Brain-derived Neurotrophic Factor-induced Potentiation of Ca2+ Oscillations in Developing Cortical Neurons
2002; Elsevier BV; Volume: 277; Issue: 8 Linguagem: Inglês
10.1074/jbc.m109139200
ISSN1083-351X
AutoresTadahiro Numakawa, Satoru Yamagishi, Naoki Adachi, Tomoya Matsumoto, Daisaku Yokomaku, Masashi Yamada, Hiroshi Hatanaka,
Tópico(s)Neurogenesis and neuroplasticity mechanisms
ResumoBrain-derived neurotrophic factor (BDNF) has been reported to exert an acute potentiation of synaptic activity. Here we examined the action of BDNF on synchronous spontaneous Ca2+ oscillations in cultured cerebral cortical neurons prepared from postnatal 2–3-day-old rats. The synchronous spontaneous Ca2+ oscillations began at approximately DIV 5. It was revealed that voltage-dependent Ca2+ channels and ionotropic glutamate receptors were involved in the synchronous spontaneous oscillatory activity. BDNF potentiated the frequency of these oscillations. The BDNF-potentiated activity reached 207 ± 20.1% of basal oscillatory activity. NT-3 and NT-4/5 also induced the potentiation. However, nerve growth factor did not. We examined the correlation between BDNF-induced glutamate release and the BDNF-potentiated oscillatory activity. Both up-regulation of phospholipase C-γ (PLC-γ) expression and the BDNF-induced glutamate release occurred at approximately DIV 5 when the BDNF-potentiated oscillations appeared. We confirmed that the BDNF-induced glutamate release occurred through a glutamate transporter that was dependent on the PLC-γ/IP3/Ca2+ pathway. Transporter inhibitors blocked the BDNF-potentiated oscillations, demonstrating that BDNF enhanced the glutamatergic transmissions in the developing cortical network by inducing glutamate release via a glutamate transporter. Brain-derived neurotrophic factor (BDNF) has been reported to exert an acute potentiation of synaptic activity. Here we examined the action of BDNF on synchronous spontaneous Ca2+ oscillations in cultured cerebral cortical neurons prepared from postnatal 2–3-day-old rats. The synchronous spontaneous Ca2+ oscillations began at approximately DIV 5. It was revealed that voltage-dependent Ca2+ channels and ionotropic glutamate receptors were involved in the synchronous spontaneous oscillatory activity. BDNF potentiated the frequency of these oscillations. The BDNF-potentiated activity reached 207 ± 20.1% of basal oscillatory activity. NT-3 and NT-4/5 also induced the potentiation. However, nerve growth factor did not. We examined the correlation between BDNF-induced glutamate release and the BDNF-potentiated oscillatory activity. Both up-regulation of phospholipase C-γ (PLC-γ) expression and the BDNF-induced glutamate release occurred at approximately DIV 5 when the BDNF-potentiated oscillations appeared. We confirmed that the BDNF-induced glutamate release occurred through a glutamate transporter that was dependent on the PLC-γ/IP3/Ca2+ pathway. Transporter inhibitors blocked the BDNF-potentiated oscillations, demonstrating that BDNF enhanced the glutamatergic transmissions in the developing cortical network by inducing glutamate release via a glutamate transporter. Neurotrophins play important roles in the survival and differentiation of the peripheral nervous system and CNS 1CNScentral nervous systemBDNFbrain-derived neurotrophic factorNMDAN-methyl d-aspartatet-PDCl-trans-pyrollidine-2,4-dicarboxylic aciddl-TBOAdl-threo-β-benzyloxyaspartateTTXtetrodotoxinMAPmitogen-activated proteinPLC-γphospholipase C-γIP3inositol 1,4,5-trisphosphatePI3 kinasephosphatidylinositol 3-kinaseGABAAγ-aminobutyric acid, type AHPLChigh pressure liquid chromatographyNGFnerve growth factorT-TBSTween 20/Tris-buffered