Brain-derived neurotrophic factor-induced potentiation of glutamate and GABA release: Different dependency on signaling pathways and neuronal activity

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 31 (2006) 70 – 84

Brain-derived neurotrophic factor-induced potentiation of glutamate and GABA release: Different dependency on signaling pathways and neuronal activity Tomoya Matsumoto,a,1 Tadahiro Numakawa,b,* Daisaku Yokomaku,a,2 Naoki Adachi,c,3 Satoru Yamagishi,c,4 Yumiko Numakawa,c Hiroshi Kunugi,b and Takahisa Taguchi a a

Neuronics Research Group, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Osaka 563-8577, Japan b Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan c Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan Received 24 February 2005; revised 30 August 2005; accepted 5 September 2005 Available online 7 October 2005

The mechanisms underlying BDNF-modulated neurotransmitter release remain elusive. Here, we found that 24-h exposure of postnatal cortical neurons to BDNF potentiated depolarization-evoked glutamate and GABA release in a protein synthesis-dependent manner. BDNFpotentiated glutamate release occurred through the PLC-; and MAPK pathways. The expression of synapsin I, synaptotagmin, and synaptophysin, but not of syntaxin or SNAP25, increased through the PLC-; and MAPK pathways. In contrast, BDNF-up-regulated GABA release and GAD65/67 expression depended on MAPK. Furthermore, neuronal activity was necessary for the up-regulation of glutamate release and synapsin I, synaptotagmin, and synaptophysin expression, but not of GABA or GAD65/67. PLC-; inhibitor attenuated BDNF-stimulated long-lasting MAPK activation. As BDNF rapidly potentiates glutamatergic transmission through PLC-; (J. Biol. Chem. 277, (2002) 6520 – 6529), PLC-;-mediated neuronal activity might sustain MAPK activation, resulting in BDNF-potentiated glutamate release. In conclusion, BDNF potentiates the excitatory and inhibitory system separately, which may be important for the regulation of synaptic plasticity. D 2005 Elsevier Inc. All rights reserved.

* Corresponding author. Fax: +81 42 346 1744. E-mail address: [email protected] (T. Numakawa). 1 Current address: Division of Pharmacology/Neurobiology, Biozentrum, University of Basel, Basel CH-4056, Switzerland. 2 Current address: Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan. 3 Current address: Neuronal Circuit Mechanisms Research Group, Brain Science Institute, RIKEN, Saitama 351-0198, Japan. 4 Current address: Department of Molecular Neurobiology, Max-PlanckInstitute of Neurobiology, Muenchen-Martinsried D-81252, Germany. Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2005.09.002

Introduction Neurotrophins, namely, nerve growth factor (NGF), brainderived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4/5, are known to bind to high-affinity Trks receptors as well as to a common low-affinity p75 receptor. Binding of NGF to TrkA, of BDNF and NT-4/5 to TrkB, or of NT-3 to TrkC (weakly to TrkB) leads to activation of the intracellular signaling pathways, including phospholipase C-g (PLC-g), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3-K) pathway (Huang and Reichardt, 2003). Activation of these pathways promotes neuronal survival and differentiation and also regulates synaptic transmission in the peripheral and central nervous system (Bibel and Barde, 2000). In particular, BDNF/TrkB is principal in the brain, indicating its importance in brain function. Indeed, BDNF is essential for the induction of long-term potentiation that is identified as a cellular basis for learning and memory (Korte et al., 1995, 1996; Patterson et al., 1996). BDNF is involved in synaptic plasticity, that is, changes in the efficacy of neurotransmitter release and postsynaptic response (Thoenen, 1995). BDNF modulates excitatory transmission in various phases. For example, BDNF acutely increases the frequency, but not the amplitude, of miniature excitatory postsynaptic currents (mEPSCs) in cultured rat hippocampal neurons (Lessmann et al., 1994; Li et al., 1998). In rat hippocampal slices, BDNF rapidly attenuates synaptic fatigue induced by high-frequency stimulation through the activation of both the MAPK and PI3-K pathways (Gottschalk et al., 1999). Treatment with BDNF for 10 min enhances depolarization-induced glutamate release from the cortical synaptosomes of rats and mice through the MAPK pathway (Jovanovic et al., 2000). These acute-phase effects of BDNF do not depend on protein synthesis. On the other hand, chronic treatment with BDNF for 12 – 72 h increases the levels of presynaptic proteins and the

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number of docked synaptic vesicles in rat hippocampal slices (Tartaglia et al., 2001). Furthermore, the frequency, but not the amplitude, of mEPSCs recorded from CA1 pyramidal neurons increases after chronic application of BDNF to hippocampal slices for several days (Tyler and Pozzo-Miller, 2001). These studies suggest that the effects of chronic exposure to BDNF are due to de novo protein synthesis. However, in contrast to the acute-phase effects of BDNF, the underlying mechanisms in the late-phase effects, including signaling pathways and activity dependency, have not been fully elucidated. In addition, comparison of the mechanisms between acute- and late-phase effects of BDNF is not sufficient. BDNF-increased presynaptic proteins could also be involved in the regulation of inhibitory transmission. Chronic treatment with BDNF for 5 days enhances depolarization-induced release of not only glutamate, but also GABA, in cultured cortical neurons (Takei et al., 1997). In contrast, BDNF application to cultured hippocampal neurons for 6 – 10 days enhances depolarization-elicited GABA release, but not glutamate release (Yamada et al., 2002). Interestingly, in both cases, BDNF increases levels of presynaptic proteins (Takei et al., 1997; Yamada et al., 2002). Thus, it is important to examine whether the signaling pathway(s) required for the up-regulation of presynaptic proteins and of GABA release are consistent. In this regard, we investigated depolarization-evoked glutamate and GABA release after BDNF application to cultured cortical neurons prepared from postnatal 2-day-old rats. Previously, we reported, using this culture, that pretreatment with BDNF for 10 min enhanced depolarization-induced glutamate release through the TrkB/PLC-g pathway, which did not depend on de novo protein synthesis (Matsumoto et al., 2001). In this study, we focused on the effect of BDNF after continuous and prolonged incubation on glutamate and GABA release and addressed the following questions: (1) Is a signaling pathway for acute-phase effect of BDNF involved in the late-phase effect of BDNF? (2) Does increase in neuronal activity during BDNF treatment play a role in the late-phase effect of BDNF? (3) Does up-regulation of presynaptic proteins equally contribute to glutamate and GABA release potentiated by BDNF? We found that chronic pretreatment with BDNF for 24 h potentiated the exocytotic release of glutamate and GABA in de novo protein synthesis-dependent manner through, however, different intracellular signaling and dependency on neuronal activity. This study is a useful tool for investigating the differences between up-regulation of the excitatory and the inhibitory system stimulated by BDNF, and for comparing the mechanisms between the acute- and late-phase effects of BDNF on neurotransmitter release.

