摘要
血管内皮生长因子(vascular endothelial growth factor,VEGF)最初发现于内皮细胞,以后又发现它也在脑内广泛表达。我们实验室以往研究结果揭示VEGF及其受体在幼年和成年大鼠脑内神经元均有表达,而且在急性分离的海马神经元,VEGF还可以通过钾通道的kv1.2蛋白酪氨酸磷酸化抑制延迟外向整合钾电流。最近,又有文献报导在海马脑片,VEGF可以抑制由电刺激兴奋性突触通路所诱导的突触后电位。
为研究VEGF对中枢神经递质传递的直接调节作用,本实验采用全细胞膜片钳技术研究了VEGF对出生后14天大鼠海马脑片CA1区谷氨酸能兴奋性突触传递的作用以及γ-氨基丁酸能抑制性突触传递的作用及其机制。实验结果如下:
一、VEGF对神经递质释放调节及其机制分析
1.为了研究VEGF对突触传递的影响,我们首先观察了VEGF对自发的谷氨酸能兴奋性突触后电流(glutamatergic spontaneous excitatory postsynaptic currents,sEPSCs)和自发的γ-氨基丁酸能抑制性突触后电流(γ-amino-butylic acid ergicspontaneous inhibitory postsynaptic currents,GABAergic sIPSCs)的作用。研究记录了30个细胞,发现VEGF具有分别促进sEPSCs频率和抑制sIPSCs频率及幅度的作用,分别占记录细胞的30%和50%。该结果表明VEGF具有快速调节突触传递的作用。为了区分VEGF的这一作用究竟是突触前还是突触后的调节效应,我们从方法学的可行性角度出发,进一步研究了VEGF对γ-氨基丁酸能抑制性突触传递的作用并进行了深入探讨。
2.为了明确VEGF是否对突触后GABA受体有直接调节作用,我们研究了VEGF对外源性GABA诱导的突触后电流的作用。外源性GABA所诱导的突触后电流的幅度可以作为突触后GABA受体反应性的指标。研究结果表明VEGF对外源性GABA诱导的突触后电流没有影响,提示VEGF对突触后GABA受体反应性没有直接调节作用。
3.为了明确VEGF是否对突触前GABA的释放有抑制效应,我们以微小的γ-氨基丁酸能抑制性突触后电流(GABAergic miniature inhibitory postsynapticcurrents,GABAergic mIPSCs)的频率作为突触前轴突末梢GABA释放的指标,观察了VEGF对GABA释放的调节效应。研究观察到VEGF显著抑制了mIPSCs的频率,并有剂量依赖性。该结果提示VEGF通过突触前水平的调节,抑制了轴突末梢GABA的释放。
4.为研究VEGF突触前抑制GABA释放的机制,我们分别用氯化钡和氯化隔阻断钾离子通道和高电压激活的钙离子通道(high-voRage-activated calciumchannels,HVA calcium channels),然后观察VEGF对mIPSCs频率的影响。研究观察到,当氯化钡存在时VEGF仍然显著抑制mIPSCs频率。而当氯化镉存在时,VEGF对mIPSCs频率的抑制作用被取消。这些结果提示VEGF是通过突触前轴突末梢电压依赖的钙离子通道(voltage-dependent calcium channels,VDCCs)抑制GABA的释放,同时也提示钾离子通道与VEGF的这一作用无关。
二、VEGF对锥体神经元高电压激活的钙电流影响的研究
以上结果提示VEGF参与了神经传递的调节,包括促进谷氨酸能兴奋性神经传递和抑制GABA能抑制性神经传递。机制分析表明VEGF通过VDCCs抑制突触前轴突末梢的GABA释放。VDCCs尤其是HVA钙离子通道不仅参与神经递质释放,而且在神经元的发育成熟和神经损伤中都起了重要作用。因此我们又研究了VEGF对HVA钙电流的直接作用。结果如下:
1.VEGF可以快速可逆地、剂量依赖地抑制大鼠海马CA1区锥体神经元HVA钙电流。
2.VEGF对HVA钙电流的电压依赖性没有影响。
该结果提示VEGF对大鼠海马CA1区锥体神经元HVA钙电流具有直接快速的、可逆的和剂量依赖的抑制作用。
结论:VEGF能快速调节突触前神经递质的释放以及抑制高电压激活的钙电流。机制分析揭示VEGF通过电压依赖的钙通道抑制突触前轴突末梢的GABA释放。
创新点:
1.首次提出VEGF具有促进谷氨酸能神经传递和抑制突触前GABA释放的作用。
2.首次提出VEGF具有快速抑制神经元细胞膜高电压激活的钙电流的作用。
3.首次提出VEGF通过抑制电压依赖的钙通道抑制突触前轴突末梢的GABA释放。
Vascular endothelial growth factor (VEGF) was originally purified from endothelial cells. It has been demonstrated that VEGF widely expresses in the mammalian brain. Our previous studies have revealed that VEGF and its receptors locate at neurons in both immature and adult rat brains, and that VEGF inhibits the delayed outward potassium currents in hippocampal neurons via enhancing tyrosine phosphorylation of potassium channel kv 1.2 protein. In addition, a recent article reported that VEGF reduced the evoked postsynaptic potentials triggered by stimulating the excitatory pathways in hippocampal slices.
To investigate whether VEGF regulates the neurotransmission, we used the whole-cell patch-clamp technique to record the glutamatergic spontaneous excitatory and GABAergic inhibitory postsynaptic currents (sEPSCs and sIPSCs) as well as the miniature inhibitory postsynaptic currents (mlPSCs) in the CAl pyramidal neurons of hippocampal slices from 14-day-old rats. The results are as follows:
1. VEGF modulates the release of neurotransmitters in the hippocampus.
(1) To study whether VEGF has any effect on neurotransmission, we separately recorded the glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) and the GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the hippocampal slices. We observed that VEGF significantly increased the frequency of sEPSCs and reduced the frequency and amplitude of sIPSCs. Among those recorded neurons, 30% and 50% of recorded neurons showed increase of sEPSCs and decrease of sIPSCs by VEGF, respectively. Then, we further analyzed whether the reduction of sIPSCs by VEGF was associated with the presynaptic or postsynaptic inhibition.