salineAMPAα-amino-3-hydroxy-5-methylisoxazole-4-proprinateBAPTA-AM1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester)CNQX6-cyano-7-nitroquinoxaline-2,3-dioneAPVd(−)2-amino-5-phosphono-valeratePpostnatalNT-3neurotrophin-3Trktropomyosin receptor kinase 1CNScentral nervous systemBDNFbrain-derived neurotrophic factorNMDAN-methyl d-aspartatet-PDCl-trans-pyrollidine-2,4-dicarboxylic aciddl-TBOAdl-threo-β-benzyloxyaspartateTTXtetrodotoxinMAPmitogen-activated proteinPLC-γphospholipase C-γIP3inositol 1,4,5-trisphosphatePI3 kinasephosphatidylinositol 3-kinaseGABAAγ-aminobutyric acid, type AHPLChigh pressure liquid chromatographyNGFnerve growth factorT-TBSTween 20/Tris-buffered salineAMPAα-amino-3-hydroxy-5-methylisoxazole-4-proprinateBAPTA-AM1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester)CNQX6-cyano-7-nitroquinoxaline-2,3-dioneAPVd(−)2-amino-5-phosphono-valeratePpostnatalNT-3neurotrophin-3Trktropomyosin receptor kinase neurons. Besides having long term effects, neurotrophins play a fundamental role in neuronal plasticity in the short term (1Thoenen H. Science. 1995; 270: 593-598Crossref PubMed Scopus (1709) Google Scholar). In particular, BDNF is essential to neuronal transmissions and activity-dependent neuronal plasticity (2Kang H. Schuman E.M. Science. 1995; 267: 1658-1662Crossref PubMed Scopus (1166) Google Scholar, 3Levine E.S. Dreyfus C.F. Black I.B. Plummer M.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8074-8077Crossref PubMed Scopus (522) Google Scholar, 4Berninger B. Poo M.m. Curr. Opin. Neurobiol. 1996; 6: 324-330Crossref PubMed Scopus (160) Google Scholar, 5Figurov A. Pozzo-Miller L.D. Olafsson P. Wang T. Lu B. Nature. 1996; 381: 706-709Crossref PubMed Scopus (943) Google Scholar, 6Korte M. Griesbeck O. Gravel C. Carroll P. Staiger V. Thoenen H. Bonhoeffer T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12547-12552Crossref PubMed Scopus (308) Google Scholar, 7Patterson S.L. Abel T. Deuel T.A. Martin K.C. Rose J.C. Kandel E.R. Neuron. 1996; 16: 1137-1145Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar, 8Akaneya Y. Tsumoto T. Kinoshita S. Hatanaka H. J. Neurosci. 1997; 17: 6707-6716Crossref PubMed Google Scholar, 9Li Y.X., Xu, Y., Ju, D. Lester H.A. Davidson N. Schuman E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10884-10889Crossref PubMed Scopus (133) Google Scholar, 10Boulanger L. Poo M.m. Nat. Neurosci. 1999; 2: 346-351Crossref PubMed Scopus (135) Google Scholar, 11Sherwood N.T. Lo D.C. J. Neurosci. 1999; 19: 7025-7036Crossref PubMed Google Scholar). Application of BDNF to cultured hippocampal neurons induced an excitatory synaptic transmission (12Lessmann V. Gottmann K. Heumann R. Neuroreport. 1994; 6: 21-25Crossref PubMed Scopus (359) Google Scholar), cation influx (13Li H.S., Xu, X.Z. Montell C. Neuron. 1999; 24: 261-273Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar), generation of action potential (14Kafitz K.W. Rose C.R. Thoenen H. Konnerth A. Nature. 1999; 401: 918-921Crossref PubMed Scopus (470) Google Scholar), and Ca2+ mobilization (15Berninger B. Garcia D.E. Inagaki N. Hahnel C. Lindholm D. Neuroreport. 1993; 4: 1303-1306Crossref PubMed Scopus (178) Google Scholar). central nervous system brain-derived neurotrophic factor N-methyl d-aspartate l-trans-pyrollidine-2,4-dicarboxylic acid dl-threo-β-benzyloxyaspartate tetrodotoxin mitogen-activated protein phospholipase C-γ inositol 1,4,5-trisphosphate phosphatidylinositol 3-kinase γ-aminobutyric acid, type A high pressure liquid chromatography nerve growth factor Tween 20/Tris-buffered saline α-amino-3-hydroxy-5-methylisoxazole-4-proprinate 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) 6-cyano-7-nitroquinoxaline-2,3-dione d(−)2-amino-5-phosphono-valerate postnatal neurotrophin-3 tropomyosin receptor kinase central