Results Chronic treatment with BDNF potentiates exocytotic release of glutamate and GABA In order to identify the mechanism underlying BDNF-mediated neurotransmitter release, we examined the effect of BDNF on the depolarization-induced release of glutamate and GABA in developing cortical cultures prepared from postnatal 2-day-old rats. We previously showed that spontaneous Ca2+ oscillations through glutamatergic transmission begin to occur 5 days after plating, and that its frequency is acutely increased by the addition of BDNF

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(100 ng/ml) to this cortical culture (Numakawa et al., 2002a). Furthermore, brief exposure to BDNF for 10 or 30 min enhances the depolarization-induced release of glutamate (Matsumoto et al., 2001; Suzuki et al., 2004), suggesting that BDNF has an acute effect on glutamatergic transmission. In this study, we tested whether longer exposure to BDNF still regulates neurotransmitter release. First, we analyzed the time-course of the effect of BDNF on glutamate release. BDNF (100 ng/ml) was applied to the cortical cultures at 5 days in vitro (DIV5) for 10 min, 1, 6, 12, 18, or 24 h. In order to compare the late-phase effect of BDNF with the acute-phase effect (Matsumoto et al., 2001), 4-aminopyridine (4AP, a K+ channel blocker) was used to depolarize cortical neurons in this study. As shown in Fig. 1Aa, pretreatment with BDNF for 10 min enhanced 4AP-induced glutamate release, which is consistent with our previous report (Matsumoto et al., 2001). Longer, continuous treatment with BDNF for 12, 18, or 24 h also potentiated 4AP-induced glutamate release, although potentiation of glutamate release was not observed 1 or 6 h after BDNF treatment (Fig. 1Aa), suggesting that the acute- and late-phase effects of BDNF have different mechanisms. The effect of BDNF on GABA release was also measured. BDNF treatment for 12, 18, or 24 h, but not for 1 or 6 h, potentiated 4AP-induced GABA release (Fig. 1Ab). In contrast with glutamate, an acute-phase (10 min) effect of BDNF on GABA release was not observed (Fig. 1Ab), suggesting that BDNF regulates the glutamatergic and the GABAergic system in a different manner. In our system, BDNF did not affect the basal release of glutamate or GABA (no stimulation) at any time point tested (Figs. 1Aa, b). Therefore, to indicate BDNF-potentiated transmitter release, the ratios of 4APinduced release to basal release are shown in the following results. To examine the late-phase effect of BDNF on glutamate and GABA release, the following experiments were performed 24 h after BDNF application, since its potentiating effects were clearly observed. Next, to address whether translation and transcription are involved in the late-phase effect of BDNF on transmitter release, we tested the effect of cycloheximide (CHX, a protein synthesis inhibitor) or actinomycin D (Act D, an RNA synthesis inhibitor). In the presence of CHX or Act D, BDNF did not potentiate 4APinduced glutamate or GABA release (Figs. 1Ba, b). We did not observe a toxic effect of CHX or Act D on cell survival (data not shown). These results suggest that chronic treatment with BDNF potentiates depolarization-induced glutamate and GABA release in de novo protein synthesis-dependent manner. To clarify whether this BDNF-potentiated release requires the activation of TrkB, the effect of K252a, a cell-permeable Trks inhibitor (Berg et al., 1992), was examined. K252a blocked BDNFpotentiated glutamate and GABA release (Figs. 1Ca, b). In contrast, K252b (200 nM), an inactive analog of K252a, had no effect on BDNF-potentiated release (data not shown). K252a strongly inhibited the phosphorylation of Trks induced by BDNF (Fig. 1Cc). We immunocytochemically confirmed the expression of TrkB in our cortical neurons (DIV5) (Matsumoto et al., 2001). In addition, we previously reported that BDNF or NT-4/5, but not NGF, strongly phosphorylated Trks, although the effect of NT-3 was much weaker than BDNF or NT-4/5 (Numakawa et al., 2002a), suggesting that TrkB is principal in our system. Thus, it is possible that BDNF potentiates glutamate and GABA release through TrkB. To check the cell population in our cultures, immunocytochemical analysis using anti-microtubule-associated protein 2 (MAP2, a neuronal marker), anti-glial fibrillary acidic protein (GFAP, a glial

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marker), and anti-glutamic acid decarboxylase 65/67 (GAD65/67, a GABAergic marker) antibodies was performed. Most of the total cells, which were estimated using DNA-binding dye DAPI, were MAP2-positive (83.5 T 1.5%, n = 5), although 7.7 T 1.3% (n = 5) were GFAP-positive (Figs. 1Da – c). As the GABAergic population was 11.3 T 1.5% (n = 5) (Figs. 1Dd – f), glutamatergic neurons may comprise most of the population.

We confirmed that 4AP-evoked release takes place via exocytosis. The 4AP-evoked release of glutamate and GABA depended on Ca2+ influx from voltage-dependent Ca2+ channels and occurred via the fusion of synaptic vesicles to the plasma membrane of the nerve terminal (Supplementary Figs. 1A, B), suggesting that 4AP-evoked release occurs via exocytosis. This raised the possibility that BDNF up-regulates the expression of

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presynaptic proteins that are essential for exocytosis (Sudhof, 1995) because BDNF-potentiated release depended on de novo protein synthesis (Fig. 1B). Pretreatment with BDNF for 24 h increased the expression of synapsin I, synaptotagmin, and synaptophysin, which are synaptic vesicle-associated synaptic proteins (SV-proteins) (Fig. 1E). On the other hand, the levels of syntaxin and SNAP25, which are plasma membrane-associated synaptic proteins, did not change (Fig. 1E), suggesting that BDNF selectively up-regulates SV-proteins. No change in the expression of class III h-tubulin (TuJ1, a neuronal marker) was observed (Fig. 1E). The up-regulation of SV-protein expression was observed at least 12 h after BDNF treatment (data not shown), when BDNFpotentiated glutamate and GABA release also began to be observed (Figs. 1Aa, b). PLC-c and MAPK pathways are involved in BDNF-up-regulated SV-protein expression and glutamate release BDNF exerts its biological effects through several signaling pathways, including the PLC-g, MAPK, and PI3-K pathways, after TrkB activation. Thus, to determine a possible signaling pathway required for the late-phase effect of BDNF, the effect of each pathway inhibitor was examined. U73122, a PLC-g inhibitor (Smith et al., 1990), which is thought to interact with a substrate-binding site of PLC-g, or U0126, a MEK inhibitor (Favata et al., 1998), abolished the increase in the levels of synapsin I, synaptotagmin, and synaptophysin induced by BDNF, although LY294002, a PI3-K inhibitor (Vlahos et al., 1994), did not (Figs. 2Aa – d). PD98059, another MEK inhibitor, also blocked BDNF-increased SV-proteins (data not shown). None of the inhibitors used affected the levels of TuJ1 (Fig. 2Aa). An inhibitory effect of U0126 or PD98059 on MAPK activation stimulated by BDNF was shown (Fig. 2Ae and Supplementary Fig. 2Ab). The effect of LY294002 on the phosphorylation of Akt (a downstream of PI3-K) was also checked (Fig. 2Af). These inhibitors had no effect on the acute activation of other signaling pathways, nor on TrkB stimulated by BDNF (see Fig. 5B), suggesting the specificity of these inhibitors. These results suggest that the up-regulation of SVproteins by BDNF takes place mainly through the activation of the PLC-g and MAPK pathways. Next, the involvement of the