(2) To study whether VEGF directly modulates the activities of postsynaptic GABA receptors, we recored the exogenous GABA-evoked postsynaptic chloride currents in hippocampal slices. The result showed that VEGF did not change the exogenous GABA-induced postsynaptic chloride currents, suggesting that VEGF-decreased sIPSCs does not relate to postsynaptic inhibition. Therefore, we further studied the effect of VEGF on the GABAergic miniature inhibitory postsynaptic currents (mlPSCs), of which frequency can reflect the release of GABA from presynaptic axonal terminals. We observed that VEGF dose-dependently reduced the mIPSC frequency. The results clearly demonstrated that the reduction of sIPSCs by VEGF was caused by inhibition of GABA release from the presynaptic axonal terminals.
(3 ) To test the mechanism of the inhibition of GABA release by VEGF, we further studied the effect of VEGF on the release of GABA in the presence of barium, a general blocker of potassium channels, and cadmium, a nonspecific blocker of high-voltage-activated calcium channels (HVA calcium channels). The result showed that cadmium not barium prevented the reduction of the mIPSC frequency by VEGF. These results indicate that VEGF suppresses the release of GABA via HVA calcium channels. Furthermore, SU1498, a specific inhibitor of Flk-1, did not influence the efficacy of VEGF-inhibited mIPSCs, suggesting that Flk-1 is not involved in the VEGF's inhibition.
Putting together, the results suggest that VEGF enhances the gluatmatergic neurotransmission, and suppresses the GABA release from presynaptic neuronal terminals via VDCCs.
2. VEGF inhibits HVA calcium currents in the CAl pyramidal neurons of hippocampal slices.
As mentioned above, the inhibition of GABA release by VEGF is associated with the activities of VDCCs, especially the HVA calcium channels. Therefore, we next studied the effect of VEGF on the HVA calcium currents in the CAl pyramidal neurons of hippocampal slices from 14-day-old rats. The results showed that VEGF rapidly, reversibly, dose-dependently decreased the HVA calcium currents, and that VEGF did not influence the voltage-dependent property of HVA calcium currents.
Collectively, VEGF can directly and specifically reduce the activities of HVA calcium channels in the hippocampal CAl pyramidal neurons.
In the present thesis, we provide the first evidence that VEGF can directly modulate the release of neurotransmitters from the presynaptic axonal terminals via voltage-dependent calcium channels.
引文
1.Ferrara,N.and W.J.Henzel,Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells.Biochem Biophys Res Commun,1989.161(2):p.851-8.
2.Ferrara,N.,H.P.Gerber,and J.LeCouter,The biology of VEGF and its receptors.Nat Med,2003.9(6):p.669-76.
3.Ogunshola,O.O.,et al.,Neuronal VEGF expression correlates with angiogenesis in postnatal developing rat brain.Brain Res Dev Brain Res,2000.119(1):p.139-53.
4.Bao,W.L.,et al.,Intraventricular vascular endothelial growth factor antibody increases infarct volume following transient cerebral ischemia.Zhongguo Yao Li Xue Bao,1999.20(4):p.313-8.
5.Yang,S.Z.,et al.,Distribution of Flk-1 and Flt-1 receptors in neonatal and adult rat brains.Anat Rec A Discov Mol Cell Evol Biol,2003.274(1):p.851-6.
6.Ogunshola,O.O.,et al.,Paracrine and autocrine functions of neuronal vascular endothelial growth factor(VEGF) in the central nervous system.J Biol Chem,2002.277(13):p.11410-5.
7.Brockington,A.,et al.,Vascular endothelial growth factor and the nervous system.Neuropathol Appl Neurobiol,2004.30(5):p.427-46.
8.Jin,K.,et al.,Vascular endothelial growth factor(VEGF) stimulates neurogenesis in vitro and in vivo.Proc Natl Acad Sci U S A,2002.99(18):p.11946-50.
9.Zhu,Y.,et al.,Vascular endothelial growth factor promotes proliferation of cortical neuron precursors by regulating E2F expression.Faseb J,2003.17(2):p.186-93.
10.Silverman,W.F.,et al.,Vascular,glial and neuronal effects of vascular endothelial growth factor in mesencephalic explant cultures.Neuroscience,1999.90(4):p.1529-41.
11.Sondell,M.,F.Sundler,and M.Kanje,Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1receptor.Eur J Neurosci,2000.12(12):p.4243-54.
12.Khaibullina,A.A.,J.M.Rosenstein,and J.M.Krum,Vascular endothelial growth factor promotes neurite maturation in primary CNS neuronal cultures. Brain Res Dev Brain Res,2004.148(1):p.59-68.
13.Sondell,M.,G.Lundborg,and M.Kanje,Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth,enhancing cell survival and Schwann cell proliferation in the peripheral nervous system.J Neurosci,1999.19(14):p.5731-40.
14.Carmeliet,P.,Blood vessels and nerves:common signals,pathways and diseases.Nat Rev Genet,2003.4(9):p.710-20.
15.Cao,L.,et al.,VEGF links hippocampal activity with neurogenesis,learning and memory.Nat Genet,2004.36(8):p.827-35.
16.Fabel,K.,et al.,VEGF is necessary for exercise-induced adult hippocampal neurogenesis.Eur J Neurosci,2003.18(10):p.2803-12.
17.Thoenen,H.,Neurotrophins and neuronal plasticity.Science,1995.270(5236):p.593-8.
18.Terlau,H.and W.Seifert,Influence of epidermal growth factor on long-term potentiation in the hippocampal slice.Brain Res,1989.484(1-2):p.352-6.
19.Abe,K.,F.J.Xie,and H.Saito,Epidermal growth factor enhances short-term potentiation and facilitates induction of long-term potentiation in rat hippocampal slices.Brain Res,1991.547(1):p.171-4.
20.Kang,H.and E.M.Schuman,Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus.Science,1995.267(5204):p.1658-62.
21.Lohof,A.M.,N.Y.Ip,and M.M.Poo,Potentiation of developing neuromuscular synapses by the neurotrophins NT-3 and BDNF.Nature,1993.363(6427):p.350-3.
