Inhibition of glutamate release by bupropion in rat cerebral cortex nerve terminals

Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 598–606

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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p

Inhibition of glutamate release by bupropion in rat cerebral cortex nerve terminals Tzu Yu Lin a,b,1, Tsung-Tair Yang c,d,1, Cheng Wei Lu a, Su-Jane Wang d,e,⁎ a

Department of Anesthesiology, Far-Eastern Memorial Hospital, Pan-Chiao, Taipei County 220, Taiwan Department of Mechanical Engineering, Yuan Ze University, Taoyuan 320, Taiwan c Department of Psychiatry, Cardinal Tien Hospital, Hsintien, Taipei 23137, Taiwan d School of Medicine, Fu Jen Catholic University, 510, Chung-Cheng Rd., Hsin-Chuang, Taipei Hsien 24205, Taiwan e Graduate Institute of Basic Medicine, and School of Medicine, Fu Jen Catholic University, Taipei Hsien 24205, Taiwan b

a r t i c l e

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Article history: Received 15 October 2010 Received in revised form 14 December 2010 Accepted 26 December 2010 Available online 7 January 2011 Keywords: Bupropion Glutamate release Presynaptic Ca2+ channels MEK Cerebral cortex Synaptosomes

a b s t r a c t Central glutamate neurotransmission has been postulated to play a role in pathophysiology of depression and in the mechanism of antidepressants. The present study was undertaken to elucidate the effect and the possible mechanism of bupropion, an atypical antidepressant, on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Result showed that bupropion exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate. The effect of bupropion on the evoked glutamate release was prevented by the chelating the intrasynaptosomal Ca2+ ions, and by the vesicular transporter inhibitor, but was insensitive to the glutamate transporter inhibitor. Bupropion decreased depolarization-induced increase in [Ca2+]C, whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of bupropion on evoked glutamate release was abolished by the N-, P- and Q-type Ca2+ channel blocker, but not by the ryanodine receptor blocker, or the mitochondrial Na+/Ca2+ exchanger blocker. In addition, the inhibitory effect of bupropion on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Western blot analyses showed that bupropion significantly decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and this effect also was blocked by MEK inhibitor. These results are the first to suggest that, in rat cerebrocortical nerve terminals, bupropion suppresses voltage-dependent Ca2+ channel and MEK/ERK activity and in so doing inhibits evoked glutamate release. This finding may provide important information regarding the beneficial effects of bupropion in the brain. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Depression, which affects about 21% of the people in the world, is one of the most common psychiatric disorders (Schechter et al., 2005). Most antidepressants increase the concentration of monoamines in the brain, such as norepinephrine, 5-hydroxytryptamine

Abbreviations: 4-AP, 4-aminopyridine; [Ca2+]C, cytosolic free Ca2+ concentration; DiSC3(5), 3-3′dipropylthiadicarbocyanine iodide; Fura-2-AM, fura-2-acetoxymethyl ester; GDH, glutamate dehydrogenase; GF109203X, bisindolylmaleimide I; HBM, HEPES buffer medium; BSA, bovine serum albumin; BAPTA-AM, 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester; MAP kinase, mitogen-activated protein kinase, MEK, mitogen-activated/extracellular signal-regulated kinase kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; PKA, protein kinase A; PKC, protein kinase C; NMDA, N-methyl-D-aspartic acid; VDCC, voltage-dependent Ca2+ channel; CNS, central nervous system. ⁎ Corresponding author. School of Medicine, Fu Jen Catholic University, 510, ChungCheng Rd., Hsin-Chuang, Taipei Hsien 24205, Taiwan. Tel.: + 886 2 29053465; fax: + 886 2 29052096. E-mail address: [email protected] (S.-J. Wang). 1 Tzu Yu Lin and Tsung-Tair Yang contributed equally to this work. 0278-5846/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.12.029

(5-HT), and dopamine (Adell et al., 2005). For example, bupropion increases the levels of dopamine and norepinephrine by blocking their reuptake into presynaptic terminals (Cooper et al., 1980; Ascher et al., 1995; Dwoskin et al., 2006; Wilkes, 2006). However, the affinity of bupropion for these 2 reuptake transporters is lower than most antidepressants (Ferris et al., 1981). Thus, it is possible that bupropion may modulate other neurotransmitters in addition to monoamines. Glutamate, a major excitatory neurotransmitter that plays an important role in many brain functions, such as synaptic plasticity, learning, and memory (Greenamyre and Porter, 1994; Danbolt, 2001), also may be affected in depression since depressed patients have high levels of glutamate in their blood plasma and brain (Sanacora et al., 2004; Kendell et al., 2005). In addition, many studies show that antidepressants decrease glutamate receptor function and glutamate release in the brain (Skolnick, 1999; Michael-Titus et al., 2000; Paul and Skolnick, 2003; Wang et al., 2003; Bonanno et al., 2005). For example, drugs that affect glutamate receptors, such as N-methyl-Daspartic acid (NMDA) receptor antagonists, metabotropic glutamate receptor agonists and antagonists, and positive modulators of αamino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA)

