Activation of the Trk Signaling Pathway by Extracellular Zinc
In certain brain regions, extracellular zinc concentrations can rise precipitously as intense neuronal activity releases large amounts of zinc from the nerve terminals. Although zinc release has been suggested to play a pathological role, its precise physiological effect is poorly understood. Here, we report that exposure to micromolar quantities of zinc for only a few minutes robustly and specifically activated tropomyosin-related kinase (Trk) receptors, most likely TrkB, in cultured cortical neurons. We further found that Trk activation by zinc is extracellularly mediated by activation of metalloproteinases, which release pro-BDNF from cells and convert pro-BDNF to mature BDNF. These results suggest that activity-dependent release of extracellular zinc leads to metalloproteinase activation, which plays a critically important role in Trk receptor activation at zinc-containing synapses. In certain brain regions, extracellular zinc concentrations can rise precipitously as intense neuronal activity releases large amounts of zinc from the nerve terminals. Although zinc release has been suggested to play a pathological role, its precise physiological effect is poorly understood. Here, we report that exposure to micromolar quantities of zinc for only a few minutes robustly and specifically activated tropomyosin-related kinase (Trk) receptors, most likely TrkB, in cultured cortical neurons. We further found that Trk activation by zinc is extracellularly mediated by activation of metalloproteinases, which release pro-BDNF from cells and convert pro-BDNF to mature BDNF. These results suggest that activity-dependent release of extracellular zinc leads to metalloproteinase activation, which plays a critically important role in Trk receptor activation at zinc-containing synapses. Trk 1The abbreviations used are: Trk, tropomyosin-related kinase; ADAM, a disintegrin and metalloproteinase; anti-pY, anti-phosphotyrosine; APMA, aminophenylmercuric acetate; BDNF, brain-derived neurotrophic factor; MMP, matrix metalloproteinase; NGF, nerve growth factor; NT-4/5, neurotrophin-4/5; p-Trk, phosphorylated Trk; MEM, minimal essential medium; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester.1The abbreviations used are: Trk, tropomyosin-related kinase; ADAM, a disintegrin and metalloproteinase; anti-pY, anti-phosphotyrosine; APMA, aminophenylmercuric acetate; BDNF, brain-derived neurotrophic factor; MMP, matrix metalloproteinase; NGF, nerve growth factor; NT-4/5, neurotrophin-4/5; p-Trk, phosphorylated Trk; MEM, minimal essential medium; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester. neurotrophin receptors are essential for the formation, maturation, and survival of diverse types of neurons during development (1Lewin G.R. Barde Y.A. Annu. Rev. Neurosci. 1996; 19: 289-317Crossref PubMed Scopus (1766) Google Scholar). Recently, Trk receptors were shown to modulate synaptic transmission and plasticity in the adult central nervous system (2Levine E.S. Dreyfus C.F. Black I.B. Plummer M.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8074-8077Crossref PubMed Scopus (517) Google Scholar, 3Kang H. Schuman E.M. Science. 1995; 267: 1658-1662Crossref PubMed Scopus (1155) Google Scholar), and TrkB activation was shown to be necessary for hippocampal CA1 long term potentiation (4Minichiello L. Korte M. Wolfer D. Kuhn R. Unsicker K. Cestari V. Rossi-Arnaud C. Lipp H.P. Bonhoeffer T. Klein R. Neuron. 1999; 24: 401-414Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar, 5Kossel A.H. Cambridge S.B. Wagner U. Bonhoeffer T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14702-14707Crossref PubMed Scopus (85) Google Scholar, 6Chen G. Kolbeck R. Barde Y.A. Bonhoeffer T. Kossel A. J. Neurosci. 1999; 19: 7983-7990Crossref PubMed Google Scholar). However, the mechanisms underlying activity-dependent activation of synaptic Trk receptors are poorly understood. The mammalian brain contains a large amount of zinc. Recent work has shown that zinc stored in glutamatergic vesicles is released into the extracellular space (7Howell G.A. Welch M.G. Frederickson C.J. Nature. 