nervous system brain-derived neurotrophic factor N-methyl d-aspartate l-trans-pyrollidine-2,4-dicarboxylic acid dl-threo-β-benzyloxyaspartate tetrodotoxin mitogen-activated protein phospholipase C-γ inositol 1,4,5-trisphosphate phosphatidylinositol 3-kinase γ-aminobutyric acid, type A high pressure liquid chromatography nerve growth factor Tween 20/Tris-buffered saline α-amino-3-hydroxy-5-methylisoxazole-4-proprinate 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) 6-cyano-7-nitroquinoxaline-2,3-dione d(−)2-amino-5-phosphono-valerate postnatal neurotrophin-3 tropomyosin receptor kinase Ca2+ appears to affect processes that are central to the development and plasticity of the CNS (16Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1240) Google Scholar), and several patterns of Ca2+ dynamics are known. Spontaneous oscillations in the intracellular Ca2+ concentration occur in developing CNS neurons, and their mechanisms are highly distinct. For example, the activation of nicotinic acetylcholine receptors is involved in the oscillatory activity of the retina, whereas a depolarization through the GABAA receptors is required for hippocampal oscillatory activity. Retinal oscillations are spatially restricted to the domains of amacrine and ganglion cells (17Wong R.O. Chernjavsky A. Smith S.J. Shatz C.J. Nature. 1995; 374: 716-718Crossref PubMed Scopus (234) Google Scholar, 18Feller M.B. Wellis D.P. Stellwagen D. Werblin F.S. Shatz C.J. Science. 1996; 272: 1182-1187Crossref PubMed Scopus (432) Google Scholar). By contrast, hippocampal activity consists of transient bursts in the intracellular Ca2+ recurring synchronously over the entire population of pyramidal and interneurons (19Garaschuk O. Hanse E. Konnerth A. J. Physiol. 1998; 507: 219-236Crossref PubMed Scopus (281) Google Scholar). In the cerebral cortex, several patterns of Ca2+ activity have been observed during cortical development, and the metabotropic glutamate receptors (mGluR), GABAA receptors, gap junctions, and ionotropic glutamate receptors are all able to mediate them. The Ca2+oscillations mediated by mGluR are triggered by mGluR agonists in neonatal or embryonic cortical mouse slices, and this Ca2+activity is not synchronized in any population of cells (20Flint A.C. Dammerman R.S. Kriegstein A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12144-12149Crossref PubMed Scopus (85) Google Scholar). In the early postnatal stage, GABAA receptor activation has been shown to be involved in Ca2+ oscillations, and again this Ca2+ activity is not synchronized (21Owens D.F. Boyce L.H. Davis M.B. Kriegstein A.R. J. Neurosci. 1996; 16: 6414-6423Crossref PubMed Google Scholar). Gap junction-mediated propagation of Ca2+ waves is not required for synaptic transmission (22Yuste R. Nelson D.A. Rubin W.W. Katz L.C. Neuron. 1995; 14: 7-17Abstract Full Text PDF PubMed Scopus (225) Google Scholar), but an IP3-related second messenger system acting via gap junctions is required for synaptic transmission (23Kandler K. Katz L.C. J. Neurosci. 1998; 18: 1419-1427Crossref PubMed Google Scholar). Recently, the large neuronal networks in slices of newborn rats revealed synchronized Ca2+ oscillations. These Ca2+ activities are mediated by AMPA and NMDA glutamate receptors and are maintained until the developmental transition of the GABAergic transmission from depolarization to hyperpolarization (24Garaschuk O. Linn J. Eilers J. Konnerth A. Nat. Neurosci. 