4 ± 14.5 ␮V; n = 6, paired t-test, p = 0.608), although applying 5 Hz stimulation alone in the absence of MK801 enhanced it (Fig. 1, left, the first NMDA-EPSP, 53.6 ± 7.5 ␮V; the last (80th) NMDA-EPSP, 69.6 ± 9.6 ␮V; n = 9, paired t-test, p = 0.004). These results demonstrate that applying the extrasynaptic procedure blocks S-NMDARs. We washed out MK-801 with normal ACSF for 30 min, and then applied TBS to selectively activate ES-NMDARs that were not blocked by MK-801 (extrasynaptic procedure). Using this extrasynaptic procedure, we investigated whether activation

Please cite this article in press as: Liu, D.-d., et al., Activation of extrasynaptic NMDA receptors induces LTD in rat hippocampal CA1 neurons. Brain Res. Bull. (2013), http://dx.doi.org/10.1016/j.brainresbull.2012.12.003

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Fig. 2. Selective activation of ES-NMDAR induced LTD. Double arrows represent TBS. (A) Selective activation of ES-NMDARs by the extrasynaptic procedure induced a gradual decrease of field EPSPs and resulted in LTD. (B) In naïve control slices, applying TBS induced short-term potentiation but not LTD. (C) In slices pretreated with MK-801 alone (without applying the 5 Hz stimulation), the same TBS failed to induce LTD. Insets, averaged sample traces recorded at 0–20 min before applying TBS (black traces) and at 50–60 min after applying TBS (red traces). (D) Summary histogram of LTD induction. *P < 0.05.

of ES-NMDARs induces LTD. As shown in Fig. 2A, selective activation of ES-NMDARs induced LTD (extrasynaptic procedure, 80.1 ± 0.4%, n = 10 slices from 10 rats), although applying the same TBS in naïve control slices only induced a short-term potentiation (Fig. 2B, 104.5 ± 0.3%, n = 8 slices from 8 rats; P < 0.05, compared with the extrasynaptic procedure). In contrast, applying MK-801 alone (but with no pairing of the 5 Hz stimulation) did not induce LTD (Fig. 2C, 102.4 ± 3.4%, n = 5 slices from 4 rats; P < 0.05, compared with the extrasynaptic procedure). The summary of LTD induction is shown in Fig. 1D (One-way ANOVAs, F(2,20), P < 0.05). 3.2. Presynaptic mechanisms were not involved in ES-NMDAR-evoked LTD induction The average amplitude of fiber volley 20 min before and 50–60 min after applying TBS was calculated and compared within all naïve control, MK-801 alone- and extrasynaptic proceduretreated groups. As shown in Fig. 3, neither of the groups showed changes in amplitude of fiber volley after applying TBS (Fig. 3, extrasynaptic procedure, before: 0.056 ± 0.009 mV; after: 0.051 ± 0.010 mV; n = 10 slices of 10 rats; naïve control, before: 0.059 ± 0.017 mV, after: 0.060 ± 0.017 mV; n = 8 slices of 8 rats; MK801 alone, before: 0.045 ± 0.013 mV, after: 0.049 ± 0.013 mV; n = 5 slices from 4 rats). Meanwhile, we also examined the presynaptic mechanisms through PPF ratio compared between applying TBS before and 60 min after in all three groups. PPF reflected an increase in presynaptic release probability that occurred when a synapse had experienced a previous stimulation event; the paired pulse ratio was a sensitive detector of changes in