T.Y. Lin et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 598–606

receptors, have antidepressant-like effects (Pittenger et al., 2007; Witkin et al., 2007; Pilc et al., 2008). These results strongly suggest that glutamate is involved in the pathophysiology of depression and in the pharmacological mechanism of antidepressants. Consequently, several glutamate-modulating drugs are being developed to treat depression (Holden, 2003). Since excessive glutamate release may be involved in the pathogenesis of depression (Sapolsky, 2000; Zarate et al., 2002), regulating its release may be an important mechanism of antidepressants, such as bupropion. However, there are no previous studies about the effect of bupropion on glutamate release in central neurons. As a result, this study investigated the effect of bupropion on glutamate release in synaptosomes and the underlying molecular mechanisms. Specifically, the effects of bupropion were tested on synaptosomes that were purified from rat cerebral cortex by measuring the release of endogenous glutamate, synaptosomal plasma membrane potential, downstream activation of voltagedependent Ca2+ channels (VDCCs), and the phosphorylation of protein kinases. 2. Methods 2.1. Chemicals and reagents DiSC3(5) and Fura-2-AM were obtained from Invitrogen (Carlsbad, CA). Bupropion, bafilomycin A1, ω-CgTX MVIIC (ω-conotoxin MVIIC) , dantrolene, DL-TBOA (DL-threo-beta-benzyl-oxyaspartate), PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one), U0126 (1,4-diamino-2,3-dicyano-1,4-bis-(2-aminophenylthio)-butadiene), CGP37157 (7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one), KN62 (1-[N,O-bis(5-isoquinolinesulphonyl)-Nmethyl-L-tyrosyl]-4-phenylpiperazine), H89 (N-[2-(p-bromocinnamylamino)-ethyl]-5-isoquinolinesulfonamide dihydrochloride), and GF109203X (bisindolylmaleimide I) were obtained from Tocris Cookson (Bristol, UK). 1, BAPTA-AM, and all other reagents were obtained from Sigma-Aldrich Co. (St. Louis, MO). Rabbit polyclonal antibodies directed against ERK1/2 and phosphor-ERK1/2 were bought from Cell Signaling Technology (Beverly, MA, USA).

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(pH 7.4), before centrifugation at 27,000 g (15,000 rpm in a JA 25.5) for 10 min. The pellets thus formed were resuspended in 3 ml of HBM, and the protein content was determined using a Bradford Protein Assay Kit (Bio-Rad, Hercules, CA, USA), based on the method of Bradford (1976), with BSA as a standard. 0.5 mg of synaptosomal suspension was diluted in 10 ml of HBM and spun at 3000 g (5000 rpm in a JA 20.1 rotor) for 10 min. The supernatants were discarded, and the synaptosomal pellets were stored on ice and used within 4–6 h. 2.4. Glutamate release assay Glutamate release from purified cerebrocortical synaptosomes was monitored online, with an assay that employed exogenous glutamate dehydrogenase (GDH) and NADP+ to couple the oxidative deamination of the released glutamate to the generation of NADPH detected fluorometrically (Nicholls, 1998; Yang and Wang, 2009). Synaptosomal pellets (0.5 mg protein) were resuspended in HBM and incubated in a stirred and thermostated cuvette maintained at 37 °C in a Perkin-Elmer LS-55 spectrofluorimeter (PerkinElmer Life and Analytical Sciences, Waltham, MA). NADP+ (2 mM), GDH (50 U/ml) and CaCl2 (1 mM) were added after 3 min. In experiments that investigated Ca2+-independent efflux of glutamate, EGTA (200 μM) was added in place of CaCl2. Other additions before depolarization were made as described in the figure legends. After a further 10 min of incubation, 4-aminopyridine (4-AP; 1 mM), high external KCl (15 mM), or ionomycin (5 μM) was added to stimulate glutamate release. Glutamate release was monitored by measuring the increase of fluorescence (excitation and emission wavelengths of 340 and 460 nm, respectively) caused by NADPH being produced by oxidative deamination of released glutamate by GDH. Data were accumulated at 2-s intervals. A standard of exogenous glutamate (5 nmol) was added at the end of each experiment, and the fluorescence response used to calculate released glutamate was expressed as nanomoles glutamate per milligram synaptosomal protein (nmol/mg). Values quoted in the text and expressed in bar graphs represent levels of glutamate cumulatively release after 5 min of depolarization. 2.5. Synaptosomal plasma membrane potential

2.2. Animals Adult male Sprague–Dawley rats (150–200 g) were employed in these studies. All animal procedures were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and were approved by the Fu Jen Institutional Animal Care and Utilization Committee. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.3. Synaptosomal preparation Synaptosomes were prepared as described previously (Nicholls, 1998; Yang and Wang, 2009). Briefly, the cerebral cortex from rats was isolated and homogenized in a medium that contained 320 mM sucrose, pH 7.4. The homogenate was spun for 2 min at 3000 g (5000 rpm in a JA 25.5 rotor; Beckman Coulter, Inc., USA) at 4 °C, and the supernatant was spun again at 14,500 g (11,000 rpm in a JA 25.5 rotor) for 12 min. The pellet was gently resuspended in 8 ml of 320 mM sucrose, pH 7.4. Two milliliters of this synaptosomal suspension was added to 3 ml Percoll discontinuous gradients that contained 320 mM sucrose, 1 mM EDTA, 0.25 mM DL-dithiothreitol, and 3, 10 and 23% Percoll, pH 7.4. The gradients were centrifuged at 32,500 g (16,500 rpm in a JA 20.5 rotor) for 7 min at 4 °C. Synaptosomes placed between the 10 and 23% percoll bands were collected and diluted in a final volume of 30 ml of HEPES buffer medium (HBM) that consisted of 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM MgCl2⋅ 6H2O, 1.2 mM Na2HPO4, 10 mM glucose, and 10 mM HEPES

The synaptosomal membrane potential can be monitored by positively charged, membrane-potential-sensitive carbocyanine dyes such as DiSC3(5). DiSC3(5) is a positively charged carbocyanine that accumulates in polarized synaptosomes that are negatively charged on the inside. At high concentrations, the dye molecules accumulate and the fluorescence is quenched. Upon depolarization, the dye moves out and hence the fluorescence increases (Akerman et al., 1987). Synaptosomes were resuspended in 1 ml HBM and incubated in a stirred and thermostated cuvette maintained at 37 °C in a PerkinElmer LS-55 spectrofluorimeter (PerkinElmer Life and Analytical Sciences, Waltham, MA). After 3 min incubation, 5 μM DiSC3(5) was added and allowed to equilibrate before the addition of CaCl2 (1 mM) after 4 min incubation. 4-AP (1 mM) was added to depolarize the synaptosomes at 10 min, and DiSC3(5) fluorescence was monitored at excitation and emission wavelengths of 646 and 674 nm, respectively. 2.6. Cytosolic free Ca2+ concentration ([Ca2+]C) [Ca2+]C was measured using the Ca2+ indicator Fura-2. Synaptosomes (0.5 mg/ml) were preincubated in HBM with 16 μM BSA in the presence of 5 μM Fura-2 and 0.1 mM CaCl2, for 30 min at 37 °C in a stirred test tube. After Fura-2 loading, synaptosomes were centrifuged in a microcentrifuge for 30 s at 3000 g (5000 rpm). The synaptosomal pellets were resuspended in HBM with BSA, and the synaptosomal suspension was stirred in a thermostated cuvette in a Perkin-Elmer LS-55 spectrofluorimeter (PerkinElmer Life and Analytical Sciences,