22.Lessmann,V.,K.Gottmann,and R.Heumann,BDNF and NT-4/5 enhance glutamatergic synaptic transmission in cultured hippocampal neurones.Neuroreport,1994.6(1):p.21-5.
23.Knipper,M.,et al.,Neurotrophin induced second messenger responses in rat brain synaptosomes.Neuroreport,1993.4(5):p.483-6.
24.Canossa,M.,et al.,Neurotrophin release by neurotrophins:implications for activity-dependent neuronalplasticity.Proc Natl Acad Sci U S A,1997.94(24):p.13279-86.
25.Holm,N.R.,et al.,Activation of calcium-dependent potassium channels in mouse[correction of rat]brain neurons by neurotrophin-3 and nerve growth factor.Proc Natl Acad Sci U S A,1997.94(3):p.1002-6.
26.Hilborn,M.D.,R.R.Vaillancourt,and S.G.Rane,Growth factor receptor tyrosine kinases acutely regulate neuronal sodium channels through the src signaling pathway J Neurosci,1998.18(2):p.590-600.
27.Wildering,W.C.,et al.,Nerve growth factor(NGF) acutely enhances high-voltage-activated calcium currents in molluscan neurons.J Neurophysiol,1995.74(6):p.2778-81.
28.Xu,J.Y.,et al.,Vascular endothelial growth factor inhibits outward delayed-rectifier potassium currents in acutely isolated hippocampal neurons.Neuroscience,2003.118(1):p.59-67.
29.Qiu,M.H.,R.Zhang,and F.Y.Sun,Enhancement of ischemia-induced tyrosine phosphorylation of Kvl.2 by vascular endothelial growth factor via activation of phosphatidylinositol 3-kinase.J Neurochem,2003.87(6):p.1509-17.
30.McCloskey,D.P.,S.D.Croll,and H.E.Scharfman,Depression of synaptic transmission by vascular endothelial growth factor in adult rat hippocampus and evidence for increased efficacy after chronic seizures.J Neurosci,2005.25(39):p.8889-97.
31.Rosenstein,J.M.and J.M.Krum,New roles for VEGF in nervous tissue-beyond blood vessels.Exp Neurol,2004.187(2):p.246-53.
32.Morrisett,R.A.and H.S.Swartzwelder,Attenuation of hippocampal long-term potentiation by ethanol:a patch-clamp analysis of glutamatergic and GABAergic mechanisms.J Neurosci,1993.13(5):p.2264-72.
33.Thomas,M.P.,D.T.Monaghan,and R.A.Morrisett,Evidence for a causative role of N-methyl-D-aspartate receptors in an in vitro model of alcohol withdrawal hyperexcitability.J Pharmacol Exp Ther,1998.287(1):p.87-97.
34.Cossart,R.,et al.,Distribution of spontaneous currents along the somato-dendritic axis of rat hippocampal CA1 pyramidal neurons.Neuroscience,2000.99(4):p.593-603.
35.Roberto,M.,et al.,Ethanol increases GABAergic transmission at both preand postsynaptic sites in rat central amygdala neurons.Proc Natl Acad Sci U S A,2003.100(4):p.2053-8.
36.De Koninck,Y.and I.Mody,Noise analysis of miniature IPSCs in adult rat brain slices:properties and modulation of synaptic GABAA receptor channels. J Neurophysiol,1994.71(4):p.1318-35.
37.Porter,N.M.,et al.,Calcium channel density and hippocampal cell death with age in long-term culture.J Neurosci,1997.17(14):p.5629-39.
38.Redman,S.,Quantal analysis of synaptic potentials in neurons of the central nervous system.Physiol Rev,1990.70(1):p.165-98.
39.Hess,P.,Calcium channels in vertebrate cells.Annu Rev Neurosci,1990.13:p.337-56.
40.Mogul,D.J.and A.P.Fox,Evidence for multiple types of Ca2+ channels in acutely isolated hippocampal CA3 neurones of the guinea-pig.J Physiol,1991.433:p.259-81.
41.Mintz,I.M.,B.L.Sabatini,and W.G.Regehr,Calcium control of transmitter release at a cerebellar synapse.Neuron,1995.15(3):p.675-88.
42.Tsien,R.W.,P.T.Ellinor,and W.A.Horne,Molecular diversity of voltage-dependent Ca2+ channels.Trends Pharmacol Sci,1991.12(9):p.349-54.
43.Dunlap,K.,J.I.Luebke,and T.J.Turner,Exocytotic Ca2+ channels in mammalian central neurons.Trends Neurosci,1995.18(2):p.89-98.
44.Augustine,G.J.,M.P.Charlton,and S.J.Smith,Calcium action in synaptic transmitter release.Annu Rev Neurosci,1987.10:p.633-93.
45.Sher,E.and F.Clementi,Omega-conotoxin-sensitive voltage-operated calcium channels in vertebrate cells.Neuroscience,1991.42(2):p.301-7.
46.Potier,B.,P.Dutar,and Y.Lamour,Different effects of omega-conotoxin GVIA at excitatory and inhibitory synapses in rat CA1 hippocampal neurons.Brain Res,1993.616(1-2):p.236-41.
47.Turner,T.J.,M.E.Adams,and K.Dunlap,Calcium channels coupled to glutamate release identified by omega-Aga-IVA.Science,1992.258(5080):p.310-3.
48.Scanziani,M.,et al.,Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus.Neuron,1992.9(5):p.919-27.
49.Doze,V.A.,G.A.Cohen,and D.V.Madison,Calcium channel involvement in GABAB receptor-mediated inhibition of GABA release in area CA1 of the rat hippocampus.J Neurophysiol,1995.74(1):p.43-53.
50.Hahm,E.T.,et al.,Opioid inhibition of GABAergic neurotransmission in mechanically isolated rat periaqueductal gray neurons.Neurosci Res,2004.50(3):p.343-54.
51.Koyama,S.,et al.,Presynaptic serotonergic inhibition of GABAergic synaptic transmission in mechanically dissociated rat basolateral amygdala neurons.J Physiol,1999.518(Pt 2):p.525-38.
