摘要
目的
铅是人们所认识的最古老的毒物之一,是一种对细胞生长,增殖,信号传导无已知必要作用的无机重金属。低浓度的铅暴露亦能引起神经系统的损伤,包括智商、听力、视力的降低和外周神经功能的损害。发育期的神经系统对铅更敏感,即使铅暴露浓度低于儿童铅中毒的下限值10μg/dl也能损伤其神经系统。这与发育期儿童的血脑屏障发育尚未完善,胃肠道对铅的吸收率高及排铅能力低等有关。婴幼儿铅暴露可引起不可逆的神经系统损害,主要表现为学习记忆能力降低和神经行为异常,并且这种损伤可持续到成年阶段,甚至终生。海马是参与学习记忆形成的重要脑区,研究者也已经证实铅通过作用于海马来影响学习记忆功能。近些年来,人们对铅对学习记忆的影响及其细胞和分子学机制做了广泛的研究。但具体的机制仍然不清楚,故开展其相关机制研究具有重要意义。
神经生长相关蛋白(GAP-43)在学习记忆过程中发挥重要作用。发育期低浓度铅暴露是否影响该蛋白在大脑皮质和海马中的表达?其影响机制如何?这些都有待于研究。据此,我们通过孕期开始给予母鼠低浓度铅暴露建立发育期仔鼠铅中毒模型,研究仔鼠不同时间点大脑皮质和海马中GAP-43的表达情况及学习记忆状况,探讨发育期慢性低浓度铅暴露对大鼠学习记忆影响的机制,为儿童铅中毒的预防提供新的理论基础和实验依据。
方法
健康2月龄雌性大鼠30只,按体重随机分成对照组、低剂量铅暴露组1、2,每组10只雌鼠。各组大鼠均伺以普通饲料,饮用双蒸水。适应性喂养1周后雌鼠和正常雄鼠1:1合笼,以次晨发现阴栓或阴道分泌物镜检发现精子者确定为妊娠0天,自怀孕第一天铅低剂量组1、2分别饮用0.05%、0.2%醋酸铅溶液,直到仔鼠出生后28天(PN28)。对照组继续饮用双蒸水。铅暴露组仔鼠断乳前通过母乳饮用醋酸铅,断乳后继续饮用相同浓度醋酸铅。PN1、PN3. PN7、PN14、PN21、PN28解剖动物取出脑海马和大脑皮质,放入液氮中,转存到-70℃冰箱中;PN28测脑铅和血铅;PN56进行水迷宫实验。取出仔鼠海马和大脑皮质标本,免疫组化、Western blot方法检测脑海马、大脑皮质GAP-43蛋白的表达的变化。
结果
1、发育期慢性低浓度铅暴露对大鼠血铅和海马、大脑皮质铅含量的影响
各慢性染铅组仔鼠血铅和脑海马、大脑皮质铅含量明显高于对照组,差异有统计学意义(P<0.01),升高程度与饮用水的铅浓度呈剂量依赖性关系。
2、发育期慢性低浓度铅暴露对大鼠学习记忆能力的影响
在水迷宫实验中,结果表明慢性铅暴露可明显损伤仔鼠的空间定位航行能力,损伤程度与饮用铅的浓度呈剂量依赖性关系。
3、发育期慢性低浓度铅暴露对脑海马和大脑皮质GAP-43蛋白表达的影响
(1)免疫组织化学结果显示:免疫组织化学结果显示,在海马CA1区、CA3区、大脑皮质PN7、PN14、PN21、PN28时和DG区PN7、PN14、PN21时各染铅组的积分光密度值与对照组比差异有统计学意义(P<0.01),即染铅组该蛋白的表达含量低于对照组,CA1区和大脑皮质PN1、PN3时及DG区PN28时低剂量组2与对照组比差异有统计学意义(P<0.05或P<0.01),CA3区PN1、PN3时各染铅组与对照组比较差异有统计学意义(P<0.01)。
(2) Western blot结果显示:与对照组相比,发育期慢性低浓度铅暴露对大鼠脑海马和大脑皮质GAP-43蛋白的表达呈现下降趋势,且其与铅暴露水平有剂量依赖性关系及发育时间表达差异。
结论
1、免疫组织化学和Western Blot结果显示由于铅暴露可导致GAP-43在脑海马和大脑皮质表达减少。
2、铅中毒可降低成年大鼠空间学习记忆能力。
3、铅降低脑海马及大脑皮质GAP-43的表达可能是铅中毒引起学习记忆能力降低的一个机制。
Objective
Lead (Pb2+) is one of the oldest poison,which is a xaenobiotic metal with no known essential function in cellular grown,proliferation,or signaling.low-level lead exposure can also lead to the damage of nervous system,including the reduce of intelligence quotient、hearing、vision and the damage of function of peripheral nerve.Developmental nervous system is more vulnerable to lead toxicity,though nervous system can be damaged with blood lead levels below 10 ug/dl that is the lower limit of lead poisoning in children.It is associated with incompletely developed blood brain barrier, Gastrointestinal high absorptivity and low capacity of the Excretion of lead. Infant' exposure to lead can lead to irreversible damage to the nervous system,mainly showing the reduce of the ability of learning and memory and neurobehavioral abnormalities,and the damage can continue to the adulthood,even the whole life. Researchers have also confirmed that lead is by way of acting on the hippocampus that is one of the key limbic regions involved in learning and memory to influence the learning and memory function.In recent years, the influence of lead on learning and memory function and its cellular and molecular mechanism have been researched widely.However, the specific mechanism is still unclear,so it is significant to study correlative mechanisms.