T.Y. Lin et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 598–606

2.7. Western blotting analysis Synaptosomes (0.5 mg protein/ml) from control and bupropiontreated groups were lysed in ice-cold Tris–HCl buffer solution, pH 7.5, that contained 20 mM Tris–HCl, 1% Triton, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM phenylmethanesulfonyl fluoride, 1 mM sodium orthovanadate and 1 μg/ml leupeptin. The lysates were sonicated for 10 s and then centrifuged at 13,000 g at 4 °C for 10 min. Equal amounts of synaptosomal proteins were loaded on a SDS polyacrylamide gel and then transferred electrophoretically to nitrocellulose membranes. The membranes were blocked with Tris-buffered saline (TBS) that contained 5% low-fat milk and incubated with appropriate primary antibodies (anti-phospho-ERK1/2, 1:2000, anti-ERK1/2, 1:1000). Following three washes with TBS, the blots were incubated with the secondary horseradish peroxidase-conjugated antibody (1:3000) at room temperature for 1 h. The blots were washed again for three times by TBS and the immunoreactive bands were detected by using the enhanced chemiluminescence method. After immunoblotting, films were scanned at 600 dpi in transmittance mode by using the scanner. The level of phosphorylation was assessed by band density, which was quantified by densitometry. 2.8. Statistical analysis Cumulative data were analyzed using Lotus 1-2-3 and MicroCal Origin. Data are expressed as mean ± S.E.M. To test the significance of the effect of a drug vs. control, a two-tailed Student's t test was used. When an additional comparison was required (such as whether a second treatment influenced the action of bupropion), a one-way analyses of variance (ANOVA) was computed. P b 0.05 was considered to represent a significant difference. 3. Results 3.1. Bupropion inhibits 4-AP-evoked glutamate release by affecting the Ca 2+ -dependent component of release rather than the Ca 2+ independent efflux due to the reversal of the glutamate transporter To examine the influence of bupropion on glutamate release, isolated nerve terminals were depolarized with the K+-channel blocker 4-aminopyridine (4-AP). 4-AP destabilizes the membrane potential and is thought to cause repetitive spontaneous Na+channel-dependent depolarization that closely approximates in vivo depolarization of the synaptic terminal, leading to the activation of voltage-dependent Ca2+ channels (VDCCs) and neurotransmitter release (Nicholls, 1998). Under control conditions, 4-AP (1 mM) evoked a glutamate release of 7.7 ± 0.1 nmol/mg/5 min from synaptosomes incubated in the presence of 1 mM CaCl2. Treatment with bupropion (50 μM) for 10 min significantly reduced 4-AP-evoked glutamate release to 4.6 ± 0.3 nmol/mg/5 min (n = 7; P b 0.01), without altering the basal release of glutamate (Fig. 1). This effect of bupropion was concentration dependent, and the IC50 value derived from a dose–response curve was approximately 85 μM (Fig. 1, inset).

The 4-AP-evoked release of glutamate from synaptosomes can be sustained by different mechanisms, including exocytosis (Ca2+dependent release) and reversal of the transporter (Ca2+-independent release) (Nicholls et al., 1987). To discriminate the effect of bupropion on these two components of endogenous glutamate release evoked by 4-AP, we performed a series of experiments as follows. First, we examined the effect of bupropion on the Ca2+independent glutamate efflux. The Ca2+-independent glutamate efflux was measured by depolarizing the synaptosomes with 4-AP (1 mM) in extracellular Ca2+-free solution that contained 50 μM BAPTA-AM, a cell-permeable Ca2+ chelator. Under these conditions, the release of glutamate evoked by 4-AP was not affected by 50 μM bupropion (Fig. 2). Second, we used DL-TBOA, a nonselective inhibitor of all excitatory amino acid transporter (EAAT) subtypes, to examine the effect of bupropion on 4-AP-evoked glutamate release. In the presence of DL-TBOA (10 μM), although 4-AP (1 mM)-evoked glutamate release was increased by the inhibitor (because of inhibition of reuptake of released glutamate) (P b 0.01), application of bupropion (50 μM) still significantly reduced the 4-AP (1 mM)-induced release of glutamate (Fig. 2). Third, the effect of bupropion on 4-AP-evoked glutamate release was examined in the presence of bafilomycin A1, which causes the depletion of glutamate in synaptic vesicles. In contrast to DL-TBOA, bafilomycin A1 (0.1 μM) reduced control 4-AP (1 mM)-evoked glutamate release (P b 0.01), and significantly blocked the inhibitory effect of bupropion (50 μM) on 4-AP (1 mM)-evoked glutamate release (Fig. 2). Thus, we concluded that the reduction of glutamate release attributable to the bupropion treatment is accounted for only by an impairment of glutamate exocytosis.