the probability of neurotransmitter release (Zucker and Regehr, 2002). The results showed that after delivering the TBS, the PPF ratio was not changed compared to before TBS delivery in three groups (Fig. 4, naïve control, before: 1.09 ± 0.046, after: 1.176 ± 0.029, paired t-test, p = 0.319, n = 3 slices of 3 rats; MK801 alone, before: 1.244 ± 0.0567, after: 1.37 ± 0.069, paired t-test, p = 0.23, n = 3 slices of 3 rats; extrasynaptic procedure, before: 1.08 ± 0.028, after: 1.134 ± 0.072, paired t-test, p = 0.532, n = 3 slices of 3 rats). In order to further investigate the possible involvement of presynaptic mechanisms in ES-NMDAR-evoked LTD, we next calculated the summation ratio of EPSP amplitude evoked by the first train (5 pulses, 100 Hz) of TBS stimulation. The ratio of peak amplitude response to the 5th pulse to that the 1st pulse was calculated. As shown in Fig. 5A, we found no significant differences in summation ratio among naïve control, MK-801 alone-, and extrasynaptic procedure-treated slices (naïve control, 2.06 ± 0.23; MK-801 alone, 1.67 ± 0.3218; extrasynaptic procedure, 2.37 ± 0.33. One-way ANOVAs, F(2,20), P = 0.61). On the other hand, we also investigated the change in EPSP amplitude during whole TBS (Fig. 5B and C). The amplitude of EPSP evoked by the 1st pulse of each train was normalized to the first train-evoked EPSP and calculated (Fig. 5B). As shown, TBS caused bell-shape changes of EPSP amplitude in all groups, and the change was significantly smaller in both MK-801 alone- and extrasynaptic procedure-treated slices than that of naïve control slices (repeated ANOVAs measurement, F(2,20), P < 0.05; naïve control vs. extrasynaptic procedure, post hoc tests, P < 0.05; naïve control vs. MK-801 alone, post hoc tests, P < 0.05). The amplitude comparison of the 1st pulse induced EPSP

Please cite this article in press as: Liu, D.-d., et al., Activation of extrasynaptic NMDA receptors induces LTD in rat hippocampal CA1 neurons. Brain Res. Bull. (2013), http://dx.doi.org/10.1016/j.brainresbull.2012.12.003

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from the 180th train and the 1st train is shown in Fig. 5C. As shown, the TBS-induced decrease of EPSP amplitude was significantly larger in both extrasynaptic procedure- and MK-801 alone-treated slices compared with naïve control slices (Fig. 5C, naïve control, 0.56 ± 0.04; MK-801 alone, 0.35 ± 0.03; extrasynaptic procedure, 0.41 ± 0.02. One-way ANOVAs, F(2,20), P = 0.004, naïve control vs. MK-801 alone, Dunn’s Method, P < 0.05, naïve control vs. extrasynaptic procedure, Dunn’s Method, P < 0.05). The decrease of EPSP

Fig. 5. Comparison of summation ratio and TBS-induced decrease of EPSP amplitude among naïve control, MK-801 alone-, and extrasynaptic procedure-treated slices. (A) left, summary histogram shows the ratio of peak amplitude after the 5th pulse to that after the 1st pulse during the first train of 100 Hz stimulation. right, sample traces of the 100 Hz stimulation-evoked EPSPs. “a” and “b” represent the peak amplitude of the 1st and 5th pulse-evoked EPSPs respectively, and b/a was calculated as the ratio. (B) The time course of TBS-evoked EPSP amplitude in naïve control (open circles), MK-801 alone- (closed triangles), and extrasynaptic procedure (closed square)-treated slices. The EPSP amplitude evoked by the 1st pulse of each 100 Hz train was normalized to the first train-evoked EPSP. (C) left, summary histogram shows the ratio of peak amplitude by the first pulse of 180th train- and that of the 1st train-evoked field EPSP. right, sample traces of the 1st train (straight line) and 180th train (dashed line) of 100 Hz stimulation-evoked EPSPs. “c” and “d” represent the peak amplitude of first pulse-evoked EPSPs from the 1st and 180th train respectively, and d/c was calculated as the ratio. *P < 0.05.

Please cite this article in press as: Liu, D.-d., et al., Activation of extrasynaptic NMDA receptors induces LTD in rat hippocampal CA1 neurons. Brain Res. Bull. (2013), http://dx.doi.org/10.1016/j.brainresbull.2012.12.003