2000; 3: 452-459Crossref PubMed Scopus (398) Google Scholar). On the other hand, very little is known about neurotrophin-dependent oscillatory activities in CNS neurons. A previous report has shown that BDNF potentiates the frequency of spontaneous Ca2+ oscillations in cultured hippocampal neurons (25Sakai N. Yamada M. Numakawa T. Ogura A. Hatanaka H. Brain Res. 1997; 778: 318-328Crossref PubMed Scopus (57) Google Scholar). However, the mechanism by which BDNF potentiates the oscillatory activity remains unclear. In the present study, we identified how BDNF potentiates synchronized spontaneous Ca2+ oscillations in cultured cerebral cortical neurons. We present the hypothesis that the BDNF potentiation of Ca2+ oscillations is mediated by glutamate, which is released in response to BDNF via a glutamate transporter. We propose that our system may be a useful tool for understanding how neurotrophins mediate the spontaneous synaptic transmission. Primary dissociated cultures were prepared from postnatal 2- or 3-day-old rat (SLC, Sizuoka, Japan) cortex as reported previously (26Hatanaka H. Tsukui H. Nihonmatsu I. Brain Res. 1988; 467: 85-95Crossref PubMed Scopus (189) Google Scholar, 27Numakawa T. Takei N. Yamagishi S. Sakai N. Hatanaka H. Brain Res. 1999; 842: 431-438Crossref PubMed Scopus (42) Google Scholar). Briefly, cells were gently dissociated with a plastic pipette after digestion with papain (90 units/ml, Worthington) at 37 °C. The dissociated cells were plated at a final density of 4 or 5 × 105/cm2 on polyethyleneimine-coated 12- and 24-well plates (4- and 2-cm2 surface area/wells, respectively; Corning). The culture medium consisted of 5% precolostrum newborn calf serum, 5% heat-inactivated horse serum, 1% rat serum, 89% of a 1:1 mixture of Dulbecco's modified Eagle's medium, and Ham's F-12 medium containing 15 mm HEPES buffer (pH 7.4), 30 nmNa2SeO3, and 1.9 mg/ml NaHCO3. Cells were stained with the anti-MAP2 (rabbit IgG, gift from Dr. H. Murofushi, University of Tokyo), anti-GLU (mouse monoclonal IgG, Sigma), and anti-glutaminase (mouse monoclonal IgM, a gift from Dr. T. Kaneko, Kyoto University) antibodies. Briefly, cells were fixed in 4% paraformaldehyde containing 0.05% Triton X at room temperature for 20 min and then incubated overnight with anti-MAP2 (1:5000) or anti-GLU (1:1000) antibodies diluted with a 0.01% Triton X solution at 4 °C. Secondary antibodies were applied at room temperature for 1 h. We used fluorescein isothiocyanate (Seikagaku Kogyo) or rhodamine (Vector Laboratories) secondary antibodies at a dilution of 1:200. For staining with the anti-glutaminase antibody, cells were fixed in 4% paraformaldehyde at room temperature for 20 min and then incubated with the anti-glutaminase antibody diluted 1:250 in a 0.3% Triton X solution. Anti-mouse IgM was used as a second antibody. For visualizing, we used a Vectastain ABC kit (Vector Laboratories) together with 0.02% (w/v) 3,3′-diaminobenzidine 4-HCl (DAB) and 0.1% (w/v) (NH4)Ni(SO4)2dissolved in 0.05 m Tris-HCl buffer (pH 7.6), containing 0.01% (v/v) H2O2. Immunoreaction was observed with a Nikon DIAPHOT-TMD microscope. The amounts of amino acids released from the cultured cortical neurons were measured as described previously (27Numakawa T. Takei N. Yamagishi S. Sakai N. Hatanaka H. Brain Res. 1999; 842: 431-438Crossref PubMed Scopus (42) Google Scholar). Briefly, the amounts released into the assay buffer (modified HEPES-buffered Krebs Ringer solution: KRH containing 130 mm NaCl, 5 mm KCl, 1.2 mm NaH2PO4, 1.8 mmCaCl2, 10 mm glucose, 1% bovine serum albumin, and 25 mm HEPES (pH 7.4)) were measured by HPLC (BAS) with a fluorescence detector (CMA280, BAS). The high K+(HK+) solution consisted of 85 mm NaCl, 50 mm KCl, 1.2 mm NaH2PO4, 1.8 mm CaCl2, 10 mm glucose, 1% bovine serum albumin, and 25 mm HEPES (pH 7.4). The Ca2+-free solution was prepared by omitting the CaCl2. Na+-free solution was prepared by the addition of sucrose or LiCl instead of NaCl. Fractions were collected at 1-min intervals in tubes and placed