52.Trettel,J.and E.S.Levine,Cannabinoids depress inhibitory synaptic inputs received by layer 2/3 pyramidal neurons of the neocortex.J Neurophysiol,2002.88(1):p.534-9.
53.Jeong,H.J.,et al.,Adenosine A1 receptor-mediated presynaptic inhibition of GABAergic transmission in immature rat hippocampal CA1 neurons.J Neurophysiol,2003.89(3):p.1214-22.
54.Armstrong,C.M.,R.P.Swenson,Jr.,and S.R.Taylor,Block of squid axon K channels by internally and externally applied barium ions.J Gen Physiol,1982.80(5):p.663-82.
55.Solessio,E.,et al.,Characterization with barium of potassium currents in turtle retinal Muller cells.J Neurophysiol,2000.83(1):p.418-30.
56.Greengard,P.,et al.,Synaptic vesicle phosphoproteins and regulation of synaptic function.Science,1993.259(5096):p.780-5.
57.Owens,D.F.,et al.,Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging.J Neurosci,1996.16(20):p.6414-23.
58.Owens,D.F.,X.Liu,and A.R.Kriegstein,Changing properties of GABA(A)receptor-mediated signaling during early neocortical development.J Neurophysiol,1999.82(2):p.570-83.
59.Delpire,E.,Cation-Chloride Cotransporters in Neuronal Communication.News Physiol Sci,2000.15:p.309-312.
60.Ganguly,K.,et al.,GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition.Cell,2001.105(4):p.521-32.
61.Ben-Ari,Y.,Excitatory actions of gaba during development:the nature of the nurture.Nat Rev Neurosei,2002.3(9):p.728-39.
62.Plotkin,M.D.,et al.,Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains.a possible mechanism underlying GABA's excitatory role in immature brain.J Neurobiol,1997. 33(6):p.781-95.
63.Yan,Y.,et al.,Inhibition of Na(+)-K(+)-Cl(-) cotransporter during focal cerebral ischemia decreases edema and neuronal damage.Brain Res,2003.961(1):p.22-31.
64.Lenart,B.,et al.,Na-K-Cl cotransporter-mediated intracellular Na+accumulation affects Ca2+ signaling in astrocytes in an in vitro ischemic model.J Neurosci,2004.24(43):p.9585-97.
65.Chen,H.,et al.,Na(+)-dependent chloride transporter(NKCC1)-null mice exhibit less gray and white matter damage after focal cerebral ischemia.J Cereb Blood Flow Metab,2005.25(1):p.54-66.
66.Ben-Ari,Y.,et al.,GABAA,NMDA and AMPA receptors:a developmentally regulated 'menage a trois'.Trends Neurosci,1997.20(11):p.523-9.
67.Barbin,G.,et al.,Involvement of GABAA receptors in the outgrowth of cultured hippocampal neurons.Neurosci Lett,1993.152(1-2):p.150-4.
68.Ben-Ari,Y.,et al.,gamma-Aminobutyric acid(GABA):a fast excitatory transmitter which may regulate the development of hippocampal neurones in early postnatal life.Prog Brain Res,1994.102:p.261-73.
69.Owens,D.F.and A.R.Kriegstein,Is there more to GABA than synaptic inhibition? Nat Rev Neurosci,2002.3(9):p.715-27.
70.Yuste,R.and L.C.Katz,Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters.Neuron,1991.6(3):p.333-44.
71.LoTurco,J.J.,et al.,GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis.Neuron,1995.15(6):p.1287-98.
72.Marty,S.,et al.,GABAergic stimulation regulates the phenotype of hippocampal interneurons through the regulation of brain-derived neurotrophic factor.Neuron,1996.16(3):p.565-70.
73.Franklin,J.L.and E.M.Johnson,Jr.,Suppression of programmed neuronal death by sustained elevation of cytoplasmic calcium.Trends Neurosci,1992.15(12):p.501-8.
74.Bading,H.,D.D.Ginty,and M.E.Greenberg,Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways.Science,1993.260(5105):p.181-6.
75.Berninger,B.,et al.,GABAergic stimulation switches from enhancing to repressing BDNF expression in rat hippocampal neurons during maturation in vitro.Development,1995.121(8):p.2327-35.
76.Obrietan,K.and A.N.van den Pol,Growth cone calcium elevation by GABA.J Comp Neurol,1996.372(2):p.167-75.
77.Toyoda,H.,et al.,Induction of NMDA and GABAA receptor-mediated Ca2+oscillations with KCC2 mRNA downregulation in injured facial motoneurons.J Neurophysiol,2003.89(3):p.1353-62.
78.Furukawa,Y.,et al.,Reduction of voltage-dependent magnesium block of N-methyl-D-aspartate receptor-mediated current by in vivo axonal injury.Neuroscience,2000.96(2):p.385-92.
79.Buzsaki,G.,Theta oscillations in the hippocampus.Neuron,2002.33(3):p.325-40.
80.Sun,M.K.,et al.,Theta rhythm of hippocampal CA1 neuron activity:gating by GABAergic synaptic depolarization.J Neurophysiol,2001.85(1):p.269-79.
81.Wong,T.,et al.,Postnatal development of intrinsic GABAergic rhythms in mouse hippocampus.Neuroscience,2005.134(1):p.107-20.
82.Yoder,R.M.and K.C.Pang,Involvement of GABAergic and cholinergic medial septal neurons in hippocampal theta rhythm.Hippocampus,2005.15(3):p.381-92.
83.Kater,S.B.,et al.,Calcium regulation of the neuronal growth cone.Trends Neurosci,1988.11(7):p.315-21.
84.Koike,T.,D.P.Martin,and E.M.Johnson,Jr.,Role of Ca2+ channels in the ability of membrane depolarization to prevent neuronal death induced by trophic-factor deprivation:evidence that levels of internal Ca2+ determine nerve growth factor dependence of sympathetic ganglion cells.Proc Natl Acad Sci USA,1989.86(16):p.6421-5.
85.Lipton,S.A.and S.B.Kater,Neurotransmitter regulation of neuronal outgrowth,plasticity and survival.Trends Neurosci,1989.12(7):p.265-70.