3.2. Bupropion does not alter the plasma membrane potential To further understand the potential mechanisms underlying the bupropion-mediated inhibition of glutamate release, we used a membrane potential-sensitive dye, DiSC3(5), to determine the effect of bupropion on synaptosomal plasma membrane potential under resting conditions and on depolarization. DiSC3(5) is a positive 12 10 8 6

Inhibition of glutamate release (% control 4-AP)

Waltham, MA). CaCl2 (1 mM) was added after 3 min and further additions were made after an additional 10 min. Fluorescence data were accumulated at excitation wavelengths of 340 and 380 nm (emission wavelength 505 nm) at 7.5-s intervals. Calibration procedures were performed as described previously (Sihra et al., 1992), using 0.1% SDS to obtain the maximal fluorescence with Fura-2 saturation with Ca2+, followed by 10 mM EGTA (Tris-buffered) to obtain minimum fluorescence in the absence of any Fura-2/Ca2+ complex. [Ca2+]C was calculated using equations described previously (Grynkiewicz et al., 1985).

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Time (sec) Fig. 1. Bupropion inhibits 4-AP-evoked glutamate release from rat cerebrocortical nerve terminals. Synaptosomes were resuspended in incubation medium at a final protein concentration of 0.5 mg/ml and incubated for 3 min before the addition of 1 mM CaCl2. To effect depolarization, 1 mM 4-AP was added after 10 min (arrow). Glutamate release was measured in the absence (control conditions) or presence of 50 μM bupropion added 10 min before the addition of 4-AP. Inset shows dose–response curve of decreases in 4-AP-evoked glutamate release in the presence of bupropion (percentage inhibition compared with controls). The numbers in parentheses indicate the number of experiments performed using independent synaptosomal preparations. The results are calculated as mean ± SEM. ⁎Differences in glutamate release after 1 mM 4-AP depolarization were significantly different in the absence and presence of bupropion (P b 0.01, Student's t test).

T.Y. Lin et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 598–606

charged carbocyanine that accumulates into the polarized synaptosome which is negatively charged on the inside, where at high concentrations, the molecules of the dye will stack and fluorescence is quenched. On depolarization, the dye moves out and hence the fluorescence increases (Akerman et al., 1987). Fig. 3 shows that 4-AP (1 mM) caused an increase in DiSC3(5) fluorescence. Preincubation with bupropion (50 μM) for 10 min before 4-AP addition did not alter the resting membrane potential and had no significant effect on the 4AP-mediated increase in DiSC3(5) fluorescence ( P N 0.05). This result indicates that the effect of bupropion on evoked glutamate release is unlikely to be due to a hyperpolarizing effect of the drug on the synaptosomal plasma membrane potential or due to an attenuation of depolarization produced by 4-AP. Confirming this, we examined the effect of bupropion on the release of glutamate evoked by an alternative secretagogue, high external [K+]. Elevated extracellular KCl depolarizes the plasma membrane by shifting the K+ equilibrium potential above the threshold potential for activation of voltagedependent ion channels. Whereas Na+ channels are inactivated under these conditions, VDCCs are activated nonetheless to mediate Ca2+ entry, which supports neurotransmitter release (Barrie et al., 1991). Addition of 15 mM KCl evoked controlled glutamate release of 9.6 ± 0.1 nmol/mg/5 min, which was reduced to 6.1 ± 0.1 nmol/mg/ 5 min in the presence of 50 μM bupropion (P b 0.01; n = 6). This phenomenon was concentration-dependent, with an IC50 value around 117 μM (Fig. 3, inset). 3.3. Bupropion reduces depolarization-induced increase in [Ca2+]C To investigate whether a reduction in [Ca2+]C is responsible for the bupropion-mediated inhibition of release, we carried out on-line fluorescent assays using a Ca2+ indicator Fura-2-AM to monitor intraterminal Ca2+ levels directly. Under control conditions, 4-AP (1 mM) caused a rise in [Ca2+]C to a plateau level of 209 ± 7 nM. Application of bupropion (50 μM) did not significantly affect basal Ca2+ levels (134 ± 5 nM), but caused a ~23% decrease in the 4-AP-evoked rise in [Ca2+]C (178± 5 nM; P b 0.01; Fig. 4A). The inhibitory effect of

P < 0.05

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bupropion was also evident with KCl (15 mM)-evoked rise in [Ca2+]C, which decreased 18% from 222 ± 5 nM in control conditions to 197 ± 6 nM in the presence of bupropion (50 μM) (Pb 0.01; Fig. 4B). Thus, a reduction of intraterminal Ca2+ seems to be associated with the inhibition of glutamate release by bupropion.

3.4. A reduction of N- and P/Q-type Ca2+ channel activity contributes to the effect of bupropion In the adult rat cerebrocortical nerve terminal preparation, the release of glutamate evoked by depolarization is expected to cause Ca2+ influx through N- and P/Q-type Ca2+ channels and Ca2+ release from internal stores (Berridge, 1998; Millan and Sanchez-Prieto, 2002). For this reason, we sought to examine which part of the Ca2+ source was involved in the effect of bupropion on 4-AP-evoked glutamate release. First, to assess the role of N- and P/Q-type Ca2+ channels, synaptosomes were preincubated with 2 μM ω-conotoxin MVIIC (ω-CgTX MVIIC), a wide-spectrum blocker of N-, P- and Q-type Ca2+ channels. Glutamate release evoked by 1 mM 4-AP (7.6± 0.1 nmol/mg/5 min) under control conditions was significantly decreased in the presence of ω-CgTX MVIIC alone (2.9± 0.1 nmol/mg/5 min) or bupropion alone (4.5 ± 0.1 nmol/ mg/5 min). Crucially, in the presence of ω-CgTX MVIIC, bupropionmediated inhibition of release was largely blocked (2.5± 0.3 nmol/mg/ 5 min; Fig. 5), indicating that N-, P- and Q-type Ca2+ channels are involved in the observed inhibition of glutamate release by bupropion. Next, a potential role of intracellular Ca2+ release in bupropionmediated inhibition of release was examined in the presence of 50 μM dantrolene, which inhibits Ca2+ release from intracellular stores by acting on ryanodine receptors on the endoplasmic reticulum (Zucchi and Ronca-Testoni, 1997). Glutamate release evoked by 1 mM 4-AP under control conditions was reduced in the presence of dantrolene (5.2 ± 0.4 nmol/mg/5 min; P b 0.01). However, in the presence of dantrolene, addition of bupropion (50 μM) caused a further inhibition of 4-AP-evoked glutamate release (3.5 ± 0.2 nmol/mg/5 min). Similar to dantrolene, CGP37157 (100 μM), a membrane-permeable blocker of mitochondrial Na+/Ca2+ exchange, decreased 4-AP (1 mM)-evoked glutamate release (4.8 ± 0.1 nmol/mg/5 min; P b 0.01), but had a nonsignificant effect on the bupropion-mediated inhibition of 4-AP (1 mM)-evoked glutamate release (3.1 ± 0.2 nmol/mg/5 min; Fig. 5). These results suggest that decrease in the Ca2+ release from