on ice by the batch method and then filtered with 0.22-μm membranes to remove cell debris. Next, the samples were treated with O-phthalaldehyde and 2-mercaptoethanol for 5 min at 12 °C before being injected into the HPLC system and analyzed using a fluorescence monitor (excitation wavelength, 340 nm; emission wavelength, 445 nm). Xestospongin C (Calbiochem) an IP3 calcium channel receptor antagonist, was applied at 1 μm for 1 h before the assay. U-73122, a PLC-γ inhibitor, was applied for 1 h at 2 μm (28Jin W., Lo, T.M. Loh H.H. Thayer S.A. Brain Res. 1994; 642: 237-243Crossref PubMed Scopus (120) Google Scholar). LY294002 (Calbiochem), a PI3 kinase inhibitor, and PD98059 (Calbiochem), a MAP kinase inhibitor, were applied for 30 min at 50 and 10 μm, respectively (29Yano H. Agatsuma T. Nakanishi S. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. Biochem. J. 1995; 312: 145-150Crossref PubMed Scopus (60) Google Scholar, 30Reiners J.J.Jr. Lee J.Y. Clift R.E. Dudley D.T. Myrand S.P. Mol. Pharmacol. 1998; 53: 438-445Crossref PubMed Scopus (125) Google Scholar).l-trans-2, 4-PDC (t-PDC) (Research Biochemicals) was applied for 20 min before the assay at 10 μm, whiledl-threo-β-benzyloxyaspartate (dl-TBOA) (a gift from Suntory Institute for Bioorganic Research, Osaka) was applied at 10 μm and tetrodotoxin (TTX) (Latoxan) at 0.5 μm. When the effects of the various drugs on BDNF-induced glutamate release were examined, the neurons were washed three times, and samples of the basal and BDNF-induced release in the presence of these drugs were collected. BDNF, NT-3, NT-4/5, and NGF were applied by bath application. Cells were cultured on polyethyleneimine-coated cover glasses (Matsunami, Osaka, Japan) attached to flexiperm (IN VITRO, Kalkberg, Osterode). Cells were washed three times with KRH and incubated for 1 h at 37 °C with 10 μm Fluo-3 AM diluted in KRH. The cells were then washed, and the cover glasses placed on an inverted microscope (TMD-300, Nikon). The dye intensity was monitored using a confocal laser microscope (RCM 8000, Nikon). Neurons were irradiated with an excitation blue light beam (488 nm) produced by an argon ion laser at a scanning frequency of one-thirtieth of a second. The emitted fluorescence was guided through a ×40 water-immersion objective to a pinhole diaphragm at 520 nm using a diachronic mirror. The intensity of emission from each neuron targeted was scanned at one-thirtieth of a second intervals with a monitor video enhancer. BAPTA-AM (100 nm) (Research Biochemicals) and thapsigargin (1 μm) (Research Biochemicals) were added 30 min prior to the application of BDNF. Xestospongin C (1 μm) and U-73122 (2 μm) were added 30 min prior to the imaging assay. dl-TBOA and t-PDC were applied 20 min before the assay at 10 μm respectively. Cells were lysed in SDS lysis buffer containing 1% SDS, 20 mm Tris-HCl (pH 7.4), 5 mm EDTA (pH 8.0), 10 mm NaF, 2 mmNa3VO4, 0.5 mm phenylarsine oxide, and 1 mm phenylmethylsulfonyl fluoride. The lysates were boiled for 3 min, and then clarified by ultracentrifugation at 60,000 × g for 30 min at 8 °C. The protein concentration of the supernatants was determined using a BCA protein assay kit (Pierce), and then 10-μg aliquots of protein were resolved by electrophoresis on 10% SDS-polyacrylamide gels. Proteins were transferred onto polyvinylidene fluoride membranes (Millipore) in 0.1m 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 (T-TBS) containing 5% nonfat dried milk at room temperature for 1 h. Membranes were then probed with 1:500 dilution of anti-phospho-Trk antibody, 1:1000 of anti-Trk antibody, 1:1000 anti-PLC-γ antibody or 1:1500 of anti-phospho-Tyr antibody in T-TBS containing 1% nonfat dried milk at room temperature for 1 h. After