1984; 308: 736-738Crossref PubMed Scopus (696) Google Scholar, 8Assaf S.Y. Chung S.H. Nature. 1984; 308: 734-736Crossref PubMed Scopus (1012) Google Scholar, 9Minami A. Takeda A. Yamaide R. Oku N. Brain Res. 2002; 936: 91-94Crossref PubMed Scopus (24) Google Scholar), predominantly following high frequency stimulation (10Vogt K. Mellor J. Tong G. Nicoll R. Neuron. 2000; 26: 187-196Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Excess zinc release has been implicated as a mechanism of neuronal death in various models of acute brain injury (11Koh J.Y. Suh S.W. Gwag B.J. He Y.Y. Hsu C.Y. Choi D.W. Science. 1996; 272: 1013-1016Crossref PubMed Scopus (921) Google Scholar, 12Choi D.W. Koh J.Y. Annu. Rev. Neurosci. 1998; 21: 347-375Crossref PubMed Scopus (672) Google Scholar). Although synaptic zinc has been suggested to play a role in hippocampal CA3 long term potentiation (13Li Y. Hough C.J. Frederickson C.J. Sarvey J.M. J. Neurosci. 2001; 21: 8015-8025Crossref PubMed Google Scholar), the precise physiological roles of zinc release are poorly understood. Zinc is known to activate Src tyrosine kinase and has been shown to up-regulate N-methyl-d-aspartate receptor activity at nontoxic concentrations (14Manzerra P. Behrens M.M. Canzoniero L.M. Wang X.Q. Heidinger V. Ichinose T. Yu S.P. Choi D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11055-11061Crossref PubMed Scopus (92) Google Scholar, 15Kim T.Y. Hwang J.J. Yun S.H. Jung M.W. Koh J.Y. Synapse. 2002; 46: 49-56Crossref PubMed Scopus (33) Google Scholar), suggesting that it may play a role in signaling events. However, the mechanism of these zinc effects has not been well characterized. Since Src kinase is activated as a downstream member of the signaling pathways of several membrane receptors, including Trk (16Iwasaki Y. Gay B. Wada K. Koizumi S. J. Neurochem. 1998; 71: 106-111Crossref PubMed Scopus (66) Google Scholar), we herein examined whether zinc release can lead to Trk activation. This could form a logical pathway for the zinc-based coupling of synaptic activity to neurotrophin signaling. Cell Cultures—Astrocytic cultures were prepared from newborn mice as previously described (17Hwang J.J. Choi S.Y. Koh J.Y. J. Neurochem. 2002; 82: 894-902Crossref PubMed Scopus (53) Google Scholar). After 2 weeks in vitro (DIV14), astrocytic cultures were used for experiments or as feeder cultures for embryonic neuronal cells. Neurons were obtained from embryonic mouse brain (embryonic day 15-16) and were plated either onto astrocytic cultures to generate mixed cultures or onto poly-l-lysine- and laminin-coated culture plates (Nunc) to generate nearly pure neuronal cultures. In the latter, cell division was halted on DIV3 by the addition of 10 μm cytosine arabinoside. More than 95% of cells in the nearly pure neuronal cultures were neurons, as identified by anti-MAP2 antibody immunocytochemistry (18Koh J.Y. Gwag B.J. Lobner D. Choi D.W. Science. 1995; 268: 573-575Crossref PubMed Scopus (326) Google Scholar). SH-SY5Y cells (1 × 106 cells/35-mm well) were cultured in a 1:1 mixture of Eagle's minimum essential medium and F-12 medium containing 10% fetal bovine serum. Cells were differentiated by treatment with 5 μm retinoic acid for 5 days in the same medium. Exposure to Zinc and Other Drugs—Brief (≤15-min) exposure of cells to zinc (as ZnCl2), Na3VO4, ionomycin, CaCl2, or aminophenylmercuric acetate (APMA) was performed in Hanks' balanced salt solution (Invitrogen) supplemented with 1.8 mm CaCl2 and 0.8 mm MgSO4. In other experiments, K252a, pyrithione, zinc entry blockers, BAPTA-AM, GM6001, or inhibitors of matrix metalloproteinase, MMPI, -II, or -III (Calbiochem), were added in MEM for 30 min prior to zinc exposure and additionally during zinc exposure. After exposure, cultures were washed with MEM and returned to the CO2 incubator. Active