Neuronal growth associated protein (GAP-43) plays a significant role in the process of learning and memory. Whether low-level lead exposure at developmental stage could influence the expression of GAP-43 in hippocampus and cerebral cortex? And what is the influence mechanism? Investigation is necessary to answer these questions. Hereby, in this paper, we built a model of lead poisoning of pup rats at developmental stage by exposing the female rats to lead from the beginning of pregnancy, by which the GAP-43 expression in hippocampus and cerebral cortex at different time points and the state of learning and memory were researched, and the mechanism of the influence that low-level lead exposure exerted on learning and memory function of rats at developmental stage was explored, in order to provide a theoretical basis and an experimental basis for the prevention of lead poisoning of children.
Methods
Female Wistar rats (n=30) aging 2 were randomly divided into three group: control group, low-dose group1(0.05% PbAc), low-dose group2(0.2% PbAc).All the groups were fed normal feeds and double distilled water. After one week adaptive feed, the rats mate according to the pattern in which a female rat mate with one male, and the next morning when vaginal plug appears or sperm was found in vaginal secretion by means of microscopic examination,it was designated as gestation day 0, denoted as GDO. Since the first day of pregnancy, low-dose group 1,2 of lead were consumed 0.05%,0.2%lead acetate solution, until the postnatal day 28 (PN28) of the pups.The control group continued to drink double distilled water with no lead. Pre-weaning the offspring of lead-exposed group drank lead acetate through breast milk,and they continued to drink the same concentration of lead acetate after weaning.At PN1,PN3,PN7,PN14,PN21,PN28,individually the hippocampus and cerebral cortex were got from pups in different groups and stored at fluid nitrogen and then stored at-70℃refrigerator;At PN28,we measured the brain lead and blood lead;Since PN56 the pups began to train in a spatial learning tast using a water maze paradigm.Removing the speciments of the hippocampus and cerebral cortex,we detected the chang of expression of the neuronal growth associated protein using Immunohistochemistry and Western blot.
Results
1、Effect of chronic developmental low-level lead exposure on the blood lead and brain(hippocampus and cerebral cortex)lead in rat.
lead levels in blood and brain of the groups of offspring in those two different lead-exposed groups were obviously higher than that in the control group,and the difference was statistically significant (P<0.01),and the extent of increasing showed in lead concentraion dependent manner.
2、Effect of chronic developmental low-level lead exposure on spatial learning and memory in rats at the developmental stage
In the Morris water maze task, results indicated that rats having drunk lead water diplayed significant impairment in their performance,and this extent of impairment showed in lead concentration dependent manner.
3、Effect of chronic developmental low-level lead exposure on GAP-43 expression in rat hippocampus and cerebral cortex
(1) Immunohistochemistry showed:there was statistical significance (P<0.01) in terms of the difference between the lead-exposed groups and the control group on the integral optical density (IOD) of hippocampal CA1 and CA3 areas, of cerebral cortex on PN7, PN14, PN 21 and PN28 and of DG area on PN7, PN14 and PN21, that was, the content of the protein expression of the lead-exposed groups was lower than that of the control group, compareing that of cerebral hippocampal CA1, and of cerebral cortex on PN1, PN3 and of DG area on PN28, the difference between low dose group 2 and the control group had statistical significance(P<0.05 or P<0.01), as well as the difference between the lead-exposed groups and the control group but that of cerebral hippocampal CA3 on PN1, PN3(P<0.01).
(2)Western Blot showed:Rat pups exposed to chronic level-lead during development did express lower levels of GAP-43 expression in hippocampus and cerebral cortex than of the control group and this exhibited in a dose-dependentand developmental expressed differences manner.
Conclusion
1、Immunohistochemistry and Western Blot results showed that chronic developmental low-level lead exposure reduced the expression of GAP-43 in the hippocampus and cerebral cortex.
2、Lead poisoning reduced the ability of learning and memory.
3、Lead on the impact of GAP-43 could be a mechanism that lead poisoning lead to a decreased capacity for learning and memory mechanism.
引文
1 Yan D, Wang L,Ma FL, et al. Developmental exposure to lead causes inherent changes on voltage-gated sodium channels in rat hippocampal CA1 neurons. Neuroscience. 2008; 153(2):436-445.