three washes with T-TBS, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or donkey anti-mouse IgG secondary antibody (Zymed Laboratories Inc. or Jackson ImmunoResearch Laboratories) and diluted 1:1000 with T-TBS at room temperature for 1 h. They were then washed at least four times with T-TBS and visualized using the ECL chemiluminescence system (Amersham Biosciences or Immunostar, Wako). The cell lysates for immunoprecipitation assay were prepared using Triton lysis buffer containing 1% Triton X-100, 20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 5 mm EDTA (pH 8.0), 10 mm NaF, 2 mmNa3VO4, 0.5 mm phenylarsine oxide, and 1 mm phenylmethylsulfonyl fluoride. Subsequently, 250-μg aliquots of protein were mixed with 1 μg of anti-PLC-γ antibody and the samples incubated at 4 °C for at least 3 h. Protein G-Sepharose (10 μl of gel) was then added and the mixtures rotated at 4 °C for 1 h. The immune complexes were pelleted by centrifugation at 10,000 × g and 4 °C for 1 min before being washed three times with the Triton buffer. After SDS-PAGE, the proteins were immunoblotted with anti-phospho-Tyr antibody. The anti-phospho-Trk antibody (31Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (471) Google Scholar) was purchased from New England Biolabs, whereas the anti-phospho-Tyr and anti-p85 antibodies were from Upstate Biotechnology. The anti-PLC-γ, anti-Trk (32Kaplan D.R. Martin-Zanca D. Parada L.F. Nature. 1991; 350: 158-160Crossref PubMed Scopus (839) Google Scholar), and anti-MAPK antibodies were from Santa Cruz Biotechnology. During the development of cultured neurons obtained from neonatal rat cerebral cortex, networks among neurons were formed, and after this synchronized spontaneous Ca2+ oscillatory activity appeared in the cultured cortical neurons. The synchronous Ca2+ oscillations in developing neurons seemed to indicate the formation of chemical synapses. To investigate the acute effect of BDNF on spontaneous neuronal activities in cultured CNS neurons, we focused on the BDNF-induced elevation in the frequency of synchronized spontaneous Ca2+ oscillations in cultured cortical neurons. First, we characterized the cortical spontaneous Ca2+ oscillations. The changes of spontaneous Ca2+ oscillations in the cultured cells during neuronal development are shown (Fig. 1,A and B). Fig. 1, A and B, respectively, gives the results from two independent series of developmental experiments. The synchronous spontaneous Ca2+oscillations begun at around DIV 5 in cultured neurons prepared from P2–3-day-old rats (Fig. 1, A and B). At DIV 6 or DIV 7, the frequencies of the Ca2+ oscillations were greater than in cultured neurons at DIV 5. The synchronous Ca2+ oscillations were observed in both neurites and soma, and the time-dependence of the oscillations in the cell bodies was similar to that in neurites (Fig. 1, C and D). Although some cells in culture did not show oscillatory activities (Fig. 1 D, c4), in this study, we focused on the synchronous spontaneous Ca2+ oscillatory activity. The quantitative data of intracellular Ca2+ were obtained from the analysis of each cell body targeted, because we could detect a much higher intensity of Ca2+ dye emission in cell bodies than in neurites. The cells that showed synchronized activity were clearly defined. To identify which cell population displays the activity, we performed an immunocytochemical staining study using anti-GLU, anti-glutaminase (a marker for glutamatergic neurons) and anti-MAP2 (a marker for neurons) antibodies after the Ca2+ imaging experiment. We confirmed that the cells in synchronized Ca2+ oscillations (Fig.2 A, aandb) were MAP2 positive (Fig. 2 A, c), indicating that they were neurons. Almost all of the neurons showing oscillations could be labeled with anti-GLU (Fig. 2 A,d). Further, we confirmed that cortical neurons displaying oscillations (Fig. 2 A, e and f) express glutaminase and this immunoreactivity was observed in the axons of neurons (Fig. 2 A, g), indicating that they were glutamatergic neurons (33Kaneko T. Kang Y. Mizuno N. J. Neurosci. 