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PLC-g and MAPK pathways in BDNF-potentiated glutamate and GABA release was examined. U0126 or PD98059 blocked the potentiation of glutamate release (Fig. 2Ba and Supplementary Fig. 2Aa). U0126 completely blocked the potentiation of GABA release (Fig. 2Bb). In contrast, LY294002 or Wortmannin (a PI3K inhibitor) did not affect BDNF-potentiated release (Figs. 2Ba, b and Supplementary Fig. 2B). U73122, but not U73343 (an inactive analog of U73122), blocked the potentiation of glutamate release (Fig. 2Ba and Supplementary Fig. 2B). U73122 did not inhibit the potentiation of GABA release (Fig. 2Bb), indicating that the PLC-g pathway is not important for GABA release. Thus, it is possible that BDNF potentiates glutamate and GABA release through different mechanisms. Neuronal activity is essential for BDNF-up-regulated SV-protein expression and glutamate release, but not GABA release BDNF acutely potentiated glutamatergic transmission in cultured cerebellar (Numakawa et al., 2001, 2003) and cortical neurons (Numakawa et al., 2002a,b). Therefore, the acute-phase effect of BDNF on neuronal activity may influence the late-phase effect (24 h). Tetrodotoxin (TTX; a Na+ channel blocker), which is expected to inhibit spontaneous neuronal activity, was added to the cultures 30 min before BDNF application because the acute-phase effect of BDNF was TTX-sensitive (Numakawa et al., 2001, 2002a). TTX completely inhibited the increase in the levels of SVproteins by BDNF (Figs. 3Aa – d). AP5 (an NMDA glutamate receptor antagonist) or CNQX (an AMPA receptor antagonist) also blocked the BDNF-increased SV-proteins (Figs. 3Aa – d), suggesting that glutamatergic activity is required. Furthermore, BDNF did not potentiate the release of glutamate in the presence of TTX, AP5, or CNQX (Fig. 3Ba). On the other hand, BDNF still had a potentiating effect on GABA release in the presence of these blockers (Fig. 3Bb), suggesting that basal neuronal activity is not necessary for BDNF-potentiated GABA release. Neuronal activity maintains MAPK activation stimulated by BDNF As shown in Fig. 3, neuronal activity was involved in BDNFup-regulated glutamate release (but not GABA) and SV-protein expression. How does neuronal activity contribute to these effects

Fig. 1. Chronic treatment with BDNF potentiates the depolarization-evoked release of glutamate and GABA and up-regulates the expression of synaptic vesicle-associated synaptic proteins in cultured cortical neurons. (A) Time-course analysis of BDNF-potentiated release of glutamate (a) and GABA (b). BDNF (100 ng/ml) was added to the cultures for 10 min, 1, 6, 12, 18, or 24 h (closed bar). Control means no application of BDNF (open bar). Depolarization was induced by 4-aminopyridine (4AP, 4 mM), a K+ channel blocker. After BDNF incubation, basal release ( 4AP) (1 min) was collected before stimulation with 4AP (+4AP) (1 min). Acute treatment with BDNF for 10 min potentiated 4AP-induced release of glutamate but not of GABA. In contrast, longer, continuous treatment with BDNF (12, 18, or 24 h) potentiated 4AP-induced release of glutamate and GABA. The data represent the mean T standard deviation (SD) (n = 4). Statistical analysis was performed using Student’s t test. ***P < 0.001, **P < 0.01, *P < 0.05 versus Control. (B) BDNF-potentiated release of glutamate (a) and GABA (b) was through translation and transcription. Cycloheximide (CHX; 1 AM), a protein synthesis inhibitor, or actinomycin D (Act D; 0.1 AM), an RNA synthesis inhibitor, was applied 60 or 30 min before BDNF application, respectively. BDNF (100 ng/ml) application was performed for 24 h in the presence of each inhibitor (closed bar). Control means no application of BDNF (open bar). The data represent the mean (ratio: 4APinduced release/basal release) T SD (n = 4). ***P < 0.001, **P < 0.01 versus Control (t test). (C) BDNF-potentiated glutamate (a) and GABA (b) release occurred through TrkB activation. K252a (200 nM), a cell-permeable Trks inhibitor, was added 30 min before BDNF application, and then the cultures were maintained for 24 h in the presence of BDNF (100 ng/ml) and K252a. Control means no application of BDNF (open bar). The data represent the mean T SD (n = 4). **P < 0.01, *P < 0.05 versus Control (t test). (c) The inhibitory effect of K252a on the activation of Trks by BDNF was examined (Western blotting). BDNF (100 ng/ml) was applied for 10 min in the presence of K252a (100 or 200 nM). K252a was added 30 min before BDNF addition. (D) A population of the cultured cortical cells was determined immunocytochemically using anti-MAP2 (a, d), GFAP (b), and GAD65/67 (e) antibodies. (c) Overlay of panels a and b. f; Overlay of panels d and e. Scale bar: 20 Am. (E) BDNF (100 ng/ml, 24 h) increased the levels of synaptic vesicle-associated synaptic proteins (synapsin I, synaptotagmin, and synaptophysin), but not of syntaxin, SNAP25, or TuJ1. Control means no application of BDNF. The amount of each protein was quantified by densitometry after Western blotting. The data represent the mean T SD (n = 3). The results were reproducible with three series of separated cultures. ***P < 0.001, **P < 0.01 versus Control (t test).

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Fig. 2. Activation of the PLC-g and MAPK pathways is required for BDNF-potentiated glutamate release and the up-regulation of synaptic vesicle-associated synaptic proteins. (A, a) BDNF up-regulated expression of synapsin I, synaptotagmin, and synaptophysin through the PLC-g and MAPK pathways. U0126 (10 AM, a MEK inhibitor) or U73122 (5 AM, a PLC-g inhibitor) suppressed the up-regulation of these proteins, but LY294002 (10 AM, a PI3-K inhibitor) did not. Inhibitors were added 30 min before BDNF application. The cultured neurons were treated with BDNF (100 ng/ml) for 24 h in the presence of each inhibitor. The level of TuJ1 was examined as a negative control. (b – d) The amount of synapsin I (b), synaptotagmin (c), and synaptophysin (d) was quantified by densitometry after Western blotting. Control means no application of BDNF (open bar). The data represent the mean T SD (n = 3). ***P < 0.001, **P < 0.01, *P < 0.05 versus Control (t test). (e, f) The inhibitory effect of U0126 on ERK1/2 (p44/42 MAPK) activation (e) or of LY294002 on Akt (PI3-K pathway) activation (f) was confirmed. These inhibitors were added 30 min before BDNF application. BDNF (100 ng/ml) was applied for 10 min in the presence of U0126 (10 AM) or LY294002 (10 AM). (B, a) BDNF-potentiated glutamate release depended on the activation of both the PLC-g and MAPK pathways. U0126 or U73122, but not LY294002, completely inhibited BDNF-potentiated glutamate release. (b) BDNF-potentiated GABA release took place through the MAPK pathway, but not PLC-g or PI3-K. BDNF and inhibitors were applied as indicated in panel A, a. The data represent the mean T SD (n = 4). ***P < 0.001, **P < 0.01 versus Control (t test).

of BDNF? BDNF-stimulated signaling may be influenced by neuronal activity. First, we examined the involvement of neuronal activity in the activation of TrkB stimulated by BDNF. TrkB activation by BDNF was not affected by TTX, AP5, or CNQX

(Figs. 4Aa and Ba). Next, downstream signaling of TrkB was determined. MAPK activation stimulated by BDNF was still maintained 24 h after BDNF exposure, although the long-lasting MAPK activation was strongly attenuated in the presence of TTX,

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Fig. 3. Neuronal activity is essential for the BDNF-potentiated release of glutamate and the up-regulation of SV-proteins but not for the release of GABA. (A, a) Neuronal activity was necessary for BDNF-increased synapsin I, synaptotagmin, and synaptophysin. TTX (0.5 AM) is a Na+ channel blocker. AP5 (10 AM) and CNQX (10 AM) are antagonists of NMDA and AMPA glutamate receptors, respectively. BDNF (100 ng/ml) was applied for 24 h in the presence of each blocker. These blockers were added 30 min before BDNF treatment. TuJ1 was not affected by these drugs. (b – d) The amount of synapsin I (b), synaptotagmin (c), and synaptophysin (d) was quantified by densitometry after Western blotting. The data represent the mean T SD (n = 3). ***P < 0.001, **P < 0.01 versus Control (t test). (B) Neuronal activity was required for the BDNF-potentiated release of glutamate (a), but not of GABA (b). BDNF and inhibitors were applied as indicated in panel A, a. The data represent the mean T SD (n = 4). ***P < 0.001, **P < 0.01, *P < 0.05 versus Control (t test).