2 Toscano CD,Guilarte TR. Lead neurotoxicity:From exposure to molecular effects. Brain Research Reviews.2005; 49(3):529-554.
3 Bellinger DC, Bellinger AM. Childhood lead poisoning:the tortuous path from science to policy. Clin. Invest.2006; 116(2):853-857.
4 Lidsky TI, Schneider JS. Lead neurotoxicity in children:basic mechanisms and clinical correlates. Brain.2003; 126(1):5-19.
5 Plusquellec P, Muckle G, Dewailly E, et al. The relation of low-level prenatal lead exposure to behavioral indicators of attention in Inuit infants in Arctic Quebec. Neurotoxicol Teratol.2007; 29(5):527-537.
6 Toscano CD, Guilarte TR,0'Callaghan JP, et al. Calcium/calmodulin-dependent protein kinase Ⅱ activity and expression are altered in the hippocampus of Pb2+-exposed rats. Brain RES.2005; 1044(1):51-58.
7 Larry IB, Aryeh R. A membrane phosphoprotein associated with neural development, axonal regenration, phospholipid metabolim, and synaptic plasticity. TINS.1987; 10(12):527-537.
8 Tolner EA, Van Vliet EA. Holtmaast AJ. et al.GAP-43mRNA and protein expression in the hippocampal and parahippocampal region during the course of epileptogenesis in rats. Eur J Neuresei.2003; 17(11):2369-2380.
9 Hassiontis M, Ashwell KW, Marotte LR, et al. GAP-43 immunoreacivity in the brain of the brain of the developing and adult wallaby [J]. Anat Embryol Berl.2002; 206(1): 97-118.
10孟金萍,胡树慧,王艳蓉,等. 儿童与成人铅中毒的差异.医学综述.2009:15(14):2087-2090.
11秦根林.我国儿童铅中毒现状及原因分析.中国学校卫生.2000:21(2):214-215.
12 Deano DW. Biology and violence. New York, NY:Cambridge University Press.1990: 55-58.
13 Larry IB, Aryeh R. GAP-43:an intrinsic determinant of neuronal development and plasticity. TINS.1997; 20(2):84-91.
14 Carulli D, Buff OA, Strata P. Reparative mechanisms in the cerebellar cortex. Prog Neurobiol.2004; 72(6):373-398.
15 Akulinin VA,Belichenko PV,Dahlstrom A. The cellular distribution of GAP-43 immunoreactivity in human neocortical areas using im munofluorescence and confocal microscopy:post-ischemic influence. Brain Res.1998; 784(1/2):341-346.
16 Routtenberg A,Cantallops 1, Zafuto S, et al. Enhanced learning after genetic overexpression of a brain growth protein. Proc Natl A cad Sci USA.2000; 97(13): 7657-7662.
17 Gerendasy D. Homeostatic tuning of Ca2+signal transduction by membrs of the calpacitin protein family. Neurosci Res.1999; 58(1):107-119.
18 Esdar C, Oelulein SA, Retnhardt S, et al. The protein kinase C(PKC) substrate GAP-43 is already expresed in neural precursor cells, culocalizeg with PKCeta and binds calmodulin. Eur Neurosci.1999; 11(2):503-516.
19 Neve RL, Coopersinith R, Mcphie DL, et al. The neuronal growth-associated protein GAP-43 interacts with rabaptin rabaptin-5 and participates in endocytosis. Neursci.1998; 18(19):7757-7767.
20 Caroni P. Actin cytoskeleton regulation through modulation of PI (4,5) P2 rafts. EMBO.2002; 20(3):4332-4336.
21 Schmitt TJ, Zawia N, Harry GJ. GAP-43 mRNA expression in the developing rat brain:alteration following lead-acetate exposure. Neurotoxicology.1996; 17 (2):407-414.
22 Scortegagna M, Chikhale E, Hanbauer 1. Efect of lead on cytoskeletal ploteins expressed in E14 mesencephalic primary cultures. Neurochem Int.1998.32(4): 353-359.
23尹杰,牛玉杰,张荣,等.孕期低水平铅暴露对子代大鼠海马神经生长相关蛋白表达的影响[J].中华劳动卫生职业病杂志.2008:26(4):208-211.
24 Fgnou DD, Tuchek JM. The biochemistry of learning and memory. Md Cell Biochem. 1995; 4(2):279-286.
25帕利军,潘建平.甲减及高碘大鼠海马组织神经生长相关蛋白的表达.中国公共卫生.2002:18(12):1433-1435.
26 Fishman MC. GAP-43:Putting constraints on neuronal plasticity. Perspective in developmental neurobiology.1996; 4(2):193-198.
1 Toscano CD,Guilarte TR. Lead neurotoxicity:From exposure to molecular effects. Brain Research Reviews.2005; 49(3):529-554.
2 Yan D, Wang L, Ma FL, et al. Developmental exposure to lead causes inherent changes on voltage-gated sodium channels in rat hippocampal CA1 neurons. Neuroscience. 2008; 153(2):436-45.