86.Larmet,Y.,A.C.Dolphin,and A.M.Davies,Intracellular calcium regulates the survival of early sensory neurons before they become dependent on neurotrophic factors.Neuron,1992.9(3):p.563-74.
87.Mattson,M.P.and S.B.Kater,Calcium regulation of neurite elongation and growth cone motility.J Neurosci,1987.7(12):p.4034-43.
88.Basarsky,T.A.,V.Parpura,and P.G.Haydon,Hippocampal synaptogenesis in cell culture:developmental time course of synapse formation,calcium influx,and synaptic protein distribution.J Neurosci,1994.14(11 Pt 1):p.6402-11.
89.Turrigiano,G.,G.LeMasson,and E.Marder,Selective regulation of current densities underlies spontaneous changes in the activity of cultured neurons.J Neurosci,1995.15(5 Pt 1):p.3640-52.
90.Rusanescu,G.,et al.,Calcium influx induces neurite growth through a Src-Ras signaling cassette.Neuron,1995.15(6):p.1415-25.
91.Spitzer,N.C.,Spontaneous Ca2+ spikes and waves in embryonic neurons:signaling systems for differentiation.Trends Neurosci,1994.17(3):p.115-8.
92.Choi,D.W.,Calcium-mediated neurotoxicity:relationship to specific channel types and role in ischemic damage.Trends Neurosci,1988.11(10):p.465-9.
93.Choi,D.W.,Calcium:still center-stage in hypoxic-ischemic neuronal death.Trends Neurosci,1995.18(2):p.58-60.
94.Campbell,L.W.,et al.,Aging changes in voltage-gated calcium currents in hippocampal CA1 neurons.J Neurosci,1996.16(19):p.6286-95.
95.Griesbeck,O.,et al.,Are there differences between the secretion characteristics of NGF and BDNF? Implications for the modulatory role of neurotrophins in activity-dependent neuronal plasticity.Microsc Res Tech,1999.45(4-5):p.262-75.
96.Nabekura,J.,et al.,Reduction of KCC2 expression and GABAA receptor-mediated excitation after in vivo axonal injury.J Neurosci,2002.22(11):p.4412-7.
97.Vaccarino,F.M.,et al.,Differential induction of immediate early genes by excitatory amino acid receptor types in primary cultures of cortical and striatal neurons.Brain Res Mol Brain Res,1992.12(1-3):p.233-41.
1.Sivilotti,L.and A.Nistri,GABA receptor mechanisms in the central nervous system.Prog Neurobiol,1991.36(1):p.35-92.
2.Benke,D.,et al.,GABAA receptor subtypes differentiated by their gamma-subunit variants:prevalence,pharmacology and subunit architecture.Neuropharmacology,1996.35(9-10):p.1413-23.
3.Nayeem,N.,et al.,Quaternary structure of the native GABAA receptor determined by electron microscopic image analysis.J Neurochem,1994.62(2):p.815-8.
4.Macdonald,R.L.and R.W.Olsen,GABAA receptor channels.Annu Rev Neurosci,1994.17:p.569-602.
5.Kaupmann,K.,et al.,Expression cloning of GABA(B) receptors uncovers similarity to metabotropic glutamate receptors.Nature,1997.386(6622):p.239-46.
6.Bowery,N.,GABAB receptors and their significance in mammalian pharmacology Trends Pharmacol Sci,1989.10(10):p.401-7.
7.Enz,R.and G.R.Cutting,Molecular composition of GABAC receptors.Vision Res,1998.38(10):p.1431-41.
8.Nusser,Z.,et al.,Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells.Proc Natl Acad Sci U S A,1996.93(21):p.11939-44.
9.Fritschy,J.M.,et al.,Independent assembly and subcellular targeting of GABA(A)-receptor subtypes demonstrated in mouse hippocampal and olfactory neurons in vivo.Neurosci Lett,1998.249(2-3):p.99-102.
10.Gunther,U.,et al.,Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.Proc Natl Acad Sci U S A,1995.92(17):p.7749-53.
11.Bamard,E.A.,et al.,International Union of Pharmacology XV.Subtypes of gamma-aminobutyric acidA receptors:classification on the basis of subunit structure and receptor function.Pharmaeol Rev,1998.50(2):p.291-313.
12.Laurie,D.J.,W.Wisden,and P.H.Seeburg,The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain.Ⅲ.Embryonic and postnatal development.J Neurosci,1992.12(11):p.4151-72.
13.Rovira,C.and Y.Ben-Ari,Developmental study of benzodiazepine effects on monosynaptic GABAA-mediated IPSPs of rat hippocampal neurons.J Neurophysiol,1993.70(3):p.1076-85.
14.Stephenson,F.A.,The GABAA receptors.Biochem J,1995.310(Pt 1):p.1-9.
15.Madl,J.E.and S.M.Royer,Glutamate dependence of GABA levels in neurons of hypoxic and hypoglycemic rat hippocampal slices.Neuroscience,2000.96(4):p.657-64.
16.Hollrigel,G.S.and I.Soltesz,Slow kinetics of miniature IPSCs during early postnatal development in granule cells of the dentate gyrus.J Neurosci,1997.17(13):p.5119-28.
17.Kneussel,M.,Dynamic regulation of GABA(A) receptors at synaptic sites.Brain Res Brain Res Rev,2002.39(1):p.74-83.
18.Moss,S.J.and T.G.Smart,Constructing inhibitory synapses.Nat Rev Neurosci,2001.2(4):p.240-50.
19.Campochiaro,P.and J.T.Coyle,Ontogenetic development of kainate neurotoxicity:correlates with glutamatergic innervation.Proc Natl Acad Sci U S A,1978.75(4):p.2025-9.
20.Cherubini,E.,J.L.Gaiarsa,and Y.Ben-Ari,GABA:an excitatory transmitter in early postnatal life.Trends Neurosci,1991.14(12):p.515-9.
21.Holmes,G.L.,R.Khazipov,and Y.Ben-Ari,New concepts in neonatal seizures.Neuroreport,2002.13(1):p.A3-8.