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amplitude, however, was not statistically different between MK801 alone- and extrasynaptic procedure-treated slices (MK-801 alone vs. extrasynaptic procedure, Dunn’s Method, P > 0.05). 4. Discussion Several studies have suggested that activation of ES-NMDARs mediates opposite intracellular signaling as compared to SNMDARs in the Ras (Kim et al., 2005), Extracellular signal-regulated kinase (ERK) (Ivanov et al., 2006), and cAMP-response element binding protein (CREB) (Hardingham et al., 2002) pathways, thus leading to neurotoxicity and promoting cell death. In the current study, we found that applying TBS did not evoke LTD either in naïve slices or in the slices pretreated with MK-801 alone (without pairing with the 5 Hz stimulation), while it did induce LTD when applied after blockade of S-NMDARs. These results validate that selective activation of ES-NMDARs induces LTD. It has been shown that TBS mimics the endogenous hippocampal firing patterns during active exploration and learning (Kamondi et al., 1998; Buzsaki et al., 2002; Tsanov and Manahan-Vaughan, 2009). Therefore, our findings that TBS efficiently activates ES-NMDARs and leads to LTD induction implicate that ES-NMDAR-evoked LTD induction plays a potential function during exploration and learning processes. Many studies have demonstrated that S- and ES-NMDARs have distinct channel kinetics in the way of rising time and decay time constants (Lozovaya et al., 1999; Chen and Diamond, 2002; Clark and Cull-Candy, 2002; Tovar and Westbrook, 2002; Pankratov and Krishtal, 2003; Lozovaya et al., 2004; Harney et al., 2008). It has been proven that the decay time of NMDA-EPSC will increase at 10 Hz but not at 5 Hz (Harris and Pettit, 2008). In addition, Lozovaya et al. (2004) showed that applying 5 Hz stimulation could only induce weak slow-down of NMDA-EPSC decay in hippocampal CA1 neurons from juvenile rats. Thus the short train of 5 Hz stimulation which we utilized in the current study activates S- but not ES-NMDARs. Applying the 5 Hz stimulation increased the amplitude of NMDA-EPSPs (Fig. 1), while the same stimulation failed to increase NMDA-EPSPs in the presence of MK-801, confirming that S-NMDARs were blocked. On the other hand, although the mean amplitude of fEPSPs during 30–32 ms after each stimulation was mainly composed of an NMDA component, it is possible that it also involved some AMPA-component when applying low-frequency stimulation. Thus applying the 5 Hz stimulation in the presence of MK-801 blocked the synaptic NMDA-component but had no effect on the remaining AMPA-component of fEPSPs, resulting in unchanged amplitude of fEPSPs during that period (Fig. 1). We validated that the induction of LTD by the extrasynaptic procedure was not accompanied with changes in fiber volley and PPF (Figs. 3 and 4), indicating that this LTD was not caused by changes in presynaptic mechanisms such as membrane depolarization, calcium influx through voltage-dependent calcium channels and the efficiency of neurotransmitter release. Changes in the paired pulse facilitation (PPF) ratio have been considered to be caused primarily by presynaptic mechanisms of calcium accumulation and changes in the probability of glutamate release in hippocampal CA1 pyramidal neurons (Manabe et al., 1993). Similar to PPF, the change in summation ratio by 5 pulses at 100 Hz could also refer to presynaptic mechanisms. Thus, our results showing no differences in summation ratio of 100 Hz stimulation-evoked EPSP amplitude among naïve control, MK-801 alone-, and extrasynaptic procedure-treated groups (Fig. 5A) further indicates that changes in presynaptic mechanisms such as calcium accumulation and the probability of glutamate release do not contribute to induction of ES-NMDAR-evoked LTD. On the other hand, TBS induced similar decreases of EPSP amplitude in MK-801 alone- and extrasynaptic procedure-treated slices (Fig. 5B and C), while only the latter showed LTD formation (Fig. 2). These results indicate that