3 Plusquellec P, Muckle G, Dewailly E, et al. The relation of low-level prenatal lead exposure to behavioral indicators of attention in Inuit infants in Arctic Quebec. Neurotoxicol Teratol.2007; 29(5):527-537.
4 Johnston MV, Goldstein GW. Selective vulnerability of the developing brain to lead. Curr Opin Neurol.1998; 11(6):689-693.
5 Malenka RC, Nicoll RA. Long-term potentiation-a decade of progress. Science.1999; 285(5435):1870-1874.
6 Zaiser AE,Miletic V. Differential effects of inorganic lead on hippocampal long-term potentiation in young rats in vivo. Brain Res.2000; 876(1/2):201-204.
7 Lasley SM, Gilbert ME. Glutamatergic components underlying Lead-induced impairments in hippocampal synaptic plasticity. Neurotoxicology.2000; 21(6): 1057-1068.
8 Malenka RC, Nicoll RA. NMDA-receptor-dependent synaptic plasticity:multiple forms and mechanisms. Trends Neurosci.1993; 16(12):521-527.
9 Zalutsky RA, Nicoll RA. Comparison of two forms of long-term potentiation in single hippocampal neurons. Science.1990; 248(4963):1619-1624.
10 Gilbert ME, Mack CM. Chronic lead exposure accelerates decay of long-term potentiation in rat dentate gyrus in vivo. Brain Res.1998; 789(1):139-149.
11 Gutowski M, Altmann L, Sveinsson K, et al. Synaptic plasticity in the CA1 and CA3 hippocampal region of pre-and postnatally lead-exposed rats. Toxicol. Lett. 1998; 95(3):195-203.
12 Kawamura Y, Manita S,Nakamura T, et al. Glutamate release increases during mossy-CA3 LTP but not during Schaffer-CA1 LTP. Eur. J. Neurosci.2004; 19(6): 1591-1600.
13 Collingridge GL, Kehl SJ, McLennan H. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. Physiol.1983; 334(2):33-46.
14 Harris EW, Cotman CW. Long-term potentiation of guinea pig mossy fiber responses is not blocked by N-methyl D-aspartate antagonists. Neurosci Lett.1986; 70(1): 132-137.
15 Hussain RJ, Parsons PJ, Carpenter DO. Effects of lead on long-term potentiation in hippocampal CA3 vary with age. Brain Res Dev Brain Res.2000; 121(2):243-252.
16 Nihei MK, Desmond NL, Mcglothan JL, et al. N-methyl-D-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning. Neuroscience.2000; 99(2):233-242.
17 Hori N, Busselberg D, Matthews MR, et al. Lead blocks LTP by an action not at NMDA receptors. Exp Neurol.1993; 119(2):192-197.
18 li XM,Gu Y,She JQ, et al. Lead inhibited N-methyl-D-aspartate receptor-independent long-term potentiation involved ryanodine-sensitive calcium stores in rat hippocampal area CA1. Neuroscience.2006; 139(2):463-473.
19 Lauri SE, Bortolotto ZA,Nistico R, et al. A role for Ca2+ stores in kainate receptor-dependent synaptic facilitation and LTP at mossy fiber synapses in the hippocampus. Neuron.2007; 39(2):327-341.
20 Mattson MP, LaFerla FM, Shan SL, et al. Calcium signaling in the ER:its role in neuronal plasticity and neurodegenerative disorders. Trends Neurosci.2000; 23(5):222-229.
21 uruichi T, Furutama D, Hakamata Y, et al. Multiple types of ryanodine receptor/Ca2+release channels are differentially expressed in rabbit brain. Neurosci.1994; 14(8):4794-4805.
22 Ozawa S, Kamiya H, Tsuzuki K. Glutamate receptors in the mammalian central nervous system. Prog. Neurobiol.1998; 54(5):581-618.
23 Monyer H, Sprengel R, Schoepfer R, et al. Heteromeric NMDA receptors:molecular and functional distinction of subtypes. Science.1992; 256(5060):1217-1221.
24 Ciabarra AM, Sullivan JM, Gahn LG, et al. Cloning and characterization of chi-1: a developmental ly regulated member of a novel class of the ionotropic glutamate receptor family. Neurosci.1995; 15(10):6498-6508.
25 Zukin RS, Bennett MV. Alternatively spliced isoforms of the NMDARI receptor subunit. Trends Neurosci.1995; 18(7):306-313.
26 Hollmann M, Heinemann S. Cloned glutamate receptors. Annu. Rev. Neurosci.1994; 17(3):31-108.
27 Monyer H, Burnashev N.Laurie DJ, et al. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron.1994; 12(3):529-540.
28 Sun L, Margolis FL, Shipley MT, et al. Identification of a long variant of mRNA encoding the NR3 subunit of the NMDA receptor:its regional distribution and developmental expression in the rat brain. FEBS Lett.1998; 441(3):392-396.