22.Cheng,C.,D.M.Fass,and I.J.Reynolds,Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated [Ca(2+)](i) increases and NMDA receptor mRNA levels.Brain Res,1999.849(1-2):p.97-108.
23.Bickler,P.E.,S.M.Gallego,and B.M.Hansen,Developmental changes in intracellular calcium regulation in rat cerebral cortex during hypoxia.J Cereb Blood Flow Metab,1993.13(5):p.811-9.
24.Marks,J.D.,J.E.Friedman,and G.G.Haddad,Vulnerability of CA1 neurons to glutamate is developmentally regulated.Brain Res Dev Brain Res,1996.97(2):p.194-206.
25.Marks,J.D.,V.P.Bindokas,and X.M.Zhang,Maturation of vulnerability to excitotoxicity:intracellular mechanisms in cultured postnatal hippocampal neurons.Brain Res Dev Brain Res,2000.124(1-2):p.101-16.
26.Voutsinos-Porche,B.,et al.,Glial glutamate transporters mediate a functional metabolic crosstalk between neurons and astrocytes in the mouse developing cortex.Neuron,2003.37(2):p.275-86.
27.Ben-Ari,Y.,Excitatory actions of gaba during development:the nature of the nurture.Nat Rev Neurosci,2002.3(9):p.728-39.
28.Ben-Ari,Y.,et al.,Interneurons set the tune of developing networks.Trends Neurosci,2004.27(7):p.422-7.
29.Ganguly,K.,et al.,GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition.Cell,2001.105(4):p.521-32.
30.Obrietan,K.and A.N.van den Pol,GABA neurotransmission in the hypothalamus:developmental reversal from Ca2+ elevating to depressing.J Neurosci,1995.15(7 Pt 1):p.5065-77.
31.Delpire,E.,Cation-Chloride Cotransporters in Neuronal Communication.News Physiol Sci,2000.15:p.309-312.
32.Plotkin,M.D.,et al.,Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains.a possible mechanism underlying GABA's excitatory role in immature brain.J Neurobiol,1997.33(6):p.781-95.
33.Hubner,C.A.,et al.,Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition.Neuron,2001.30(2):p.515-24.
34.Stein,V.,et al.,Expression of the KCl cotransporter KCC2 parallels neuronal maturation and the emergence of low intracellular chloride.J Comp Neurol,2004.468(1):p.57-64.
35.Rodriguez,E.,et al.,Perception's shadow.long-distance synchronization of human brain activity.Nature,1999.397(6718):p.430-3.
36.Owens,D.F.and A.R.Kriegstein,Is there more to GABA than synaptic inhibition? Nat Rev Neurosci,2002.3(9):p.715-27.
37.Bolea,S.,et al.,Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices.J Neurophysiol,1999.81(5):p.2095-102.
38.Toyoda,H.,et al.,Induction of NMDA and GABAA receptor-mediated Ca2+oscillations with KCC2 mRNA downregulation in injured facial motoneurons.J Neurophysiol,2003.89(3):p.1353-62.
39.Leinekugel,X.,et al.,Ca2+ oscillations mediated by the synergistic excitatory actions of GABA(A) and NMDA receptors in the neonatal hippocampus.Neuron,1997.18(2):p.243-55.
40.Ben-Ari,Y.,et al.,Giant synaptic potentials in immature rat CA3 hippocampal neurones.J Physiol,1989.416:p.303-25.
41.Leinekugel,X.,Developmental patterns and plasticities:the hippocampal model.J Physiol Paris,2003.97(1):p.27-37.
42.Leinekugel,X.,et al.,Giant depolarizing potentials:the septal pole of the hippocampus paces the activity of the developing intact septohippocampal complex in vitro.J Neurosci,1998.18(16):p.6349-57.
43.Risold,P.Y.and L.W.Swanson,Structural evidence for functional domains in the rat hippocampus.Science,1996.272(5267):p.1484-6.
44.Stent,G.S.,A physiological mechanism for Hebb's postulate of learning.Proc Natl Acad Sci U S A,1973.70(4):p.997-1001.
45.Garaschuk,O.,E.Hanse,and A.Konnerth,Developmental profile and synaptic origin of early network oscillations in the CA1 region of rat neonatal hippocampus.J Physiol,1998.507(Pt 1):p.219-36.
46.Kandler,K.and L.C.Katz,Neuronal coupling and uncoupling in the developing nervous system.Curr Opin Neurobiol,1995.5(1):p.98-105.
47.Yuste,R.,et al.,Neuronal domains in developing neocortex:mechanisms of coactivation.Neuron,1995.14(1):p.7-17.
48.Wong,R.O.,et al.,Early functional neural networks in the developing retina.Nature,1995.374(6524):p.716-8.
49.Feller,M.B.,et al.,Requirement for cholinergic synaptic transmission in the propagation of spontaneous retinal waves.Science,1996.272(5265):p.1182-7.
50.Behar,T.N.,et al.,GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms.J Neurosci,1996.16(5):p.1808-18.
51.Behar,T.N.,et al.,GABA receptor antagonists modulate postmitotic cell migration in slice cultures of embryonic rat cortex.Cereb Cortex,2000.10(9):p.899-909.
52.Barbin,G.,et al.,Involvement of GABAA receptors in the outgrowth of cultured hippocampal neurons.Neurosci Lett,1993.152(1-2):p.150-4.
53.Ben-Ari,Y.,et al.,gamma-Aminobutyric acid(GABA):a fast excitatory transmitter which may regulate the development of hippocampal neurones in early postnatal life.Prog Brain Res,1994.102:p.261-73.
54.Desarmenien,M.G.and N.C.Spitzer,Role of calcium and protein kinase C in development of the delayed rectifier potassium current in Xenopus spinal neurons.Neuron,1991.7(5):p.797-805.
55.Gu,X.and N.C.Spitzer,Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients.Nature,1995.375(6534):p.784-7.
56.LoTurco,J.J.,et al.,GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis.Neuron,1995.15(6):p.1287-98.
57.Rusanescu,G.,et al.,Calcium influx induces neurite growth through a Src-Ras signaling cassette.Neuron,1995.15(6):p.1415-25.