AP5, or CNQX (Figs. 4Ab and Bb). In contrast, BDNF-activated PLC-g or Akt was not greatly influenced by these blockers (Figs. 4Bc, d). These results suggest that the long-lasting activation of MAPK stimulated by BDNF depends on glutamatergic neuronal activity. Long-lasting activation of MAPK is dependent on PLC-c pathway-mediated neuronal activity Glutamatergic neuronal activity was required for the longlasting maintenance of MAPK activation (Fig. 4). Therefore, we examined the role of the PLC-g pathway because this pathway is important for the BDNF-potentiated glutamatergic system in the acute phase (Matsumoto et al., 2001; Numakawa et al., 2001, 2002a). BDNF-stimulated MAPK activation was dramatically shortened in the presence of U73122 (Figs. 5A and Bb), although

the initial activation of MAPK triggered by BDNF was not blocked. The activation of TrkB, PLC-g, or Akt induced by BDNF was not greatly affected by U73122 (Figs. 5Ba, c, d). U0126 or LY294002 completely blocked the activation of MAPK or Akt, respectively (Figs. 5Bb, d). U0126 or LY294002 did not significantly affect the activation of TrkB or the other signaling cascades (Figs. 5Ba – d). These results suggest that neuronal activity through the PLC-g pathway is involved in the long-lasting activation of MAPK. Up-regulation of GAD65/67 through MAPK activation stimulated by BDNF may be involved in BDNF-potentiated GABA release We found that chronic treatment with BDNF potentiated exocytotic glutamate and GABA release, which seems to be consistent with the observation that SV-proteins increased after

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Fig. 4. Long-lasting activation of MAPK induced by BDNF requires neuronal activity. Time-course of activation of Trks, ERK1/2 (p44/42 MAPK), PLC-g 1, and Akt stimulated by BDNF. The effect of TTX (0.5 AM), AP5 (10 AM), or CNQX (10 AM) was examined. BDNF (100 ng/ml) was applied for 10 min, 1, 3, 6, 9, 12, or 24 h in the presence of each blocker. These blockers were added 30 min before BDNF addition. (A) (a) Activation of Trks stimulated by BDNF was not affected by any of these blockers. TuJ1 is shown as a negative control. (b) The BDNF-stimulated activation of ERK1/2 was strongly attenuated by TTX, AP5, or CNQX. (B) Trks (a), ERK1/2 (b1, b2), PLC-g 1 (c), and Akt (d) activation was quantified by densitometry after Western blotting. Marked suppression of ERK1/2 activation (b1, b2) was observed in the presence of TTX (red), AP5 (blue), or CNQX (green). Trks (a), PLC-g 1 (c), or Akt (d) activation was not attenuated. The data represent the mean T SD (n = 3).

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Fig. 5. The PLC-g pathway is involved in the long-lasting activation of MAPK. Time-course of Trks, ERK1/2, PLC-g 1, and Akt activation stimulated by BDNF with each pathway inhibitor. BDNF application (100 ng/ml) was conducted for 10 min, 1, 3, 6, 9, 12, or 24 h in the presence of each blocker. These blockers were added 30 min before BDNF application. (A) Long-lasting ERK1/2 activation by BDNF was shortened in the presence of U73122 (5 AM). TuJ1 is a negative control. (B) Time-course of Trks (a), ERK1/2 (b1, b2), PLC-g 1 (c), or Akt (d) activation analyzed by densitometry. (a) A significant decrease in Trks activation was not observed with any of these inhibitors. (b1, b2) U73122 (5 AM) (red) decreased BDNF-stimulated ERK1/2 activation. U0126 (10 AM) (blue) completely blocked ERK1/2 activation. LY294002 (10 AM) (green) did not affect ERK1/2 activation. (c) None of these inhibitors changed PLC-g activation. (d) LY294002 blocked the activation of Akt, but the other inhibitors did not. The data represent the mean T SD (n = 3).

BDNF treatment (Figs. 1A, E). However, as shown in Figs. 2 and 3, there are differences in dependency on the signaling pathway and neuronal activity between the up-regulation of GABA release and SV-protein expression, raising the possibility that a mechanism other than the up-regulation of SV-proteins contributes to BDNF-potentiated GABA release. We then examined the upregulation of GAD65/67, which synthesizes GABA (Soghomonian and Martin, 1998). Pretreatment with BDNF for 24 h significantly up-regulated GAD65/67 expression. TTX, AP5, or CNQX did not block the up-regulation of GAD65/67 (Figs. 6Aa, b). The increase in the levels of GAD65/67 was blocked by U0126, but not U73122 or LY294002 (Figs. 6Ba, b). These results are consistent with the characteristics of BDNF-potentiated GABA release (Figs. 2Bb and 3Bb). Next, we investigated whether BDNF

increases the number of GABAergic neurons in our system because BDNF promotes the differentiation of GABAergic neurons (Mizuno et al., 1994). Immunocytochemical analysis using anti-GAD65/67 antibody showed that BDNF did not change the number of GAD65/67-positive cells [the relative values (GAD65/67-positive cells/MAP2-positive cells) with and without BDNF were 12.8 T 2.7% and 12.0 T 1.7%, respectively; P > 0.5 (n = 6)]. Thus, BDNF-up-regulated GAD65/67 expression may not be due to GABAergic differentiation. As expected, the intensity of immunoreactivity for GAD65/67 in an individual cell body was increased by BDNF (Fig. 6C). The increase in GAD65/ 67 intensity by BDNF required MAPK activation, but not neuronal activity (Fig. 6C). In neurites, GAD65/67 immunoreactivity was clearly detected after BDNF application, while no

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signal was observed in GAD65/67-positive cells of non-treated cultures (Supplementary Figs. 3A, B). The increase in GAD65/67 intensity at neurites was also blocked by U0126, but not TTX (data not shown). The GAD65/67 immunoreactivity was uniform in DIV6 neurons (Supplementary Fig. 3A), although it is expected that GAD-positive puncta can be observed in neurites at a more mature stage (Yamada et al., 2002; Kohara et al., 2003). These results suggest that BDNF promotes the production of GABA through GAD65/67 expression, which might result in potentiating GABA release.