29 Das S, Sasaki YF, Rothe T, et al. Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A. Nature.1998; 393(6683):377-381.
30 Luo J, Bosy TZ, Wang Y, et al. Ontogeny of NMDA R1 subunit protein expression in five regions of rat brain. Brain Res. Dev.1996; 92(1):10-17.
31 Riva MA, Tascedda F,Molteni R, et al. Regulation of NMDA receptor subunit mRNA expression in the rat brain during postnatal development. Brain Res. Mol. Brain Res.1994; 25(3/4):209-216.
32 Zhong J, Carrozza DP,Williams K, et al. Expression of mRNAs encoding subunits of the NMDA receptor in developing rat brain. Neurochem.1995; 64(2):531-539.
33 Williams K. Ifenprodil discriminates subtypes of the N-methyl-daspartate receptor: selectivity and mechanisms at recombinant heteromeric receptors. Mol. Pharmacol. 1993; 44(4):851-859.
34 Guilarte TR, McGlothan JL. Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure. Brain Res.1998; 790(1/2): 98-107.
35 Nihei MK, Desmond NL. N-methyl-d-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning. Neuroscience.2000; 99(2):233-242.
36 Nihei MK, Guilarte TR. Molecular changes in glutamatergic synapses induced by Pb2+:association with deficits of LTP and spatial learning. Neurotoxicology.2001; 22(5):635-643.
37 Toscano CD, Hashemzadeh-Gargari H. Developmental Pb2+-exposure alters NMDAR subtypes and reduces CREB phosphorylation in the rat brain. Brain Res. Dev. Brain Res.2002; 139(2):217-226.
38 Williams K, Russell SL, Shen YM, et al. Developmental switch in the expression of NMDA receptors occurs in vivo and in vitro. Neuron.1993; 10(2):267-278.
39 Pina-Crespo JC, Gibb AJ. Subtypes of NMDA receptors in newborn rat hippocampal granule cells. Physiol.2002;541(pt1):41-64.
40 Tovar KR, Westbrook GL. The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. Neurosci.1999; 19 (10): 4180-4188.
41 Robert A, Howe JR. Development of glutamatergic synaptic activity in cultured spinal neurons. Neurophysiol.2000; 83(2):659-670.
42 Rumbaugh G, Vicini S. Distinct synaptic and extrasynaptic NMDA receptors in developing cerebellar granule neurons. Neurosci.1999; 19(24):10603-10610.
43 Flint AC, Maisch US. NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex. Neurosci.1997; 17(7):2469-2476.
44 Guilarte TR, Miceli RC. Age-dependent effects of lead on[3H]MK-801 binding to the NMDA receptor-gated ionophore:in vitro and in vivo studies. Neurosci. Lett.
1992; 148(1/2):27-30.
45 Hashemzadeh-Gargari H, Guilarte TR. Divalent cations modulateNmethyl-d-aspartate receptor function at the glycine site. Pharmacol. Exp. Ther.1999; 290(3):1356-1362.
46 Scheetz AJ, Constantine-Paton M. Modulation of NMDA receptor function: implications for vertebrate neural development. FASEB J.1994; 8(10):745-752.
47 Nihei MK, Guilarte TR. NMDAR-2A subunit protein expression is reduced in the hippocampus of rats exposed to Pb2+ during development. Brain Res. Mol. Brain Res. 1999; 66(1/2):42-49.
48 Liu L, Wong TP. Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science.2004; 304(5673):1021-1024.
49 Wu GY, Deisseroth K. Activity-dependent CREB phosphorylation:convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway. Proc. Natl. Acad. Sci. U. S. A..2001; 98(5):2808-2813.
50 Tao X, Finkbeiner S, Arnold DB, et al. Ca2+influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism. Neuron.1998; 20(4): 709-726.
51 Bressler J, Kim KA,Chakraborti T, et al. Molecular mechanisms of lead neurotoxicity. Neurochem. Res.1999; 24(4):595-600.
52 Habermann E, Crowell K, Janicki P. Lead and other metals can substitute for Ca2+ in calmodulin. Arch. Toxicol.1983; 54(1):61-70.
53 Malinow R, Madison DV, Tsien RW. Persistent protein kinase activity underlying long-term potentiation. Nature.1988; 335(6193):820-824.
54 Abeliovich A, Paylor R, Chen C, et al. PKC gamma mutant mice exhibit mild deficits in spatial and contextual learning. Cell.1993; 75(7):1263-1271.
55 Tomsig JL, Suszkiw JB. Multisite interactions between Pb2+and protein kinase C and its role in norepinephrine release from bovine adrenal chromaffin cells. Neurochem.1995; 64(6):2667-2673.
56 Lu H, Guizzetti M, Costa LG. Inorganic lead activates the mitogen-activated protein kinase kinase-mitogen-activated protein kinase-p90(RSK) signaling pathway in human astrocytoma cells via a protein kinase C-dependent mechanism. Pharmacol. Exp. Ther.2002; 300(3):818-823.