58.Kornatsu,Y.and M.Iwakiri,Long-term modification of inhibitory synaptic transmission in developing visual cortex.Neuroreport,1993.4(7):p.907-10.
59.Fox,K.,The critical period for long-term potentiation in primary sensory cortex.Neuron,1995.15(3):p.485-8.
60.Crair,M.C.and R.C.Malenka,A critical periodfor long-term potentiation at thalamocortical synapses.Nature,1995.375(6529):p.325-8.
61.Kirkwood,A.,H.K.Lee,and M.F.Bear,Co-regulation of long-term potentiation and experience-dependent synaptic plasticity in visual cortex by age and experience.Nature,1995.375(6529):p.328-31.
62.Durand,G.M.,Y.Kovalchuk,and A.Konnerth,Long-term potentiation and functional synapse induction in developing hippocampus.Nature,1996.381(6577):p.71-5.
63.Leinekugel,X.,et al.,Correlated bursts of activity in the neonatal hippocampus in vivo.Science,2002.296(5575):p.2049-52.
64.Buzsaki,G.and J.J.Chrobak,Temporal structure in spatially organized neuronal ensembles:a role for interneuronal networks.Curr Opin Neurobiol,1995.5(4):p.504-10.
65.Draguhn,A.,et al.,Electrical coupling underlies high-frequency oscillations in the hippocampus in vitro.Nature,1998.394(6689):p.189-92.
66.Usher,M.and N.Donnelly,Visual synchrony affects binding and segmentation in perception.Nature,1998.394(6689):p.179-82.
67.O'Keefe,J.and M.L.Recce,Phase relationship between hippocampalplace units and the EEG theta rhythm.Hippocampus,1993.3(3):p.317-30.
68.Shen,J.,et al.,The effect of aging on experience-dependent plasticity of hippocampalplace cells.J Neurosci,1997.17(17):p.6769-82.
69.Skaggs,W.E.and B.L.McNaughton,Replay of neuronal firing sequences in rat hippocampus during sleep following spatial experience.Science,1996.271(5257):p.1870-3.
70.Paulsen,O.and E.I.Moser,A model of hippocampal memory encoding and retrieval:GABAergic control of synaptic plasticity.Trends Neurosci,1998.21(7):p.273-8.
71.Cobb,S.R.,et al.,Synchronization of neuronal activity in hippocampus by individual GABAergic interneurons.Nature,1995.378(6552):p.75-8.
72.Buhl,E.H.,K.Halasy,and P.Somogyi,Diverse sources of hippocampal unitary inhibitory postsynaptic potentials and the number of synaptic release sites.Nature,1994.368(6474):p.823-8.
73.Csicsvari,J.,et al.,Oscillatory coupling of hippocampal pyramidal cells and interneurons in the behaving Rat.J Neurosci,1999.19(1):p.274-87.
74.Dutar,P.,et al.,The septohippocampal pathway:structure and function of a central cholinergic system.Physiol Rev,1995.75(2):p.393-427.
75.Vertes,R.P.and B.Kocsis,Brainstem-diencephalo-septohippocampal systems controlling the them rhythm of the hippocampus.Neuroscience,1997.81(4):p.893-926.
76.Sun,M.K.,et al.,Them rhythm of hippocampal CA1 neuron activity:gating by GABAergic synaptic depolarization.J Neurophysiol,2001.85(1):p.269-79.
77.Pearce,R.A.,Physiological evidence for two distinct GABAA responses in rat hippocampus.Neuron,1993.10(2):p.189-200.
78.Freund,T.F.and G.Buzsaki,lnterneurons of the hippocampus.Hippocampus,1996.6(4):p.347-470.
79.Miles,R.,et al.,Differences between somatic and dendritic inhibition in the hippocampus.Neuron,1996.16(4):p.815-23.
80.Kapur,A.,et al.,Regulation of the NMDA component of EPSPs by different components of postsynaptic GABAergic inhibition:computer simulation analysis inpiriform cortex.J Neurophysiol,1997.78(5):p.2546-59.
81.Banks,M.I.,J.A.White,and R.A.Pearce,Interactions between distinct GABA(A) circuits in hippocampus.Neuron,2000.25(2):p.449-57.
82.Ouardouz,M.and J.C.Lacaille,Properties of unitary IPSCs in hippocampal pyramidal cells originating from different types of interneurons in young rats.J Neurophysiol,1997.77(4):p.1939-49.
83.Sik,A.,et al.,Hippocampal CA1 interneurons.an in vivo intracellular labeling study.J Neurosci,1995.15(10):p.6651-65.
84.Fukuda,T.,et al.,Dense GABAergic input on somata of parvalbumin-immunoreactive GABAergic neurons in the hippocampus of the mouse.Neurosci Res,1996.26(2):p.181-94.
85.Steegrnaier,M.,et al.,The E-selectin-ligand ESL-1 is a variant of a receptor for fibroblast growth factor Nature,1995.373(6515):p.615-20.
86.Roche,K.W.,W.G.Tingley,and R.L.Huganir,Glutamate receptor phosphorylation and synaptic plasticity.Curr Opin Neurobiol,1994.4(3):p.383-8.
87.Siegelbaum,S.A.,Channel regulation.Ion channel control by tyrosine phosphorylation.Curr Biol,1994.4(3):p.242-5.
88.Kim,H.G.,et al.,Neurotrophin 3 potentiates neuronal activity and inhibits gamma-aminobutyratergic synaptic transmission in cortical neurons.Proc Natl Acad Sci U S A,1994.91(25):p.12341-5.
89.Thoenen,H.,Neurotrophins and neuronal plasticity.Science,1995.270(5236):p.593-8.
90.Sommer,C.,et al.,Exogenous brain-derived neurotrophic factor prevents postischemic downregulation of[3H]muscimol binding to GABA(A) receptors in the cortical penumbra.Brain Res Mol Brain Res,2003.111(1-2):p.24-30.
91.Brunig,I.,et al.,BDNF reduces miniature inhibitory postsynaptic currents by rapid downregulation of GABA(A) receptor surface expression.Eur J Neurosci,2001.13(7):p.1320-8.