BDNF potentiates glutamate and GABA release in mature cortical neurons The question is raised concerning whether the biological responses of glutamatergic or GABAergic neurons to BDNF are due to neuronal maturation or synaptic plasticity in the mature stage because our cortical cultures at DIV5 are still developing (Numakawa et al., 2002a). Therefore, the response of matured cortical cultures at DIV14 was tested. As shown in Fig. 7, the potentiation of glutamate and GABA release occurred 24 h after BDNF exposure of this matured culture. The up-regulation of SVproteins (synapsin I and synaptotagmin) and GAD65/67 was also observed (Table 1). In contrast, BDNF had little effect on the expression of membrane-associated synaptic proteins (syntaxin and SNAP25) and TuJ1 (Table 1). BDNF-potentiated glutamate release was through the PLC-g and MAPK pathways (Fig. 7A), while GABA release was dependent on the MAPK pathway (Fig. 7B). Furthermore, the BDNF-potentiated release of glutamate, but not of GABA, was AP5-sensitive (Figs. 7A, B). The dependencies of SV-proteins or GAD65/67 expression on signaling and neuronal activity were consistent with those of glutamate or GABA release, respectively (Table 1). These results suggest that BDNF-potentiated glutamate and GABA release occur in the mature stage through the same mechanism as that in the premature stage. In addition, the expression of postsynaptic proteins after BDNF treatment was examined. The levels of AMPA (GluR1 and GluR2/ 3), NMDA receptors (NR2B), or postsynaptic density protein 95 kDa (PSD-95) were up-regulated 24 h after BDNF treatment at DIV5 (Table 2a and Supplementary Fig. 4A). In contrast, BDNF did not change the expression of these proteins in mature cortical neurons (Table 2b and Supplementary Fig. 4B), suggesting that BDNF has a different role in the expression of these postsynaptic proteins between immature and mature neurons.

Discussion In this study, we found that chronic pretreatment with BDNF potentiated depolarization-induced glutamate and GABA release in

Fig. 6. Up-regulation of GAD65/67 by BDNF occurs through the MAPK pathway. (A) (a) Up-regulation of GAD65/67 expression after BDNF exposure was not dependent on neuronal activity. TTX (0.5 AM), AP5 (10 AM), or CNQX (10 AM) had no effect on BDNF-increased GAD65/67 expression. These blockers were added 30 min before BDNF addition. BDNF (100 ng/ml) was incubated for 24 h in the presence of each blocker. TuJ1 is shown as a control. (b) Densitometric analysis of panel (a). The data represent the mean T SD (n = 3). ***P < 0.001, **P < 0.01 versus Control (t test). (B) (a) MAPK activation was required for BDNF-increased GAD65/67. The increase in GAD65/67 expression stimulated by BDNF was blocked by U0126 (10 AM) but not U73122 (5 AM) or LY294002 (10 AM). BDNF (100 ng/ml) was applied for 24 h in the presence of each inhibitor. These inhibitors were treated 30 min before BDNF application. (b) Densitometric analysis of panel (a). The data represent the mean T SD (n = 3). ***P < 0.001 versus Control (t test). (C) The intensity of GAD65/ 67 immunoreactivity at a GABAergic cell body was increased by BDNF. Compared with non-treatment (n = 48, selected cell number), BDNF (100 ng/ml) treatment for 24 h markedly increased the mean intensity of GAD65/ 67 immunoreactivity at the single cell level (n = 50, selected cell number). U0126 (10 AM) (n = 50) but not TTX (0.5 AM) (n = 54), blocked the BDNF effect. The data represent the mean T SD. ***P < 0.001 versus Control (t test).

T. Matsumoto et al. / Mol. Cell. Neurosci. 31 (2006) 70 – 84

Fig. 7. BDNF potentiates glutamate and GABA release in mature cortical neurons. BDNF (100 ng/ml) was applied to the cultured cells at DIV14 for 24 h. U0126 (10 AM), U73122 (5 AM), or AP5 (10 AM) was added for 30 min before BDNF treatment. (A) BDNF potentiated 4AP-induced glutamate release through the MAPK and PLC-g pathways. AP5 completely blocked BDNF-potentiated glutamate release, suggesting that glutamatergic activity is required. (B) Activation of the MAPK pathway, but not of PLC-g, was essential for the potentiation of GABA release. AP5 had no effect, suggesting that BDNF-potentiated GABA release occurs in an activity-independent manner. The data represent the mean T SD (n = 4). ***P < 0.001, **P < 0.01, *P < 0.05 versus Control (t test).

cultured cortical neurons through, however, different mechanisms, including (1) signaling pathways, (2) BDNF-increased proteins, and (3) activity dependency (Fig. 8). The BDNF-potentiated

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glutamate release was through the PLC-g and MAPK pathways, while the GABA release required MAPK activation. BDNFincreased SV-proteins were through the PLC-g and MAPK pathways. In contrast, the dependency of BDNF-increased GAD65/67 coincided with that of GABA release. Neuronal activity was necessary for the effect of BDNF on the release of glutamate, but not of GABA. Although chronic treatment with BDNF potentiated 4APevoked glutamate release, the basal release did not change (Fig. 1A). Previous reports indicated the potentiating effect of BDNF on basal neuronal activity. BDNF and NT-3 treatment for 1 – 3 days strongly increased mEPSC frequency in cultured hippocampal neurons prepared from embryonic Day 16 (E16) rats cultured for 2 weeks, while a small but significant increase in amplitude was observed (Vicario-Abejon et al., 1998). In contrast, in hippocampal neurons prepared from postnatal Day 0 (P0) rats, the frequency of mEPSCs did not greatly increase 4 – 7 days after BDNF treatment at DIV3, while its amplitude increased (McLean Bolton et al., 2000). In hippocampal slices prepared from P7 rats, BDNF increased mEPSCs frequency, but not the amplitude, in CA1 pyramidal neurons within 12 – 14 DIV (Tyler and Pozzo-Miller, 2001). In cortical cultures from P4 – 6 rats, BDNF treatment for 2 days from DIV7 – 9 did not affect the frequency of mEPSCs in either pyramidal cells or interneurons, while the amplitude of mEPSCs was increased in interneurons, but not in pyramidal cells (Rutherford et al., 1998). Thus, neuronal maturity, brain region, or neuronal cell types might contribute to these different responses to BDNF. The fusion of synaptic vesicles to the plasma membrane at the nerve terminal is an essential step in exocytosis. In this step, synaptic vesicle-associated synaptic proteins (SV-proteins; synapsin I, synaptotagmin, synaptobrevin, synaptophysin, and so on) and plasma membrane-associated synaptic proteins (PM-proteins; syntaxin and SNAP25, etc.) are important (Sudhof, 1995). In this study, the levels of SV-proteins (synapsin I, synaptotagmin, and synaptophysin), but not of PM-proteins (syntaxin and SNAP25), were up-regulated 24 h after BDNF treatment (Fig. 1E and Table 1). Similarly, it has been reported that BDNF application (at DIV9) to a hippocampal slice culture obtained from P7 rats for 48 h augmented the levels of SV-proteins (synaptotagmin, synaptophysin, and synaptobrevin, but not synapsin I), but not of PM-proteins (syntaxin and SNAP25) (Tartaglia et al., 2001). The increase in synaptotagmin, but not other SV-proteins, was via de novo protein synthesis (Tartaglia et al., 2001). In contrast, the longer application of BDNF to cultured cortical neurons (prepared from E17 rats) for 5 days (Takei et al., 1997) or to cultured hippocampal neurons

Table 1 The expression of synapsin I, synaptotagmin, syntaxin, SNAP25, GAD65/67, and TuJ1 24 h after BDNF application to cultured cortical neurons at DIV14