1995; 15: 8362-8377Crossref PubMed Google Scholar). To examine the involvement of glutamate receptors and voltage-dependent Ca2+channels in the spontaneous Ca2+ oscillations, several antagonists were applied. Cd2+, a voltage-dependent Ca2+ channel blocker, reduced the Ca2+ oscillations (Fig. 2 B, a). APV (a NMDA receptor inhibitor, 10 μm) and CNQX (an AMPA receptor inhibitor, 10 μm) also blocked the Ca2+ oscillations (Fig. 2 B,b). In an extracellular Ca2+-free condition, no spontaneous Ca2+ oscillations were observed (Fig.2 B, c). TTX (a Na+ channel blocker) also abolished the oscillations (Fig. 2 B, d). In contrast, the GABA receptor antagonists, bicuculline (10 μm) and/or saclofen (10 μm) did not block the spontaneous Ca2+ activity. The frequency of oscillations at DIV 6 in the presence of both bicuculline and saclofen was 109 ± 13.1% of nontreated basal activity. These results suggested that voltage-dependent Ca2+ channels and ionotropic glutamate receptors were involved in the synchronous spontaneous Ca2+ oscillations of cultured cortical neurons. To test the possibility that BDNF enhanced the synaptic connectivity, we examined the acute action of BDNF on the spontaneous Ca2+ activity. Application of BDNF potentiated the frequency of the synchronous Ca2+oscillations at DIV 6 (Fig.3 A, top). Although in some cases, we observed a resting cortical culture at around DIV 5 or later, BDNF was still capable of triggering continuous Ca2+ oscillations in the resting neurons (data not shown). In contrast, at DIV 3 BDNF induced a transient increase in intracellular Ca2+, but it did not initiate continuous Ca2+ oscillations (Fig. 3 A, bottom), indicating that the BDNF-potentiated Ca2+ oscillations in cultured cortical neurons required in vitro maturation. The BDNF-potentiated Ca2+ oscillations were sustained for 15–20 min (Fig. 3 B). In some cases, the oscillatory activity was sustained for longer (40 min). NT-3 and NT-4/5 also potentiated the activity, but NGF did not (Fig. 3 C). BDNF and NT-4/5 enhanced the oscillatory activity by the same amount degree. The effect of NT-3 was less than that of BDNF. In the presence of K252a, BDNF did not potentiate the Ca2+ oscillations, although K252a alone did not influence the basal activity (Fig.3 C). Cortical neurons are known to express TrkB and TrkC but not TrkA receptors. As expected, BDNF, NT-3, and NT-4/5 significantly induced tyrosine phosphorylation of Trk receptors (Fig. 3 D), but NGF did not. These results strongly suggested that the neurotrophin-potentiated Ca2+ oscillations required the Trk activation. It was revealed that the cortical spontaneous Ca2+ oscillations were mediated by glutamate. Previously, we showed that BDNF triggered a rapid glutamate release from the neurons. To test for a correlation between the two phenomena, we examined the BDNF-induced glutamate release during neuronal development (Fig. 4 A). BDNF did not induce glutamate release in early cultured cortical neurons (at DIV 3 and 4). On the other hand, after 5 days in vitro culture or more (DIV 6–9), BDNF induced significant glutamate release within 1 min after exogenous application. These results indicated that the two phenomena, the BDNF-potentiated Ca2+ oscillations and the BDNF-induced glutamate release, occurred over the same period ofin vitro maturation. The HK+-evoked glutamate release was also observed at DIV 5 or later. The extent of HK+-evoked