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neither TBS-induced short-term plasticity nor presynaptic mechanisms contribute to the induction of ES-NMDAR-evoked LTD. Therefore, an important question is how does the activation of ES-NMDARs induce LTD? It is well established that NMDARs are heteromers, composed of NR1 and NR2 or NR3 subunits (Moriyoshi et al., 1991; Monyer et al., 1992; Hollmann and Heinemann, 1994). Strong evidence shows that the specific subunit composition of NMDARs determines their biophysical properties (Erreger et al., 2005), because the different intracellular C-termini of NR2 subunits serve as critical scaffolds for different complex intracellular signal transduction cascades (Sprengel et al., 1998). Besides this, several studies have shown that NR2A subunit-containing NMDARs are critical for LTP induction (Li et al., 2009; Zhang et al., 2009; Jin and Feig, 2010) while the NR2B subunit mainly regulates LTD or attenuates LTP formation (Kohr et al., 2003; Liu et al., 2004). Other reports, however, had also shown the contrary role of NR2B in LTD induction: Overexpression of NR2B-containing NMDARs had no effect on induction of LTD but did have an effect on LTP induction in hippocampal CA1 neurons (Foster et al., 2002; Wang et al., 2009), while reduction of NR2B-containing NMDARs caused impaired LTP but not LTD (Gardoni et al., 2009); In prefrontal cortex, Cui et al. reported that the NR2B-containing NMDAR is critical for induction of LTP but not LTD (Cui et al., 2011); Shin et al. also found that in the lateral nucleus of the amygdala, NR2B plays an important role in LTP induction (Shin et al., 2006). Interestingly, studies from cultured neurons have indicated that the NR2A subunit is preferentially localized at the synaptic region while the NR2B subunit is located at extrasynaptic regions in mature synapses (Tovar and Westbrook, 1999). Taking these findings together, Hardingham et al. proposed that NMDAR-induced responses depend on their synaptic or extrasynaptic locations (Hardingham and Bading, 2010). Thus, our finding that selective activation of extrasynaptic NMDARs alone induces LTD raised a possibility that extrasynaptic NR2B-containing NMDARs contribute to LTD induction. The roles of NR2B or NR2A subunits in ES-NMDAR-mediated LTD induction remain to be further studied. On the other hand, several reports showed that triheteromeric NR1/NR2A/NR2B receptors also co-express with diheteromeric NMDARs (Chazot et al., 1994; Luo et al., 1997; Kew et al., 1998). A recent study has even claimed that triheteromeric NR1/NR2A/NR2B receptors are the major type of NMDARs in adult hippocampal synapses (Rauner and Kohr, 2011). Therefore, in order to investigate the functions of extrasynaptic NR2B-contained NMDARs, it is needed to inhibit both diheteromeric NR1/NR2B receptors and triheteromeic NR1/NR2A/NR2B receptors. Previous studies showed that the selective NR2B subunit antagonist ifenprodil (Kiss et al., 2012) exhibited low sensitivity to NR1/NR2A/NR2B receptors (Hatton and Paoletti, 2005). The extent of inhibition of RO25-6981 (Fischer et al., 1997) and CP-101, 606 (Chazot et al., 2002), both of which are selective antagonists for NR2B, to NR1/NR2A/NR2B receptors is still unclear. Therefore, necessary investigations of the contribution of the NR2B subunit on LTD induction will rely on the development of better selective antagonists for both diheteromeric NR1/NR2B and triheteromeric NR1/NR2A/NR2B receptors in future studies. Recently, an important discovery about the contributions of synaptic and extrasynaptic NMDARs to induction of synaptic plasticity has been reported by Papouin et al. (2012). They show that D-serine and glycine act as coagonists for activation of synapticand extrasynaptic NMDARs respectively and studied the contributions of synaptic and extrasynaptic NMDARs to LTD induction through degrading D-serine and glycine respectively. They found that LTD relies on activation of both synaptic and extrasynaptic NMDARs. Taken together with our observations in the current study that activation of extrasynaptic NMDARs induces LTD, these findings strongly suggest extrasynaptic NR2B-containing NMDARs play

Please cite this article in press as: Liu, D.-d., et al., Activation of extrasynaptic NMDA receptors induces LTD in rat hippocampal CA1 neurons. Brain Res. Bull. (2013), http://dx.doi.org/10.1016/j.brainresbull.2012.12.003