MMPs were purchased from Calbiochem. Immunoprecipitation and immunoblots—For immunoprecipitation, cell lysates or media were centrifuged. Aliquots of supernatant were incubated overnight with the appropriate antibody and then precipitated on Protein A-agarose beads at 4 °C for 1 h. The immunoprecipitates were washed three times with lysis buffer, and proteins were separated by 6-15% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp.) for Western blot analyses. The SuperSignal West Dura Extended Duration Substrate (Pierce) and AutoChemi System (UVP BioImaging) were used to visualize the immunoreactive bands. To increase the signals for BDNF and pro-BDNF, cultures were infected for 36 h with 5 × 109 plaque-forming units/ml of adenovirus encoding mouse prepro-BDNF (Ad-BDNF). Antibodies—Anti-BDNF antibody (sc-546; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used for immunoprecipitation of both pro-BDNF and BDNF. Other utilized antibodies included the function-blocking anti-BDNF antibody (Promega); anti-pan-Trk, -TrkA, -TrkB, -TrkC, and -p75NTR antibodies (Santa Cruz Biotechnology); anti-phosphotyrosine (anti-pY) antibody (Upstate Biotechnology, Inc., Lake Placid, NY); anti-NT-3, -NT-4, and -nerve growth factor (NGF) antibodies (R&D); anti-Src and -p-Src antibodies (BIOSOURCE); and antiextracellular signal-regulated kinase, -Akt, and -p-Akt antibodies (Cell Signaling Technology). Newport Green 2,7-dichlorofluorescein Diacetate-Zinc Staining—Cells were preloaded with 5 μm Newport Green 2,7-dichlorofluorescein diacetate (Molecular Probes, Inc., Eugene, OR) in serum-free MEM for 30 min in the CO2 incubator. After exposure to 10 μm zinc for 15 min, cultures were viewed under a fluorescence microscope and photographed. MMP Zymography—Culture media or cell lysates were centrifuged at 12,000 rpm for 5 min. The resulting supernatants were incubated with 50 μl of gelatin-Sepharose for 60 min and centrifuged at 12,000 rpm for 2 min. Each pellet was washed three times with phenylmethylsulfonyl fluoride-containing PBS and electrophoresed using zymography sample buffer and zymogram gels (Novex). The EnzCheck Gelatinase Assay Kit (Molecular Probes) was used for in situ zymography according to the provided protocol. After exposure to zinc, cells were washed with MEM and incubated with 40 μg of DQ™-gelatin-fluorescein isothiocyanate for 1 h at 37 °C. Cells were then viewed by fluorescence microscopy and photographed. In Vitro Treatment of pro-BDNF with MMPs—Ad-BDNF-infected cortical cells were suspended in ice-cold hypotonic lysis medium (1 mm NaHCO3, 5 mm MgCl2, 100 μm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml leupeptin) and lysed by sonication. To remove nuclei and unbroken cells, samples were sedimented at 600 × g for 10 min, and membrane fractions were separated further by centrifugation at 100,000 × g for 1 h. Membrane proteins (25 μg) were incubated with 2 μg/ml of MMP-2, -3, -7, or -9 (Calbiochem) in reaction buffer (50 mm Tris-Cl, pH 5.6, 150 mm NaCl, 5 mm CaCl2) for 3 h. Immunoblots for BDNF were performed as described above. Immunoprecipitates with anti-pan-Trk antibody were prepared from protein preparations of mouse cortical cultures that had been exposed to 10 μm zinc for 15 min in serum-free medium. The anti-Trk immunoprecipitates were Western blotted with anti-TrkA, anti-TrkB, anti-TrkC, or anti-pY antibodies. The results revealed that zinc exposure markedly increased the level of phosphorylated Trk (p-Trk) (Fig. 1A). On the other hand, the level of p75NTR was very low in cortical cells as compared with that in PC12 cells (Fig. 1A). Since cortical cells abundantly expressed TrkB but not TrkA or TrkC (barely detectable), the protein band recognized by the anti-pY antibody was most likely that of phosphorylated TrkB. An increase in Trk phosphorylation was first detected at 3 μm zinc and reached a maximum at 30-300 μm zinc (Fig. 1B). Thus, an intermediate concentration of 10 μm was used in subsequent experiments, unless otherwise