Synapsin I Synaptotagmin Syntaxin SNAP25 GAD65/67 TuJ1

Control

BDNF

1.00 1.00 1.00 1.00 1.00 1.00

2.01 2.27 1.04 1.04 2.09 1.03

T T T T T T

0.19* 0.18* 0.06 0.05 0.05* 0.10

U0126

U0126 + BDNF

U73122

1.05 T 0.15 0.95 T 0.11 0.98 T 0.09 0.97 T 0.11 0.94 T 0.18 0.92 T 0.08

1.11 T 0.98 T 1.00 T 0.99 T 0.94 T 0.92 T

0.96 0.97 0.94 0.95 0.85 1.04

0.11 0.11 0.09 0.11 0.25 0.13

T T T T T T

0.05 0.04 0.05 0.05 0.19 0.17

U73122 + BDNF 0.97 1.03 0.88 0.88 1.97 1.06

T T T T T T

0.04 0.12 0.02 0.03 0.31* 0.24

AP5 0.91 0.89 1.05 1.00 0.86 0.91

AP5 + BDNF T T T T T T

0.05 0.09 0.14 0.10 0.17 0.15

1.06 T 1.01 T 1.06 T 0.97 T 1.90 T 0.95 T

0.14 0.12 0.18 0.13 0.12* 0.11

The cultured neurons were treated with BDNF (100 ng/ml) at DIV14. Twenty-four hours after BDNF treatment, the cell lysates were collected. U0126 (10 AM), U73122 (5 AM), or AP5 (10 AM) was added 30 min before BDNF application, respectively. The amount of each protein was quantified by densitometry after Western blotting. Control means no application of BDNF. The changes in protein levels are expressed as a ratio to the control. The data represent the mean T SD (n = 4). * P < 0.001 versus Control (t test).

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Table 2a The expression of postsynaptic proteins 24 h after BDNF application to cultured cortical neurons at DIV5

GluR1 GluR2/3 NR2B PSD-95

Control

BDNF

1.00 1.00 1.00 1.00

2.75 2.65 1.41 1.37

T T T T

0.22* 0.92* 0.04* 0.04*

The cortical cultures were treated with BDNF (100 ng/ml) at DIV5. Twenty-four hours after BDNF treatment, the cell lysates were collected. The amount of each protein was quantified by densitometry after Western blotting. Control means no application of BDNF. The changes in protein levels are expressed as a ratio to the control. The data represent the mean T SD (n = 4). * P < 0.001 versus Control (t test).

(from E18 rats) for 7 – 10 days (Yamada et al., 2002) induced the increase in both SV- and PM-proteins. These results suggest that BDNF up-regulates the levels of SV-proteins before up-regulating the levels of PM-proteins. Several reports showed that BDNF regulates the density of the spine (McAllister et al., 1995; Shimada et al., 1998; Tyler and Pozzo-Miller, 2001, 2003; Alonso et al., 2004; Ji et al., 2005). Thus, it is possible that an increase in the number of synapses during neuronal maturation results in BDNFpotentiated transmitter release. In our cortical cultures, treatment with BDNF at DIV5 increased the expression of postsynaptic proteins including AMPA (GluR1 and GluR2/3), NMDA (NR2B) glutamate receptors, and PSD-95 as well as that of SV-proteins (Fig. 1E, Table 2a, and Supplementary Fig. 4A). On the other hand, only SV-proteins, but not these postsynaptic proteins, were upregulated by BDNF treatment at DIV14 (Tables 1 and 2b, and Supplementary Fig. 4B). As the potentiation of glutamate release also occurred in DIV14 neurons (Fig. 7A), it is possible that BDNF exerts potentiating effects on transmitter release through the upregulation of presynaptic machinery. With regard to inhibitory transmission, BDNF application to hippocampal neurons in slice (Tanaka et al., 1997) or in primary culture (Brunig et al., 2001) rapidly reduced the amplitude of spontaneous or miniature inhibitory postsynaptic currents (IPSCs), while their frequency did not change, suggesting that BDNF does not affect presynaptic function in the acute phase. Our results that BDNF had no effect on the basal and exocytotic release of GABA in the acute phase (Fig. 1Ab) support these previous studies. In contrast, BDNF acutely induced the increase in depolarizationinduced GABA release, but not in basal release, from P23 rat visual cortical synaptosomes (Sala et al., 1998), suggesting a difference in the role of BDNF between immature and mature GABAergic systems. On the other hand, chronic treatment with BDNF (several days) enhanced depolarization-induced GABA release in cultured cortical (Takei et al., 1997) or hippocampal neurons (Yamada et al., 2002). In both cases, BDNF increased the levels of presynaptic proteins, suggesting its contribution to GABA release. However, we observed the inconsistency of signaling and activity dependencies between BDNF-up-regulated SV-protein expression and GABA release (Figs. 2 and 3), suggesting the involvement of other proteins. We found that BDNF increased GAD65/67 expression at the cellular level (Fig. 6C and Supplementary Fig. 3), which is consistent with a previous report (Yamada et al., 2002). Taking into account the consistency of signaling and activity dependencies, the up-regulation of GAD65/ 67 plays, at least in part, a role in BDNF-potentiated GABA

release. The role of BDNF in GABAergic maturation (Marty et al., 1997) might be involved because our culture at DIV5 is still developing (Numakawa et al., 2002a). Indeed, BDNF accelerated GABAergic maturation through GAD65 expression in the visual cortex of a BDNF-overexpressed mouse (Huang et al., 1999). However, BDNF-potentiated GABA release was also observed in a more mature culture through the same mechanism as that in the premature stage (Fig. 7 and Table 1), suggesting that BDNF could modulate inhibitory transmission in an established cortical circuit. BDNF-increased GAD65/67 expression was observed in not only cell body but also in neurites (Supplementary Fig. 3). Therefore, it is possible that additional GABA synthesis by BDNF at the synaptic sites is involved in BDNF-potentiated GABA release. It is interesting to examine a change in vesicular GABA transporter (VGAT) expression, since VGAT is functionally and structurally coupled with GAD proteins to facilitate the efficient vesicular transport of GABA (Jin et al., 2003). Neuronal activity has a close connection with BDNF-mediated synaptic transmission. In our system, neuronal activity was necessary for the BDNF-potentiated release of glutamate but not of GABA (Fig. 3B). What role does neuronal activity play? Neuronal activity may affect the activation of the signaling cascades after BDNF stimulation. The suppression of neuronal activity by TTX, NMDA glutamate receptor antagonists, or under low-level extracellular Ca2+ (0.1 mM) strongly reduced the levels of activated MAPK in cultured cortical neurons (Chandler et al., 2001), suggesting that glutamate-mediated neuronal activity maintains the MAPK pathway. Our results support this finding because the maintenance of BDNF-activated MAPK requires glutamatergic neuronal activity (Fig. 4). TrkB activation was involved in the translocation of activated MAPK into the nucleus observed 30 – 60 min after theta burst stimulation (Patterson et al., 2001). Thus, it is interesting to examine whether the long-lasting activation of MAPK by BDNF occurs in the nucleus and whether the translocation of activated MAPK into the nucleus is important for BDNF-potentiated transmitter release via de novo protein synthesis. With regard to GABAergic activity, it is expected that a blockade of the GABAergic system resulting in activation of glutamatergic transmission would enhance BDNF-potentiated glutamate release. However, GABA receptor antagonists (bicuculline and saclofen) did not affect basal glutamatergic activity in our system (Numakawa et al., 2002a), implying that GABAergic activity during BDNF exposure has less effect on the potentiation of glutamate release after BDNF exposure. Interestingly, the long-lasting activation of MAPK stimulated by BDNF depended on PLC-g activation (Fig. 5) as well as glutamatergic neuronal activity (Fig. 4). MAPK might be downTable 2b The expression of postsynaptic proteins 24 h after BDNF application to cultured cortical neurons at DIV14