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Fig. 2. Bupropion-mediated inhibition of 4-AP-induced glutamate release is due to a decrease in physiological exocytotic vesicular release. Glutamate release was induced by 1 mM 4-AP in the absence or presence of 50 μM bupropion, 50 μM BAPTA (without CaCl2), 50 μM BAPTA (without CaCl2) and 50 μM bupropion, 10 μM DL-TBOA, 10 μM DLTBOA and 50 μM bupropion, 0.1 μM bafilomycin A1, or 0.1 μM bafilomycin A1 and 50 μM bupropion. BAPTA, DL-TBOA, or bafilomycin A1 were added 20 min before depolarization, while bupropion was added 10 min before depolarization. The numbers in parentheses indicate the number of experiments performed using independent synaptosomal preparations. The results are calculated as mean ± SEM (P b 0.05, ANOVA followed by two-tailed Student's t test).

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Time (sec) Fig. 3. Bupropion fails to affect synaptosomal membrane potential. Synaptosomal membrane potential was monitored with 5 μM DiSC3(5) on depolarization with 1 mM 4-AP, in the absence (control) or presence of 50 μM bupropion added 10 min before depolarization. Inset shows dose–response curve of decreases in KCl (15 mM)-evoked glutamate release in the presence of bupropion (percentage inhibition compared with controls). The numbers in parentheses indicate the number of experiments performed using independent synaptosomal preparations. The results are calculated as mean ± SEM.

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T.Y. Lin et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 598–606

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Fig. 5. Bupropion-mediated inhibition of 4-AP-evoked glutamate release is abolished by N- and P/Q-type Ca2+ channel blockade. Glutamate release was induced by 1 mM 4-AP, in the absence (control) or presence of 50 μM bupropion, 2 μM ω-CgTX MVIIC, 2 μM ωCgTX MVIIC and 50 μM bupropion, 50 μM dantrolene, 50 μM dantrolene and 50 μM bupropion, 100 μM CGP37157, or 100 μM CGP37157 and 50 μM bupropion. Bupropion was added 10 min before depolarization, while the other drugs were added 30 min before depolarization. The numbers in parentheses indicate the number of experiments performed using independent synaptosomal preparations. The results are calculated as mean ± SEM (P b 0.05, ANOVA followed by two-tailed Student's t test).

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10 min before ionomycin addition did not alter glutamate release (3.1± 0.1 nmol/mg/5 min; Fig. 6). This indicates that the bupropion-mediated inhibition of glutamate release is not the consequence of a direct action on release events downstream of Ca2+ influx.

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Time (sec) Fig. 4. Bupropion attenuates the 4-AP- and KCl-induced increase in cytosolic Ca2+ concentration ([Ca2+]C). [Ca2+]C was monitored using Fura-2. The synaptosomes were stimulated using 1 mM 4-AP (A) or 15 mM KCl (B) in the absence (control) or presence of 50 μM bupropion, which was added 10 min before stimulation. The numbers in parentheses indicate the number of experiments performed using independent synaptosomal preparations. The results are calculated as mean ± SEM. ⁎Differences in [Ca2+]C levels after 1 mM 4-AP or 15 mM KCl depolarization were significantly different in the absence and presence of bupropion (P b 0.01, two-tailed Student's t test). Differences in basal [Ca2+]C levels in the absence and presence of bupropion were not significantly different (P N 0.05).

intracellular stores appears not to mediate the inhibitory effect of bupropion on glutamate release. 3.5. Bupropion fails to alter the release induced by the Ca2+ ionophore ionomycin Although the foregoing data indicate a correlation of the inhibitory effect of bupropion on glutamate release with a suppression of voltage-dependent Ca2+ channels (VDCCs), there remains the possibility that bupropion could affect targets downstream of Ca2+ entry to also inhibit glutamate release. To address this possibility, we examined the effect of bupropion on the release evoked by the Ca2+ ionophore ionomycin. Ionomycin induces the release of vesicular glutamate in a manner that is independent of plasma membrane depolarization, and therefore independent of the activity of VDCCs (Sihra et al., 1992). Thus, this treatment allows the assessment of only those influences directly affecting exocytotic release machinery, without the involvement of upstream ion-channel function. Fig. 6 shows that ionomycin (5 μM) induced the release of 3.2 ± 0.1 nmol/ mg/5 min. Preincubation of synaptosomes with 50 μM bupropion for