indicated. Because our cultures contained both neurons and astrocytes, we examined whether astrocytes were required for the zinc-dependent increase in TrkB phosphorylation. Nearly pure neuronal or astrocytic cultures (>95% of either cell type) were prepared and exposed to 10 μm zinc for 15 min. Western blots showed markedly increased levels of p-Trk in neuronal cultures treated with zinc compared with untreated control cultures (Fig. 1C). In contrast, zinc treatment of astrocytic cultures did not alter the low levels of endogenous TrkB or p-Trk. Since mixed cultures were more resistant to wash injury than nearly pure neuronal cultures, mixed cultures were used in subsequent experiments, unless otherwise indicated. The of in the level of p-Trk was examined (Fig. and a exposure was we that as as 1 min the addition of 10 μm zinc, p-Trk levels were in treated cultures than in control cultures. 5 and 15 min, the level of p-Trk was markedly The level of p-Trk returned to 30 min of zinc, that effect was of exposure, the levels of p-Trk at 15 min and then Since μm zinc was to the effect of BDNF G. M. PubMed Scopus Google Scholar), we examined the effect of zinc on Trk phosphorylation. In our cortical culture 10 or μm zinc Trk phosphorylation by a exposure to 10 BDNF (Fig. However, in the same cells, a exposure to 10 or μm zinc was found to activate Since zinc is a known of tyrosine J.M. R. L.M. J. Cell 1999; 21: PubMed Scopus Google Scholar), we examined the that zinc the phosphorylation levels of tyrosine kinase We examined the phosphorylation of growth factor receptor and growth receptor zinc After immunoprecipitation with the appropriate Western blots with anti-pY antibody in the phosphorylation of growth factor receptor or growth receptor control and cultures (Fig. cortical cultures treated with a of tyrosine G. S. J. J. P. B. G. C. J. 272: Full Text Full Text PDF PubMed Scopus Google Scholar), did not alter the levels of p-Trk (Fig. These the that zinc-dependent phosphorylation of Trk receptors likely TrkB in cortical is to we examined the that increase the level of p-Trk. levels were increased using the We found that treatment with did not increase the levels of p-Trk, at B.J. Canzoniero L.M. Koh J.Y. M. Choi D.W. 1999; PubMed Scopus Google concentrations (Fig. In the extracellular concentration to 10 mm did not increase levels of p-Trk (Fig. However, treatment with 10 μm zinc was to increase the level of p-Trk in cultures. Trk the of receptor activation, is a of An of Trk tyrosine kinase, P. M. Google Scholar), Trk phosphorylation by zinc (Fig. downstream of Trk receptors, as Src tyrosine extracellular and 2000; PubMed Scopus Google Scholar), were phosphorylated in cultures. Treatment with phosphorylation of of the downstream (Fig. these results that zinc signaling downstream of We whether zinc on Trk receptors or extracellularly by cultures with a zinc that zinc entry into cells Chung Hsu C.Y. Res. 1999; PubMed Scopus Google Scholar). Exposure to zinc and markedly increased zinc the effect of zinc as by fluorescence with the zinc Newport Green (Fig. Because the addition of zinc-dependent Trk it that zinc (Fig. of zinc entry known zinc with a mixture of and Koh J.Y. Choi D.W. Neuron. Full Text PDF PubMed Scopus Google Scholar, Canzoniero L.M. Yu S.P. Koh J.Y. G.A. Choi D.W. J. Neurosci. PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, L.M. Choi D.W. J. Neurosci. 