GluR1 GluR2/3 NR2B PSD-95

Control

BDNF

1.00 1.00 1.00 1.00

0.98 0.96 1.00 1.02

T T T T

0.09 0.05 0.10 0.02

The cortical cultures were treated with BDNF (100 ng/ml) at DIV14. Twenty-four hours after BDNF treatment, the cell lysates were collected. The amount of each protein was quantified by densitometry after Western blotting. Control means no application of BDNF. The changes in protein levels are expressed as a ratio to the control. The data represent the mean T SD (n = 4).

T. Matsumoto et al. / Mol. Cell. Neurosci. 31 (2006) 70 – 84

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Fig. 8. A model of BDNF-potentiated glutamate and GABA release in the acute and late phases. We previously reported that BDNF potentiated glutamatergic transmission in a PLC-g pathway-dependent manner in the acute phase (green) (Matsumoto et al., 2001; Numakawa et al., 2002a). The present study showed that chronic pretreatment with BDNF up-regulated synaptic vesicle-associated synaptic protein (SV-protein) expression and potentiated glutamate release in both a PLC-g and MAPK pathway-dependent manner (blue). Neuronal activity was necessary for these BDNF-potentiated effects (blue). Interestingly, longlasting activation of MAPK induced by BDNF required not only neuronal activity but also PLC-g activation. Thus, it is possible that BDNF-potentiated neuronal activity through the PLC-g pathway (gray arrow) is important for the longer activation of MAPK (red arrow), which might be involved in the latephase effect of BDNF on glutamate release. Potentiation of GABA release also occurred in the late phase (dark blue). Neuronal activity was not necessary for GABA release. An increase in the levels of GAD65/67 depends on MAPK pathway activation, which is consistent with the characteristics of BDNF-potentiated GABA release.

stream of PLC-g, since protein kinase C, downstream of PLC-g (Kim et al., 2000), activated raf-1, a direct activator of MEK (Kolch et al., 1993). However, a PLC-g inhibitor failed to suppress the initial activation of MAPK by BDNF (Fig. 5b), suggesting that these pathways are independent. PLC-g activation was required for transient BDNF secretion induced by neurotrophins in the hippocampal neurons (Canossa et al., 2001). Thus, BDNF-induced secretion of BDNF through the PLC-g pathway might be involved in the prolonged activation of MAPK. However, TrkB and PLC-g were activated at most for 12 h, and their activation did not depend on neuronal activity, which is not consistent with the characteristics of MAPK activation (Fig. 4). We have previously shown that BDNF rapidly potentiated glutamatergic transmission through the PLC-g pathway (Numakawa et al., 2002a). Thus, we propose that the enhancement of glutamatergic activity, which is caused through the PLC-g pathway in the acute phase, sustains MAPK activation stimulated by BDNF, which might be essential for the potentiation of glutamate release after chronic exposure to BDNF (Fig. 8). BDNF may exert accurate effects on glutamatergic transmission through multiple signaling pathways. The effect of BDNF on cortical excitability has been reported (Rutherford et al., 1998). The application of cortical neurons at DIV7 – 9 (prepared from P4 – 6 rats) to BDNF (25 ng/ml) for 2 days increased the amplitude of mEPSCs and the ratio of the firing rate in the interneurons, but not the pyramidal cells. The frequency of mEPSCs in the pyramidal cells and interneurons did not change (Rutherford et al., 1998), suggesting that BDNF postsynaptically up-regulates the excitability of interneurons. If exposure to BDNF was conducted for a longer time in our system, such postsynaptic regulation might have been observed, although experimental conditions including the age of the animals, the cell density of cultures, and the ratio of excitatory to inhibitory neurons were different (Rutherford et al., 1997). In this study, BDNF up-

regulated exocytotic release from glutamatergic and GABAergic neurons through a different mechanism. It is possible that these modulations of the glutamatergic and GABAergic system by BDNF are important for the fine regulation of the cortical system.

Experimental methods Cell culture Primary dissociated cultures were prepared from the cerebral cortex of postnatal 2-day-old rats (Wister ST; SLC, Shizuoka, Japan) as reported previously (Matsumoto et al., 2001; Numakawa et al., 2002a). Dissected cortical tissues were collected in an icecold L15 medium (Life Technologies, Inc., Maryland, United States of America) supplemented with 0.6% glucose. The tissues were then gently dissociated by pipetting after digestion with papain (90 U/ml) (Worthington Biochemical Corp. Co., New Jersey, USA) at 37-C for 20 min. The dissociated cells were plated at a final density of 5  105 cells/cm2 on polyethyleneimine-coated 6-, 12-, and 24-well plates (9.6-, 3.8-, and 2-cm2 surface area/well, respectively) (Becton Dickinson Labware, New Jersey, USA). The culture medium consisted of 5% precolostrum newborn calf serum (Mitsubishi Kasei Co., Tokyo, Japan), 5% heat-inactivated horse serum (Life Technologies, Inc.), and 90% of a 1:1 mixture of Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) and Ham’s F-12 medium (Life Technologies, Inc.) containing 30 nM Na2SeO3, 1.9 mg/ml NaHCO3, and 15 mM HEPES (pH 7.4). Detection of amino acid neurotransmitters The amount of amino acids was measured as described previously (Numakawa et al., 1999, 2000). The amount of