3.6. The bupropion-mediated inhibition of glutamate release involves the MAP kinase/ERK pathway Various kinases, including mitogen-activated protein (MAP) kinase, protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and Ca2+/calmodulin-dependent kinase II (CaMKII), have been shown to regulate glutamate release at the presynaptic level (Sihra and Pearson, 1995; Pereira et al., 2002; Millan et al., 2003; Chang and Wang., 2009; Yang and Wang, 2009). To assess what kind of protein kinase signaling pathway participated in the bupropionmediated inhibition of evoked glutamate release, we performed occlusion experiments with protein kinase inhibitors. We first used PD98059 to specifically inhibit the activation of mitogen-activated/ extracellular signal-regulated kinase kinase (MEK), the immediate upstream regulator of MAP kinase (Alessi et al., 1995). Fig. 7 shows that control glutamate release evoked by 1 mM 4-AP was reduced by 50 μM PD98059 (P b 0.01). Although 4-AP-evoked glutamate release was significantly reduced in the presence of 50 μM bupropion (P b 0.01), this effect was abolished by the pretreatment with PD98059, with the release measured in the presence of PD98059 and bupropion being similar to that obtained in the presence of PD98059 alone. We also used another MEK inhibitor, U0126. As with PD98059, although U0126 (50 μM) attenuated control 4-AP (1 mM)evoked glutamate release (3.5 ± 0.1 nmol/mg/5 min; P b 0.01), it also occluded the inhibitory effect of bupropion on 4-AP (1 mM)-evoked glutamate release (3.1 ± 0.2 nmol/mg/5 min) (Fig. 7). However, in the presence of staurosporine at concentrations (1 μM) that inhibit PKC and PKA activities (Wilkingson and Hallam, 1994), the inhibitory action of bupropion on 4-AP-evoked glutamate release was unaffected (Fig. 7). Similar results were observed with the PKC inhibitor GF109203X (10 μM), the PKA inhibitor H89 (10 μM), or the CaMKII inhibitor KN62 (50 μM) (Fig. 7). The application of staurosporine (1 μM), GF109203X (10 μM), H89 (100 μM) or KN62 (50 μM) alone

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Time (sec) Fig. 6. Lack of bupropion effect on ionomycin-induced glutamate release. Glutamate release was induced by 5 μM ionomycin in the absence (control) or presence of 50 μM bupropion, added 10 min before depolarization. The numbers in parentheses indicate the number of experiments performed using independent synaptosomal preparations. The results are calculated as mean ± SEM. Glutamate release in the presence of bupropion was not significantly different from that in control (P N 0.05).

significantly reduced the 4-AP (1 mM)-evoked glutamate release (P b 0.01; Fig. 7). These results suggest that bupropion-inhibited glutamate release involves a MAP kinase pathway. To further authenticate the role of MAP kinase in the observed inhibition of glutamate release by bupropion, we performed western blotting to examine the effect of bupropion on the phosphorylation of MAP kinase/extracellular signal-regulated kinase 1 and 2 (ERK1/2). Fig. 8 shows the results of analysis performed on extracts of synaptosomes depolarized with 4-AP in the presence of external Ca2+ and treated with bupropion (50 μM). The results showed that depolarization of synaptosomes with 4-AP (1 mM) markedly increased ERK1/2 phosphorylation levels. When synaptosomes were pretreated with bupropion (50 μM) for 10 min before depolarization with 4-AP, a significant decrease in the 4-AP-induced ERK1/2 phosphorylation was observed (Pb 0.05; Fig. 8). In addition, the MEK inhibitor PD98059 (50 μM) significantly reduced 4-AP (1 mM)-induced ERK1/2 phosphorylation (Pb 0.05). Furthermore, in the presence of PD98059, the action of bupropion on 4-AP (1 mM)-induced ERK1/2 phosphorylation was prevented (Fig. 8). 4. Discussion Although monoamine-based therapies are the primary current treatment approaches, numerous lines of evidences have implicated the glutamate system in the pathogenesis of depression, including high levels of glutamate both in plasma and the brain of depressed patients (Sanacora et al., 2004; Kendell et al., 2005), antidepressantlike activity induced by glutamate receptor antagonists in animal models (Skolnick, 2002), and reduction of glutamate release and glutamate receptor function by antidepressants (Skolnick, 1999; Michael-Titus et al., 2000; Paul and Skolnick, 2003; Wang et al., 2003; Bonanno et al., 2005). As a consequence, affecting glutamate neurotransmission such as inhibiting glutamate release at nerve terminals or inhibiting glutamate receptor function may be one of the targets in the clinical action of antidepressants. Bupropion is used clinically an atypical antidepressant with a mixed neurophamacological profile. The mechanisms underlying the antidepressant effects of bupropion are, however, not fully clarified. Therefore, the purpose of this study was to investigate the relationship between antidepressant bupropion and presynaptic modulation of glutamate release and to determine the underlying molecular mechanisms. By using a preparation of nerve terminals from rat cerebral cortex and

Glutamate Release (nmol/mg)

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Fig. 7. Bupropion-mediated inhibition of 4-AP-evoked glutamate release is occluded by MEK inhibitors. Glutamate release was induced by 1 mM 4-AP, in the absence (control) or presence of 50 μM bupropion, 50 μM PD98059, 50 μM PD98059 and 50 μM bupropion, 50 μM U0126, 50 μM U0126 and 50 μM bupropion, 1 μM staurosporine, 1 μM staurosporine and 50 μM bupropion, 10 μM GF109203X, 10 μM GF109203X and 50 μM bupropion, 10 μM H89 or 10 μM H89 and 50 μM bupropion. PD98059, U0126, staurosporine, GF109203X, or H89 was added 40 min before depolarization, while bupropion was added 10 min before depolarization. The numbers in parentheses indicate the number of experiments performed using independent synaptosomal preparations. The results are calculated as mean ± SEM (P b 0.05, ANOVA followed by two-tailed Student's t test).