1999; 19: PubMed Google did not zinc-dependent Trk phosphorylation (Fig. the addition of an prior to zinc exposure did not Trk phosphorylation (Fig. these results that the of zinc on Trk receptors is most likely we examined the mechanism for Trk activation by extracellular zinc. Since activation of Trk is Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar), we examined whether Trk activation by zinc the of as brain-derived neurotrophic factor or antibodies BDNF or zinc-dependent Trk phosphorylation. The anti-BDNF antibody the effect to a than the antibody (Fig. a of the antibodies Trk phosphorylation (Fig. In contrast, treatment with or antibodies at concentrations that Trk activation in cortical cultures and PC12 cells, had effect on Trk phosphorylation (Fig. Because BDNF to a in Trk phosphorylation as compared with NT-4/5, we on BDNF as the neurotrophin to Trk phosphorylation in and in the levels of BDNF and pro-BDNF in cultures. In cells, zinc treatment the levels of pro-BDNF in cells, but pro-BDNF mature BDNF could be detected in media by immunoprecipitation (Fig. to increase the signals for BDNF and pro-BDNF, cultures were infected with adenovirus containing the encoding mouse prepro-BDNF (Ad-BDNF). Immunoblots of anti-BDNF immunoprecipitates from infected cultures showed that the level of pro-BDNF in cells with the levels of pro-BDNF and mature BDNF in the media increased compared with control levels (Fig. we examined the of BDNF release by zinc in cortical cultures (Fig. with the of Trk activation by zinc, the levels of pro-BDNF and mature BDNF in the media increased at 15 min and then we examined whether the BDNF from neuronal cells. Nearly pure cortical neuronal cultures and SH-SY5Y cells were infected with in the mixed cortical cultures, exposure to zinc the levels of pro-BDNF in cell lysates and increased the levels of pro-BDNF and mature BDNF in the media (Fig. that neurons could be a of BDNF. Since zinc can neurons D.W. Koh J.Y. Annu. Rev. Neurosci. 1998; 21: 347-375Crossref PubMed Scopus (672) Google Scholar), we examined the that BDNF release is by release of proteins from cells. exposure to μm in neuronal death and release exposure to 10 or μm zinc for 15 min did not the of cell death or release 1 h (Fig. In release into the a used of neuronal cell death (18Koh J.Y. Gwag B.J. Lobner D. Choi D.W. Science. 1995; 268: 573-575Crossref PubMed Scopus (326) Google Scholar), was in cortical cultures 1 h exposure to μm zinc for 15 min as compared with wash In contrast, as neurons membrane a exposure to μm release to the media that zinc treatment releases BDNF min (Fig. it is that BDNF release was by cell The zinc-dependent release of pro-BDNF and the increase of mature BDNF levels in the media the that of the released pro-BDNF is to BDNF in the extracellular as MMPs are for the of pro-BDNF to BDNF R. P. Science. 2001; PubMed Scopus Google Scholar). with the activation of metalloproteinase activity by zinc, zymography of samples showed that the activity of and in the media obtained from cortical cultures was than that from control cultures (Fig. showed that 10 μm zinc increased activity to and activity to that of the μm zinc increased the to and the to 3 for for of with In situ zymography using isothiocyanate showed that metalloproteinase activity was increased in cultures compared with control cultures (Fig. metalloproteinase and inhibitors for -II, and -III zinc-dependent Trk phosphorylation (Fig. the addition of the metalloproteinase APMA, to cultures in the of zinc increased the levels of p-Trk (Fig. treatment with and in cortical as well as and to R. P. Science. 2001; PubMed Scopus Google increased the levels of p-Trk to (Fig. membrane fractions (25 μg) obtained from cortical cultures were incubated for 3 h with or μg/ml levels of pro-BDNF and of mature BDNF increased in (Fig. suggesting that MMPs can the with the report R. P. Science. 2001; PubMed Scopus Google Scholar), and were more at pro-BDNF to BDNF under our in vitro In addition to effects on Trk phosphorylation by zinc, metalloproteinase inhibitors both the of pro-BDNF levels in cells and the increase of pro-BDNF and mature BDNF levels in media of cultures infected with (Fig. the metalloproteinase (APMA) the pro-BDNF levels in cells and increased the levels of pro-BDNF and mature BDNF in the media (Fig. these results suggest that MMPs zinc-dependent Trk activation. it is likely that in addition to BDNF MMPs pro-BDNF release to the media from cells. extracellular zinc concentrations in the brain are well 1 μm (10Vogt K. Mellor J. Tong G. Nicoll R. Neuron. 