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glutamate and GABA released from the cultured cortical neurons into the assay buffer (KRH buffer; modified HEPES-buffered Krebs Ringer solution containing 130 mM NaCl, 5 mM KCl, 1.2 mM NaH2PO4, 1.8 mM CaCl2, 10 mM glucose, 1% bovine serum albumin (BSA), and 25 mM HEPES (pH 7.4)), was measured using high performance liquid chromatography (HPLC; Shimadzu Co., Kyoto, Japan). After the cortical neurons were cultured for 5 days or 14 days, BDNF (100 ng/ml) was applied. Exposure to BDNF was performed for 10 min, 1, 3, 6, 9, 12, 18, or 24 h. Subsequently, we collected samples after three washes with the KRH buffer. Firstly, the KRH buffer was collected without stimulation, that is, the amount of glutamate and GABA in the sample was considered as basal release. Next, cell depolarization was induced for 1 min, and the KRH buffer was collected (a depolarization-induced release). Depolarization was triggered with 4-aminopyridine (4 mM) (4AP; Sigma, Missouri, USA), a K+ channel blocker, because 4AP induces Ca2+-dependent neurotransmitter release (Tibbs et al., 1989). The cultured neurons were treated with each inhibitor as follows. Cycloheximide (1 AM) (CHX; Sigma) or actinomycin D (0.1 AM) (Act D; Sigma) was added 60 or 30 min prior to BDNF stimulation, respectively. Exposure to BDNF was maintained for 24 h in the presence or absence of each inhibitor. Similarly, K252a (200 nM) (Alexis Co., Switzerland) or K252b (200 nM) (Calbiochem-Novabiochem GmbH, Schwalbach, Germany) was applied to the cultured neurons 30 min before BDNF treatment. U0126 (10 AM) (Promega Corp., Wisconsin, USA), PD98059 (50 AM) (Calbiochem-Novabiochem GmbH), U73122 (5 AM) (Sigma), U73343 (5 AM) (Sigma), LY294002 (10 AM) (Calbiochem-Novabiochem GmbH), or Wortmannin (10 AM) (Wort; Sigma) was applied 30 min before BDNF treatment. Tetrodotoxin (0.5 AM) (TTX; Latoxan Valence, France), d-( )-2-amino-5-phosphonopentanoic acid (10 AM) (AP5; Sigma), or 6-cyano-7-nitroquinoxaline-2,3-dione (10 AM) (CNQX; Sigma) was added 30 min before BDNF application. These inhibitors had almost no effect on cell survival (data not shown), which was determined using MTT assay (Mosmann, 1983; Hansen et al., 1989). For the experiment with a Ca2+-free solution, the cultured neurons were washed three times with a Ca2+-free solution containing EGTA (3 mM) after BDNF treatment (24 h). Tetanus toxin (10 nM) (TeNT; List Biological Laboratories Inc., California, USA) or cadmium chloride (100 AM) (CdCl2; Sigma) was applied to the cultured cells for 4 h or 30 min after BDNF exposure (24 h), respectively. When the effects of these drugs on release were examined, the cultures were washed three times using KRH containing each drug. The collected samples were treated with O-phthalaldehyde and 2-mercaptoethanol for 5 min at 15-C. The samples were then injected into the HPLC system and analyzed using a fluorescence monitor (RF10A-xl; Shimadzu Co., excitation wavelength, 350 nm; emission wavelength, 450 nm). The results in the figures are represented as a ratio: depolarization-evoked release/basal release. We confirmed that none of the inhibitors used in the assay affected the basal release of glutamate or GABA (data not shown). All experiments for this analysis were performed using 3 – 5 separate cultures to confirm reproducibility. Representative data from a sister culture are shown in the figures. N indicates the well number of a plate. Immunocytochemistry The cultured cells were stained with anti-microtubule-associated protein 2 (MAP2), anti-glial fibrillary acidic protein (GFAP),

and anti-glutamic acid decarboxylase 65/67 (GAD65/67) antibodies. At 5 or 6 days in culture, the cells were fixed with 4% paraformaldehyde in 0.2 M sodium phosphate solution (pH 7.4) at room temperature for 20 min and then washed with phosphatebuffered saline (PBS). The fixed cells were permeabilized with PBS containing 0.2% Triton X-100 for 3 min, followed by 30-min incubation at room temperature in the blocking solution (3% BSA in PBS). Anti-MAP2 (Sigma), anti-GFAP (Dakocytomation Denmark A/S, Glostrup, Denmark), or anti-GAD65/67 (Sigma) antibody was incubated at room temperature for 1 h at a dilution of 1:1000, respectively. After washing with PBS, secondary antibodies were applied to the neurons at room temperature for 1 h: FITC- or TRITC-conjugated anti-mouse IgG (Molecular probes, Oregon, USA; 1:1000) or anti-rabbit IgG (Molecular probes; 1:1000) was used. Immunoreactivity was monitored using a fluorescence microscope (BX60; OLYMPUS Co., Tokyo, Japan). To determine the intensity of immunoreactivity, densitometric measurements were conducted using image analysis software (ImageJ 1.32j; NIH, USA). N indicates the number of measured cells or sections at neurites from three separate cultures. For cell counting, we subsequently co-labeled cell nuclei using 4V,6diamidino-2-phenylindole (DAPI; 10 AM; Sigma) for 5 min at room temperature. In this analysis, n indicates the well number of a plate. Immunoblotting At 6 or 15 days in culture, the cells were lysed in a lysis buffer containing 1% sodium dodecyl sulfate (SDS), 20 mM Tris – HCl (pH 7.4), 5 mM EDTA (pH 8.0), 10 mM NaF, 2 mM Na3VO4, 0.5 mM phenylarsine oxide, and 1 mM phenylmethylsulfonyl fluoride. The lysates were boiled for 3 min and then clarified by ultracentrifugation at 60,000g for 30 min at 8-C. The concentration of proteins in the supernatants was determined using a BCA protein assay kit (Pierce, Chemical Co., Illinois, USA), and 10 Ag aliquots of the protein was then resolved on electrophoresis on 7.5% or 10% SDS-polyacrylamide gels. The proteins were transferred onto polyvinylidene fluoride membranes (Millipore Corp., Massachusetts, USA) in 0.1 M Tris base, 0.192 M glycine, and 20% methanol using a semi-dry electrophoretic transfer system. The membranes were blocked with 0.1% Tween 20/Tris-buffered saline (TBST) containing 3% BSA at room temperature for 20 – 30 min. The membranes were then probed with primary antibodies as follows. The membranes were incubated with anti-synapsin I antibody (Chemicon International Inc., California, USA) at a dilution of 1:2000 with TBST containing 1% BSA at room temperature for 1 h. Similarly, antisynaptotagmin (1:1000; Transduction Laboratories, Kentucky, USA), anti-synaptophysin (1:250; Boehringer Mannheim GmbH, Mannheim, Germany), anti-syntaxin (1:10000; Sigma), antiSNAP25 (1:2000; Synaptic Systems, Gottingen, Germany), anti-phospho-TrkA (1:1000; Cell Signaling Technology Inc., Massachusetts, USA), anti-TrkB (1:1000; Transduction Laboratories), anti-phospho-PLC-g 1 (1:500; Cell Signaling Technology Inc.), anti-PLC-g 1 (1:1000; Santa Cruz Biotechnology Inc., California, USA), anti-phospho-Akt (1:1000; Cell Signaling Technology Inc.), anti-Akt (1:1000; Cell Signaling Technology Inc.), anti-phospho-p44/42 MAPK (1:1000; Cell Signaling Technology Inc.), anti-ERK1 (1:1000; Santa Cruz Biotechnology Inc.), anti-GAD65/67 (1:500; Sigma), anti-GluR1 (1:1000; Chemicon International Inc.), anti-GluR2/3 (1:1000; Chemicon

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International Inc.), anti-NR2B (1:1000; Sigma), anti-PSD-95 (1:1000; Upstate, NY, USA), or anti-class III h-tubulin (TuJ1) antibody (1:4000; Berkeley Antibody Company, California, USA) was used. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated donkey anti-mouse IgG or goat anti-rabbit IgG secondary antibodies (Zymed Laboratories Inc., California, USA) diluted 1:4000 or 1:3000 with TBST containing 1% BSA at room temperature for 1 h, respectively. The signals were visualized using an ECL chemiluminescence system (ECL plus; Amersham Biosciences, New Jersey, USA). Representative data from a sister culture are shown. To quantify the amount of proteins after Western blotting, we measured the density of immunoblots using image analysis software (Science Lab 98 Image Gauge; Fuji Photo Film Co. Ltd., Tokyo, Japan). The changes in protein expression are indicated as a ratio that was normalized to a control in each experiment. N indicates the number of experiments performed with 3 – 5 separate cultures.

Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at doi:10.1016/j.mcn.2005.09.002.

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