57 Olivi L, Sisk J, Bressler J. The involvement of lipid activators of protein kinase C in the induction of ZIF268 in PC12 cells exposed to lead. Neurochem. Res.2003; 28(1):65-71.
58 Westerink RH, Klompmakers AA, Westenberg HGM, et al. Signaling pathways involved in Ca2+-and Pb2+-induced vesicular catecholamine release from rat PC12 cells. Brain Res.2002; 957(1):25-36.
59 CreminJr JD, Smith DR. In vitro vs. in vivo Pb effects on brain protein kinase C activity. Environ. Res.2002; 90(3):191-199.
60 KawakamiY, Kitaura J,Hartman SE, et al. Regulation of protein kinase Cβ1 by two protein-tyrosine kinase. Btk ans Syk. Proc. Natl. Acad. Sci.2000; 97(13): 7423-7428.
61 Xu SZ.Bullock L, Shan CJ, et al. PKC isoforms were reduced by lead in the developing rat brain. Devl Neuroscience.2005; 23(1):53-64.
62 Bayer KU, Lohler J, Schulman K, et al. Developmental expression of the CaM kinase II isoforms:ubiquitous gamma-and delta-CaM kinase II are the early isoforms and most abundant in the developing nervous system. Brain Res. Mol. Brain Res. 1999; 70(1):147-154.
63 Vallano ML, Beaman-Hall CM, Mathur A, et al. Astrocytes express specific variants of CaM KII delta and gamma, but not alpha and beta, that determine their cellular localizations. Glia.2000; 30(2):154-164.
64 Hudmon A, Schulman H. Neuronal Ca2+/calmodulin-dependent protein kinase II:the role of structure and autoregulation in cellular function. Annu. Rev. Biochem. 2002; 71(1):473-510.
65 Hardingham N, Glazewski S, Pakhotin P, et al. Neocortical long-term potentiation and experience dependent synaptic plasticity require alpha-calcium/calmodulindep-endent protein kinase Ⅱ autophosphorylation Neurosci.2003; 23(11):4428-4436
66 Salinas JA, Huff NC. Lead and conditioned fear to contextual and discrete cues. Neurotoxicol. Teratol.2002; 24(4):541-550.
67 Westerink RH, Klompmakers AA, Wwstenberg HG, et al. Signaling pathways involved in Ca2+-and Pb2+-induced vesicular catecholamine release from rat PC12 cells. Brain Res.2002; 957(1):25-36.
68 Toscano CD, Callaghan JP, et al. Calcium/calmodulin-dependent protein kinase Ⅱ activity and expression are altered in the hippocampus of Pb2+-exposed rats. Brain Research.2005; 1044(1):51-58.
69 Nihei MK, Desmond NL, McGlothan JL, et al. N-methyl-d-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning. Neuroscience.2000; 99(2):233-242.
70 Cammarota M, Bevilaqua LR, Viola H, et al. Participation of CaMKII in neuronal plasticity and memory formation. Cell. Mol. Neurobiol.2002; 22(3):259-267.
71 Hashemzadeh-Gargari H, Guilarte TR. Divalent cations modulate N-methyl-d-aspartate receptor function at the glycine site. Pharmacol. Exp. Ther.1999; 290(3):1356-1362.
72 Thomas GM, Huganir RL. MAPK cascade signalling and synaptic Plasticity. Nat. Rev, Neurosci.2004; 5(3):173-183.
73 Adams JP, Sweatt JD. Molecular psychology:roles for the ERK MAP kinase cascade in memory. Annu. Rev. Pharmacol. Toxicol.2002; 42(2):135-163.
74 Giovannini MG, Blitzer RD, et al. Mitogen-activated protein kinase regulates early phosphorylation and delayed expression of Ca2+/calmodulin-dependent protein kinase Ⅱ in long-term potentiation. Neurosci.2001; 21(18):7053-7062.
75 Ferrer I, Blanco R, Carmona M, et al. Phosphorylated mitogenactivated protein kinase (MAPK/ERK-P), protein kinase of 38 kDa (p38-P), stress-activated protein kinase (SAPK/JNK-P), and calcium/calmodulin-dependent kinase II (CaM kinase II) are differentially expressed in tau deposits in neurons and glial cells in tauopathies. Neural Transm.2001; 108(12):1397-1415.
76 Lu H, Guizzetti M, Costa LG. Inorganic lead activates the mitogen-activated protein kinase kinase-mitogen-activated protein kinase-p90(RSK) signaling pathway in human astrocytoma cells via a protein kinase Odependent mechanism. Pharmacol. Exp. Ther.2002; 300(3):818-823.
77 Ramesh GT, Manna SK, Aggrawal BB, et al. Lead exposure activates nuclear factor kappa B, activator protein-1, c-Jun N-terminal kinase and caspases in the rat brain. Toxicol. Lett.2001; 123(2-3):195-207.