2000; 26: 187-196Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 12Choi D.W. Koh J.Y. Annu. Rev. Neurosci. 1998; 21: 347-375Crossref PubMed Scopus (672) Google Scholar). However, following intense neuronal zinc concentrations can rise as high as several to of μm (7Howell G.A. Welch M.G. Frederickson C.J. Nature. 1984; 308: 736-738Crossref PubMed Scopus (696) Google Scholar, 8Assaf S.Y. Chung S.H. Nature. 1984; 308: 734-736Crossref PubMed Scopus (1012) Google Scholar, K. Mellor J. Tong G. Nicoll R. Neuron. 2000; 26: 187-196Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 12Choi D.W. Koh J.Y. Annu. Rev. Neurosci. 1998; 21: 347-375Crossref PubMed Scopus (672) Google Scholar). The results suggest that in extracellular zinc levels lead to activation of Trk receptors, TrkB. Although further may be to the mechanism of Trk activation, our suggest that the extracellular activation of MMPs is a for Trk activation. Zinc treatment releases pro-BDNF from cells in an to increased pro-BDNF levels in the In levels of mature BDNF increased in the which may R. P. Science. 2001; PubMed Scopus Google Scholar). Since pro-BDNF can activate TrkB S. J. 2001; Full Text Full Text PDF PubMed Scopus Google as well as and mature BDNF TrkB R. P. Science. 2001; PubMed Scopus Google Scholar), both may to the TrkB activation in the Although p75NTR can be additionally activated by the release of pro-BDNF, our cortical neurons p75NTR at very low levels J.Y. Koh J.Y. J. Neurosci. 2000; PubMed Google Scholar), and the effect of pro-BDNF release may be the activation of TrkB at in is zinc treatment pro-BDNF release from cells. In cells, zinc has been shown to activate MMPs and release growth a growth factor receptor J.M. R. D. L.M. J. Cell PubMed Scopus Google Scholar). However, in cortical cell cultures, release of pro-BDNF is pro-BDNF is not a membrane protein and in released pro-BDNF as compared with may be to the matrix MMPs and a disintegrin and metalloproteinase in the cell membrane are of that can in the extracellular Because of activation of MMPs and by a of the by as zinc H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). However, in certain or of the and MMPs in the of M. B. J. A. Science. 2002; PubMed Scopus Google Scholar). Although the did not the mechanism of metalloproteinase activation by zinc, the extracellular of zinc that it may activate either by to the or by pathways M. B. J. A. Science. 2002; PubMed Scopus Google Scholar, S. S. J. 1996; 98: PubMed Scopus Google or be to the precise activation that specifically zinc-dependent Trk activation were not identified in the most MMP inhibitors several including our to that zinc and which are the in mouse cortical cultures, it is that in play μm zinc has been shown to by BDNF in PC12 cells G. M. PubMed Scopus Google Scholar). However, in our cortical cultures, μm zinc only TrkB activation by BDNF. μm zinc activated TrkB, that activation of Trk by zinc its effect on The of the in was than that previously G. M. PubMed Scopus Google Scholar), to in cell is that zinc concentration on the media the or of as and various the zinc concentrations in our experiments may been than the In TrkB activation has been to play a role in synaptic plasticity as hippocampal long term potentiation (4Minichiello L. Korte M. Wolfer D. Kuhn R. Unsicker K. Cestari V. Rossi-Arnaud C. Lipp H.P. Bonhoeffer T. Klein R. Neuron. 1999; 24: 401-414Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar, 5Kossel A.H. Cambridge S.B. Wagner U. Bonhoeffer T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14702-14707Crossref PubMed Scopus (85) Google Scholar, 6Chen G. Kolbeck R. Barde Y.A. Bonhoeffer T. Kossel A. J. Neurosci. 1999; 19: 7983-7990Crossref PubMed Google Scholar). The that zinc release may play an important role in the activity-dependent activation of TrkB or other Trk receptors at zinc-containing glutamatergic synapses. This work an important for the of synaptic zinc in to activity-dependent synaptic We for the of and B. J. Gwag and for