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an important role in LTD induction. These findings also provided us with a potential method to investigate the role of extrasynaptic NR2B subunits in LTD induction through degrading glycine in the extrasynaptic area in coming studies. The results obtained suggest that activation of ES-NMDARs by TBS evokes LTD. Considering that activation of ES-NMDARs takes place mainly during certain pathological conditions related to massive release of glutamate, for instance stroke, anoxic events, brain trauma and epilepsies, the ES-NMDAR-mediated LTD may participate in the pathogenesis of various neuronal diseases. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The work was supported by the grants from the National Science Foundation of China (nos. 31271198 and 81121001), the Shanghai Committee of Science and Technology (no. 11ZR1415900), and State Key Laboratory of Medical Neurobiology, Fudan University (no. 10–12). References Buzsaki, G., Csicsvari, J., Dragoi, G., Harris, K., Henze, D., Hirase, H., 2002. Homeostatic maintenance of neuronal excitability by burst discharges in vivo. Cerebral Cortex 12 (9), 893–899. Chavis, P., Westbrook, G., 2001. Integrins mediate functional pre- and postsynaptic maturation at a hippocampal synapse. Nature 411 (6835), 317–321. Chazot, P.L., Coleman, S.K., Cik, M., Stephenson, F.A., 1994. Molecular characterization of N-methyl-d-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule. Journal of Biological Chemistry 269 (39), 24403–24409. Chazot, P.L., Lawrence, S., Thompson, C.L., 2002. Studies on the subtype selectivity of CP-101,606: evidence for two classes of NR2B-selective NMDA receptor antagonists. Neuropharmacology 42 (3), 319–324. Chen, S., Diamond, J.S., 2002. Synaptically released glutamate activates extrasynaptic NMDA receptors on cells in the ganglion cell layer of rat retina. Journal of Neuroscience 22 (6), 2165–2173. Clark, B.A., Cull-Candy, S.G., 2002. Activity-dependent recruitment of extrasynaptic NMDA receptor activation at an AMPA receptor-only synapse. Journal of Neuroscience 22 (11), 4428–4436. Cui, Y., Jin, J., Zhang, X., Xu, H., Yang, L., Du, D., Zeng, Q., Tsien, J.Z., Yu, H., Cao, X., 2011. Forebrain NR2B overexpression facilitating the prefrontal cortex longterm potentiation and enhancing working memory function in mice. PLoS ONE 6 (5), e20312. Dudek, S.M., Bear, M.F., 1992. Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-d-aspartate receptor blockade. Proceedings of the National Academy of Sciences of the United States of America 89 (10), 4363–4367. Erreger, K., Dravid, S.M., Banke, T.G., Wyllie, D.J., Traynelis, S.F., 2005. Subunitspecific gating controls rat NR1/NR2A and NR1/NR2B NMDA channel kinetics and synaptic signalling profiles. Journal of Physiology 563 (Pt 2), 345–358. Fischer, G., Mutel, V., Trube, G., Malherbe, P., Kew, J.N., Mohacsi, E., Heitz, M.P., Kemp, J.A., 1997. Ro 25-6981, a highly potent and selective blocker of N-methyld-aspartate receptors containing the NR2B subunit. Characterization in vitro. Journal of Pharmacology and Experimental Therapeutics 283 (3), 1285–1292. Foster, K.A., McLaughlin, N., Edbauer, D., Phillips, M., Bolton, A., Constantine-Paton, M., Sheng, M., 2002. Distinct roles of NR2A and NR2B cytoplasmic tails in longterm potentiation. Journal of Neuroscience 30 (7), 2676–2685. Gardoni, F., Mauceri, D., Malinverno, M., Polli, F., Costa, C., Tozzi, A., Siliquini, S., Picconi, B., Cattabeni, F., Calabresi, P., Di Luca, M., 2009. Decreased NR2B subunit synaptic levels cause impaired long-term potentiation but not long-term depression. Journal of Neuroscience 29 (3), 669–677. Groc, L., Bard, L., Choquet, D., 2009. Surface trafficking of N-methyl-d-aspartate receptors: physiological and pathological perspectives. Neuroscience 158 (1), 4–18. Hardingham, G.E., Bading, H., 2010. Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders. Nature Reviews Neuroscience 11 (10), 682–696. Hardingham, G.E., Fukunaga, Y., Bading, H., 2002. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nature Neuroscience 5 (5), 405–414. Harney, S.C., Jane, D.E., Anwyl, R., 2008. Extrasynaptic NR2D-containing NMDARs are recruited to the synapse during LTP of NMDAR-EPSCs. Journal of Neuroscience 28 (45), 11685–11694.

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