78 Cordova FM, Rodrigues AL, Giacomelli MBO, et al. Lead stimulates ERK1/2 and p38MAPK phosphorylation in the hippocampus of immature rat. Brain Res.2004; 998 (1):65-72.
79 Leal RB, Cordova FM, Herd L, et al. Lead-stimulated p38MAPK-dependent Hsp27 phosphorylation. Toxicol. Appl. Pharmacol.2002; 178(1):44-51.
80 Chandler LJ, Sutton G, Dorairaj NR, et al. N-methyl-daspartate receptor-mediated bidirectional control of extracellular signal-regulated kinase activity in cortical neuronal cultures. Biol. Chem.2001; 276(4): 2627-2636.
81 Friguls B, Petegnief V, Justicia C, et al. Activation of ERK and Akt signaling in focal cerebral ischemia:modulation by TGF-alpha and involvement of NMDA receptor. Neurobiol. Dis.2002.11(3):443-456.
82 Kalluri HS, Ticku MK. Regulation of ERK phosphorylation by ethanol in fetal cortical neurons. Neurochem. Res.2003; 28(5):765-769.
83 Paul S, Nairn AC, Wang P, et al. NMDA-mediated activation of the tyrosine phosphatase STEP regulates the duration of ERK signaling. Nat. Neurosci.2002; 6(1):34-42.
84 Ehlers MD. Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system. Nat. Neurosci.2003; 6(3):231-242.
85 Krapivinsky G, Krapivinsky L, Manasian Y, et al. The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRFl. Neuron.2003; 40(4):775-784.
86 Lonze BE, Ginty DD. Function and regulation of CREB family transcription factors in the nervous system. Neuron.2002; 35(4):605-623.
87 Hardingham GE, Bading H. The Yin and Yang of NMDA receptor signalling. Trends Neurosci.2003; 26(2):81-89.
88 Montminy MR, Bilezikjian LM. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature.1987; 328(6126):175-178.
89 Chrivia JC, Kwok RP, Lamb N, et al. Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature.1993; 365(6449):855-859.
90 Guzowski JF, McGaugh JL. Antisense oligodeoxynucleotidemediated disruption of hippocampal cAMP response element binding protein levels impairs consolidation of memory for water maze training. Proc. Natl. Acad. Sci. U. S. A..1997; 94(6): 2693-2698.
91 Toscano CD, Hashemzadeh-Gargari H, Jennifer L, et al. Developmental Pb2+-exposure alters NMDAR subtypes and reduces CREB phosphorylation in the rat brain. Brain Res. Dev. Brain Res.2002; 139(2):217-226.
92 Toscano CD, McGlothan JL, Guilarte TR. Lead exposure alters cyclic-AMP response element binding protein phosphorylation and binding activity in the developing rat brain. Brain Res. Dev. Brain Res.2003; 145(2):219-228.
93 Hardingham GE, Fukunaga Y, Bading H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat. Neurosci.2002; 5(5): 405-414.
94 Toscano CD, Guilarte TR. Lead neurotoxicity:From exposure to molecular effects. Brain Research Reviews.2005; 49(3):529-554.
95 Nihei MK, Guilarte TR. NMDAR-2A subunit protein expression is reduced in the hippocampus of rats exposed to Pb2+during development. Brain Res. Mol. Brain Res.1999; 66(1-2):42-49.
96 Guilarte TR, McGlothan JL. Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure. Brain Res.1998; 790(1-2): 98-107.
97 Lau GC, Saha S, Faris R, et al. Up-regulation of NMDAR1 subunit gene expression in cortical neurons via a PKA-dependent pathway. Neurochem.2004; 88(3):564-575.
98 Impey S, Mark M, Villacres EC, et al. Induction of CRE-mediated gene expression by stimuli that generate long-lasting LTP in area CA1 of the hippocampus. Neuron. 1996; 16(5):973-982.
99 Mizuno M, Yamada K, Maekawa N, et al. CREB phosphorylation as a molecular marker of memory processing in the hippocampus for spatial learning. Behav. Brain Res. 2002; 133(2):135-141.
100 Viola H, Furman M, Izquierdo I, et al. Phosphorylated cAMP response element-binding protein as a molecular marker of memory processing in rat hippocampus:effect of novelty. Neurosci.2000; 20(23):1-5.
101 Athos J, Impey S, Pineda VV, et al. Hippocampal CRE-mediated gene expression is required for contextual memory formation. Nat. Neurosci.2002; 5(11): 1119-1120.
102 Yuan J, Yankner BA. Apoptosis in the nervous system. Nature.2000; 407(6805): 802-813.
103 Savolainen KM, Loikkanen J, Eerikainen S, et al. Interactions of excitatory neurotransmitters and xenobiotics in excitotoxicity and oxidative stress:Glutamate and lead. Toxicol lett.1998; 102-103(2):363-367.