92.Kneussel,M.and H.Betz,Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites:the membrane activation model.Trends Neurosci,2000.23(9):p.429-35.
93.Wan,Q.,et al.,Recruitment of functional GABA(A) receptors to postsynaptic domains by insulin.Nature,1997.388(6643):p.686-90.
94.Shuaib,A.,et al.,Insulin elevates hippocampal GABA levels during ischemia.This is independent of its hypoglycemic effect.Neuroscience,1995.67(4):p. 809-14.
95.Griesbeck,O.,et al.,Are there differences between the secretion characteristics of NGF and BDNF? Implications for the modulatory role of neurotrophins in activity-dependent neuronal plasticity.Microsc Res Tech,1999.45(4-5):p.262-75.
96.Moss,S.J.and T.G.Smart,Modulation of amino acid-gated ion channels by protein phosphorylation.Int Rev Neurobiol,1996.39:p.1-52.
97.Krishek,B.J.,et al.,Regulation of GABAA receptor function by protein kinase C phosphorylation.Neuron,1994.12(5):p.1081-95.
98.Sciancalepore,M.and E.Cherubini,Protein kinase A-dependent increase in frequency of miniature GABAergic currents in rat CA3 hippocampal neurons.Neurosci Lett,1995.187(2):p.91-4.
99.McDonald,B.J.,et al.,Adjacent phosphorylation sites on GABAA receptor beta subunits determine regulation by cAMP-dependent protein kinase.Nat Neurosci,1998.1(1):p.23-8.
100.Inoue,M.,et al.,Intracellular calcium ions decrease the affinity of the GABA receptor.Nature,1986.324(6093):p.156-8.
101.Martina,M.,G.Kilic,and E.Cherubini,The effect of intracellular Ca2+ on GABA-activated currents in cerebellar granule cells in culture.J Membr Biol,1994.142(2):p.209-16.
102.Chen,Q.X.and R.K.Wong,Suppression of GABAA receptor responses by NMDA application in hippocampal neurones acutely isolated from the adult guinea-pig.J Physiol,1995.482(Pt 2):p.353-62.
103.Stelzer,A.and H.Shi,Impairment of GABAA receptor function by N-methyl-D-aspartate-mediated calcium influx in isolated CA1 pyramidal cells.Neuroscience,1994.62(3):p.813-28.
104.Lyons,H.R.,et al.,Distinct signal transduction pathways for GABA-induced GABA(A) receptor down-regulation and uncoupling in neuronal culture.a role for voltage-gated calcium channels.J Neurochem,2001.78(5):p.1114-26.
105.Wang,J.H.,Short-term cerebral ischemia causes the dysfunction of interneurons and more excitation of pyramidal neurons in rats.Brain Res Bull,2003.60(1-2):p.53-8.
106.Wang,X.,et al.,Profiles of glutamate and GABA efflux in core versus peripheral zones of focal cerebral ischemia in mice.Neurosci Lett,2001.313(3):p.121-4.
107.Shuaib,A.,et al.,GABA and glutamate levels in the substantia nigra reticulata following repetitive cerebral ischemia in gerbils.Exp Neurol,1997.147(2):p.311-5.
108.Iqbal,S.,et al.,Neuroprotective effect of tiagabine in transient forebrain global ischemia:an in vivo microdialysis,behavioral,and histological study.Brain Res,2002.946(2):p.162-70.
109.Phillis,J.W.,CI-966,a GABA uptake inhibitor,antagonizes ischemia-induced neuronal degeneration in the gerbil.Gen Pharmaeol,1995.26(5):p.1061-4.
110.Lyden,P.D.,et al.,Synergistic combinatorial stroke therapy.A quantal bioassay of a GABA agonist and a glutamate antagonist.Exp Neurol,2000.163(2):p.477-89.
111.Kristensen,B.W.,J.Noraberg,and J.Zimmer,The GABAA receptor agonist THIP is neuroprotective in organotypic hippocampal slice cultures.Brain Res,2003.973(2):p.303-6.
112.Grabb,M.C.,et al.,Preconditioned resistance to oxygen-glucose deprivation-induced cortical neuronal death:alterations in vesicular GABA and glutamate release.Neuroscience,2002.115(1):p.173-83.
113.Francis,A.and W.Pulsinelli,The response of GABAergic and cholinergic neurons to transient cerebral isehemia.Brain Res,1982.243(2):p.271-8.
114.Frahm,C.,C.Haupt,and O.W.Witte,GABA neurons survive focal ischemic injury.Neuroseience,2004.127(2):p.341-6.
115.Chen,Q.,et al.,Excitotoxic cell death dependent on inhibitory receptor activation.Exp Neurol,1999.160(1):p.215-25.
116.Nunez,J.L.,J.J.Alt,and M.M.McCarthy,A new model for prenatal brain damage.I.GABAA receptor activation induces cell death in developing rat hippocampus.Exp Neurol,2003.181(2):p.258-69.
117.Nabekura,J.,et al.,Reduction of KCC2 expression and GABAA receptor-mediated excitation after in vivo axonal injury.J Neurosci,2002.22(11):p.4412-7.
118.Kang,T.C.,et al.,Changes in Na(+)-K(+)-Cl(-) cotransporter immunoreactivity in the gerbil hippocampus following transient ischemia.Neurosci Res,2002.44(3):p.249-54.
119.Beck,J.,et al.,Na-K-Cl cotransporter contributes to glutamate-mediated excitotoxicity.J Neurosci,2003.23(12):p.5061-8.
120.Yan,Y.,et al.,Inhibition of Na(+)-K(+)-Cl(-) cotransporter during focal cerebral ischemia decreases edema and neuronal damage.Brain Res,2003.961(1):p.22-31.
121.Chen,H.,et al.,Na(+)-dependent chloride transporter(NKCC1)-null mice exhibit less gray and white matter damage after focal cerebral ischemia.J Cereb Blood Flow Metab,2005.25(1):p.54-66.
122.Lenart,B.,et al.,Na-K-Cl cotransporter-mediated intracellular Na+accumulation affects Ca2+ signaling in astrocytes in an in vitro ischemic model.J Neurosci,2004.24(43):p.9585-97.