examining the release of endogenous glutamate, we showed that bupropion rapidly reduced depolarization-evoked glutamate release in a dose dependent manner. Furthermore, this is the first report of a significant effect of this antidepressant on the central glutamate system. Several possible mechanisms for this effect are discussed below. The release of glutamate as a result of the depolarization of isolated nerve terminals has 2 components. The first component is a Ca2+-dependent exocytosis of synaptic vesicles that contain glutamate. The second component is a Ca2+-independent increase in glutamate efflux by the glutamate transporter after prolonged depolarization (Nicholls et al., 1987). In this study, we found that bupropion did not affect 4-AP-evoked glutamate release in the absence of extracellular Ca2+, which suggested that bupropion does not affect glutamate release by reversing the direction of the plasma membrane glutamate transporter. This result is consistent with the finding that bupropion inhibited glutamate release in the presence of DL-TBOA, a nonselective inhibitor of all EAAT subtypes. Furthermore, bafilomycin A1, which depletes the glutamate content of synaptic vesicles, abolished the inhibitory effect of bupropion on 4-AP-evoked glutamate release. Together, these results demonstrated that the Ca2+-dependent component of glutamate release is the molecular basis for the bupropion-mediated inhibition of 4-AP-evoked glutamate release. The inhibition of Ca2+-dependent glutamate release by bupropion could be due to an alteration of the plasma membrane potential, a direct inhibition of the Ca2+ channel coupled to the exocytosis of glutamate, or an effect on some other part of the release machinery. The first possibility is unlikely for 3 reasons. First, bupropion did not have a significant effect on the synaptosomal plasma membrane potential either in the resting state or after depolarization with 4-AP, which indicated a lack of an effect on K+ conductance. Second, bupropion significantly inhibited both 4-AP and KCl-evoked glutamate release. Since 4-AP-evoked glutamate release involves Na+ and

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Fig. 8. Bupropion significantly decreases 4-AP-induced phosphorylation of ERK1/2, and this effect is abolished by the MEK inhibitor PD98059. Phosphorylation of ERK1/2 was detected in synaptosomal lysates by western blotting using anti-phospho-ERK1/2 antibody. Purified synaptosomes were incubated at 37 °C for 2 min in HBM that contained 1.2 mM CaCl2, in the absence (control) or presence of 1 mM 4-AP, 1 mM 4-AP and 50 μM bupropion, 1 mM 4-AP and 50 μM PD98059 or 1 mM 4-AP and 50 μM PD98059 and 50 μM bupropion. PD98059 was added 40 min before 4-AP addition, while bupropion was added 10 min before depolarization. Data are expressed as a percentage of the phosphorylation obtained in the controls in the absence of 4-AP stimulation. The numbers in parentheses indicate the number of experiments performed using independent synaptosomal preparations. The results are calculated as mean ± SEM. Significant differences in the absence and presence of bupropion were as indicated (using ANOVA followed by two-tailed Student's t test).

Ca2+ channels whereas KCl-evoked glutamate release only involves Ca2+ channels (Barrie et al., 1991; Nicholls, 1998), it is not likely that Na+ channels are involved in the modulation of glutamate release by bupropion. Third, bupropion did not affect the 4-AP-evoked Ca2+independent glutamate release, which is only dependent on the membrane potential (Attwell et al., 1993). These results suggested that the bupropion-mediated decrease in evoked glutamate release is not due to a reduction in synaptosomal excitability as a result of modulation of Na+ or K+ ion channels. Consequently, bupropion must act further downstream in the stimulus-evoked exocytosis process. Our results suggested that bupropion inhibits 4-AP- and KCl-evoked glutamate release by decreasing intracellular Ca2+ levels. In synaptic terminals, a depolarization-induced increase in [Ca2+]C, coupled with glutamate release, is mediated by Ca2+ influx through N- and P/Q-type VDCCs and Ca2+ release from intracellular stores, such as the endoplasmic reticulum and mitochondria (Berridge, 1998; Millan and Sanchez-Prieto, 2002). In this study, the inhibition of glutamate release by bupropion was highly sensitive to the inhibition of N- and P/Q-type VDCCs, which are involved in triggering glutamate release from synaptosomes (Vazquez and Sanchez-Prieto, 1997; Millan and Sanchez-Prieto, 2002). This suggested that N- and P/Q-type VDCCs are involved in the modulation of glutamate release by bupropion. However, since the suppression of N- and P/Q-type VDCC activity did not completely abolish the inhibitory effect of bupropion on 4-AP-evoked glutamate release (about 9% of the activity remained), we cannot rule out the involvement of other types of Ca2+ channels. In contrast, we could exclude the effect of a reduction in the release of Ca2+ from