glutamate release was greater than that of the BDNF-induced one at DIV 7 and DIV 9. If glutamate release induced by BDNF modulates the Ca2+oscillations frequency, then an increase in the concentration of glutamate in the extracellular solution should shift the concentration dependence of the BDNF-potentiated effect on the oscillations. BDNF at 5 ng/ml alone induced transient Ca2+ increases but rarely potentiated the frequency of Ca2+ oscillations (Fig.4 B, c). Glutamate (1 μm) also triggered transient Ca2+ increases without any frequency potentiation (Fig. 4 B, b). However, in the presence of glutamate (1 μm) and 5 ng/ml BDNF, the oscillatory activity was potentiated to the same level as that induced by BDNF alone at 100 ng/ml (Fig. 4 B, d ande). In contrast, the oscillations induced by co-application of glutamate (1 μm) with BDNF at 100 ng/ml did not result in any further potentiation over that produced by 100 ng/ml of BDNF alone (Fig. 4 C). These results suggested that the BDNF-potentiated spontaneous Ca2+ oscillations were indeed because of the BDNF-induced glutamate release. Application of glutamate at higher concentrations (50 or 100 μm) induced a burst of intracellular Ca2+ increases and resulted in termination of the spontaneous oscillations (data not shown). Thus, we compared the sensitivities to BDNF (5 or 100 ng/ml) with or without glutamate (1 μm). We previously reported that the BDNF-induced glutamate release from cultured cortical neurons was dependent on intracellular Ca2+ (34Takei N. Numakawa T. Kozaki S. Sakai N. Endo Y. Takahashi M. Hatanaka H. J. Biol. Chem. 1998; 273: 27620-27624Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and that the PLC-γ/IP3 pathway was essential for the glutamate release in cultured cerebellar neurons (35Numakawa T. Matsumoto T. Adachi N. Yokomaku D. Kojima M. Takei N. Hatanaka H. J. Neurosci. Res. 2001; 66: 96-108Crossref PubMed Scopus (50) Google Scholar). As shown in TableI, we confirmed the Ca2+dependence of the BDNF-induced release in cultured cortical neurons. In the extracellular Ca2+-free condition, BDNF was still able to induce the glutamate release. On the other hand, BAPTA-AM (an intracellular Ca2+ chelator) or thapsigargin (which causes a depletion of the intracellular Ca2+ pools) completely abolished the BDNF-induced release. Furthermore, we examined the PLC-γ/IP3 pathway contribution to the BDNF-induced release. Xestospongin C, a cell-permeable IP3 receptor antagonist, completely blocked the BDNF-induced release. U-73122, an inhibitor of PLC-γ, also blocked the release. On the other hand, PD98059, a MAP kinase inhibitor, and LY294002, a PI3 kinase inhibitor, had no effect (data not shown). These results suggested that the PLC-γ/IP3/Ca2+ pathway is essential for the BDNF-induced glutamate release from cultured cortical neurons as previously shown in cultured cerebellar neurons (35Numakawa T. Matsumoto T. Adachi N. Yokomaku D. Kojima M. Takei N. Hatanaka H. J. Neurosci. Res. 2001; 66: 96-108Crossref PubMed Scopus (50) Google Scholar).Table IBDNF induces glutamate release in an intracellular Ca2+-dependent mannerNoneCa2+-freeBAPTA-AMThapsigarginXestosponginCU-73122×10−10 mol/wellBasal33.7 ± 4.9849.4 ± 8.1448.1 ± 8.4747.6 ± 6.0236.2 ± 4.1232.9 ± 2.98BDNF84.5 ± 10.2**96.0 ± 20.3**41.8 ± 3.3937.9 ± 5.5139.3 ± 6.8137.8 ± 4.89Cultured cortical neurons (at DIV 6) were treated with BDNF for 1 min by bath application at a concentration of 100 ng/ml. Basal release was measured by collecting for 1 min before the application. BAPTA-AM (100 nm) and thapsigargin (1 μm) were added 30 min prior to BDNF, while U-73122 (2 μm) and xestospongin C (1 μm) were applied to the cultured neurons for 1 h before the assay. Amino acid relea
Referência(s)