intracellular stores because dantrolene, an inhibitor of intracellular Ca2+ release from the endoplasmic reticulum, and CGP37157, a mitochondrial Na+/Ca2+ exchange blocker, did not affect the inhibitory effect of bupropion on 4-AP-evoked glutamate release. Furthermore, when ionomycin was used as a secretagogue, which bypassed potential targets of bupropion that are downstream of Ca2+ influx, bupropion did not inhibit glutamate release. As a result, bupropion is likely to act at the level of Ca2+ entry. Although we do not have direct evidence that bupropion acts on presynaptic Ca2+ channels, our results implied that bupropion inhibits glutamate release by suppressing Ca2+ influx through N- and P/Q-type VDCCs. Many studies have shown that protein kinases, such as MAP kinase, PKC, PKA, and CaMKII, are involved in the regulation of presynaptic function and glutamate release (Sihra and Pearson, 1995; Millan et al., 2003; Lin et al., 2009; Lu et al., 2010). As a result, it is likely that a protein kinase signaling pathway is involved in the modulation of glutamate release by bupropion. Our investigation showed that: (1) MEK (MAP kinase kinase) inhibitors blocked the inhibitory effect of bupropion on glutamate release; (2) PKC, PKA, and CaMKII inhibitors did not have any effect on bupropion-mediated inhibition of glutamate release; and (3) PD98059, a MEK inhibitor, prevented the inhibitory effect of bupropion on 4-AP-induced phosphorylation of ERK1/2. These results indicated that bupropionmediated inhibition of glutamate release in cerebrocortical nerve terminals is associated with the suppression of MAP kinase/ERK activity. However, a question arises how suppression of MAP kinase/ ERK pathway might be involved in the action of bupropion. Studies have identified a presynaptic mechanism by which ERK-dependent phosphorylation of synapsin I modulates neurotransmitter release in neuronal culture (Jovanovic et al., 2000; Chi et al., 2003). Synapsin I is localized to presynaptic terminals and tethers synaptic vesicles to cytoskeleton located in the distal reserve pool. Phosphorylation of synapsin I by MAP kinase/ERK promotes dissociation of synaptic vesicles from the actin cytoskeleton, which increases the number of vesicles that are available at the active zone for neurotransmitter release (Jovanovic et al., 1996, 2000; Yamagata et al., 2002; Schenk et al., 2005). Therefore, further studies are needed to determine whether the bupropion inhibits glutamate release by decreasing MAP kinase/ ERK-dependent phosphorylation of synapsin I and the availability of synaptic vesicles. Regarding the mechanisms mediating the antidepressant effects of bupropion, it generally is thought to increase the levels of dopamine and norepinephrine by blocking reuptake of these neurotransmitters to presynaptic terminals (Cooper et al., 1980; Ascher et al., 1995; Dwoskin et al., 2006; Wilkes, 2006). However, the action of bupropion for these reuptake transporters is weak (Ferris et al., 1981), its mechanisms of action remain to be elucidated. In fact, several studies have point out the involvement of dopaminergic (D1, D2 and D3) and serotonergic (5-HT2A) receptors in the antidepressant effects of bupropion (Yamada et al., 2004; Kitamura et al., 2008, 2010). In addition, as a hyperfunction of the glutamate system has been shown to occur in depression (Palucha and Pilc, 2007), it can be speculated that reduced glutamate release from nerve terminals is related to the antidepressant effects of bupropion. Indeed, several clinical used antidepressants drugs, such as tricyclic antidepressant drugs, selective serotonin reuptake inhibitors and monoamine oxidase inhibitors, have been shown to reduce glutamate release. Such effects were observed in many brain areas, including cerebral cortex (Prikhozhan et al., 1990; Golembiowska and Dziubina, 2000; Wang et al., 2003; Bonanno et al., 2005). In the current study, we demonstrate that bupropion is able to inhibit glutamate release from cerebral cortex synaptosomes at 50 μM. This is consistent with findings of Cooper et al., who has demonstrated that high dose bupropion (50 and 100 mg/kg) increased the locomotor activity and antagonized dopamine depletions produced by 6-hydroxydopamine in rat (Cooper et al., 1980).

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Moreover, bupropion, at 30 and 40 mg/kg, have been shown to decrease the firing rate of norepinephrine or dopamine neurons in the brain of rats, reverse the reserpine-induced immobility in forced swim test, or increase the extracellular concentrations of dopamine in the mouse brain (Cooper et al., 1994; Dong and Blier, 2001; Dhir and Kulkarni, 2007). However, the mentioned-above effects of bupropion were also observed at low dose range, 2.5–10 mg/kg (Dhir and Kulkarni, 2007; Kitamura et al., 2008, 2010). These discrepancies are not clear, but may be due to different experimental models used. On the other hand, the effect of bupropion in different brain regions shows many differences. A recent study reported that bupropion, administered into the nucleus accumbens rather than the medial prefrontal cortex, decreased the immobility time in the forced swimming test (Kitamura et al., 2010). The present study shows significant effects in the cerebral cortex, but future investigations could also explore other vulnerable regions, such as hippocampus and prefrontal cortex. 5. Conclusion The main finding of the present study is that, in rat cerebrocortical nerve terminals, bupropion effects a decrease in the Ca2+ influx through N- and P/Q-type Ca2+ channels, which subsequently reduces MAP kinase/ERK activity to cause a decrease in evoked glutamate release. More importantly, this study provides for the first time evidence that bupropion acts on the central glutamate system. This effect of bupropion action might account for some of its antidepressant activity. Further investigations are needed to explore the clinical applications of our findings and to continue to elucidate the mechanisms of the effects of bupropion in the brain. Disclosure/conflicts of interest We have no conflicts of interest to disclose. Acknowledgments This work was supported by grants from the Far-Eastern Memorial Hospital of Taiwan, Republic of China (FEMH-99-C-031, FEMH-99-C036) and the Cardinal Tien Hospital (CTH-99-1-2A41). References Adell A, Castro E, Celada P, Bortolozzi A, Pazos A, Artigas F. Strategies for producing faster acting antidepressants. Drug Discov Today 2005;10:578–85. Akerman KE, Scott IG, Heikkila JE, Heinonen E. Ionic dependence of membrane potential and glutamate receptor-linked responses in synaptoneurosomes as measured with a cyanine dye, DiS-C2-(5). J Neurochem 1987;48:552–9. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD98059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J Biol Chem 1995;270:27489–94. Ascher JA, Cole JO, Colin JN, Feighner JP, Ferris RM, Fibiger HC, et al. Bupropion: a review of its mechanism of antidepressant activity. J Clin Psychiatry 1995;56:395–401. Attwell D, Barbour B, Szatkowski M. Nonvesicular release of neurotransmitter. Neuron 1993;375:645–53. Barrie AP, Nicholls DG, Sanchez-Prieto J, Sihra TS. An ion channel locus for the protein kinase C potentiation of transmitter glutamate release from guinea pig cerebrocortical synaptosomes. J Neurochem 1991;57:1398–404. Berridge MJ. Neuronal calcium signaling. Neuron 1998;21:13–26. Bonanno G, Giambelli R, Raiter L, Tiraboschi E, Zappettini S, Musazzi L, et al. Chronic antidepressants reduce depolarization-evoked glutamate release and protein interactions favoring formation of SNARE complex in hippocampus. J Neurosci 2005;25:3270–9. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. Chang Y, Wang SJ. Inhibitory effect of glutamate release from rat cerebrocortical nerve terminals by resveratrol. Neurochem Int 2009;54:135–41. Chi P, Greengard P, Ryan TA. Synaptic vesicle mobilization is regulated by distinct synapsin I phosphorylation pathways at different frequencies. Neuron 2003;38:69–78.

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