104 Haupt S, Berger M, Goldberg Z, et al. Apoptosis-the p53 network. Cell Sci.2003; 116(pt20):4077-4085
105 Slee EA,O'Connor DJ, Lu X, et al. To die or not to die:how does p53 decide. Oncogene.2004; 23(16):2809-2818.
106 Degterev A, Boyce M, Yuan J. A decade of caspases. Oncogene.2003; 22(53): 8543-8567.
107 Woo M, Hakem R, Soengas MS, et al. Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Develop.1998; 12(6): 806-819.
108 Xu J, Ji LD, Xu LH, et al. Lead-induced apoptosis in PC 12 cells:Involvement of p53,Bcl-2 family and caspase-3. Toxicology Letters.2006; 166(2):160-167.
109 He L, Poblenz AT, Medrano CJ, et al. Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore. Biol. Chem.2000; 275(16):12175-12184.
110 Sharifi AM, Mousavi SH, Bakhshayesh M, et al. Study of correlation between leadinduced cytotoxicity and nitric oxide production in PC12 cells. Toxicol. Lett. 2005; 160(1):43-48.
111 Chen L, Yang X, Jiao H, et al. Tea catechins protect against lead-induced ROS formation, mitochondrial dysfunction, and calcium dysregulation in PC12 cells. Chem. Res. Toxicol..2003; 16(9):1155-1161.
112 Sharif i AM, Baniasadi S, Jorjani M, et al. Investigation of acute lead poisoning on apoptosis in rat hippocampus in vivo. Neurosci. Lett.2002; 329(1):45-48.
113 Loikkanen J, Chvalova K, Naarala J, et al. Pb2+-induced toxicity is associated with p53-independent apoptosis and enhanced by glutamate in GT1-7 neurons. Toxicol. Lett.2003; 144(2):235-246.
114 Chao SL, Moss JM, Harry GJ, et al. Lead-induced alterations of apoptosis and neurotrophic factor mRNA in the developing rat cortex, hippocampus, and cerebellum. Biochem Mol Toxicol.2007; 21(5):265-272.
115 Minichiello L, Klein R. TrkB and TrkC neurotrophin receptors cooperate in promoting survival of hippocampal and cerebellar granule neurons. Genes Dev 1996; 10(22):2849-2858.
116 Gould E, Woolley CS, McEwen BS. Naturally occurring cell death in the developing dentate gyrus of the rat. Comp Neurol.1991; 304(3):408-418.
117OrtizC, Cardemil IL. Heat-shock responses in two leguminous plants:a comparative study. Exp Bot.2001; 52(361):1711-1719.
118 Qian Y, Harris ED, Zheng Y, et al. Lead targets GRP78, a molecular chaperone, in 06 rat glioma cells. Toxicolog and Applied Pharmacology.2000; 163(3):260-266.
119 Qian YC, Zheng Y, Ramos KS, et al. GRP78 compartmentalized redistribution in Pb-treated glia:role of GRP78 in leadinduced oxidative stress. Neurotoxicology. 2005; 26(2):267-275.
120 White LD, Cory-Slechta DA, Gilbert ME, et al. New and evolving concepts in the neurotoxicology of lead. Toxicology Applied Pharmacology.2007; 225(1):1-27.
121 Qian YC, Zheng Y, Weber D, et al. A 78-kDa glucose-regulated protein is involved in the decrease of interleukin-6 secretion by lead treatment from astrocytes. Cell Physiol.2007; 293(3):C897-C905.
122 Lidsky TI, Schneider JS. Lead neurotoxicity in children:basic mechanisms and clinical correlates. Brain.2003; 126(pt1):5-19.
123 Heidmets LT, Zharkovsky T, Jurgenson M, et al. Early postnatal, low-level lead exposure increases the number of PSA-NCAM expressing cells in the dentate gyrus of adult rat hippocampus. Neurotoxicology.2006,27(1):39-43.
124 Minana R, Climent E, Barettino D, et al. Alcohol exposure alters the expression pattern of neural cell adhesion molecules during brain development. Neurochem. 2000; 75(3):954-964.
125 Crossin KL, Krushel LA. Cellular signaling by neural cell adhesion molecules of the immunoglo-bulin in superfamily. Develop Dynamics.2000; 216:260-279.
126 Edelman GM, Crossin KL. Cell adhesion molecules:implications for molecular histology. Annu Rev Biochem.1991; 60:155-90.
127 Kojima N, Kono M, Yoshida Y, et al. Biosynthesis and expression of polysialic acid on the neural cell adhesion molecule is predominantly directed by ST8Sia II/STX during in vitro neuronal differentiation. Biol Chem.1996;271(36): 22058-22062.
128 Eckhardt M, Muhlenhoff M, Bethe A, et al. Molecular characterization of eukaryotic polysialyltiransferase-1. Nature.1995; 373(6516):715-718.
129 Sadoul R, Hirn M, Deagostini-Bazin H, et al. Adult and embryonic mouse neural cell adhesion molecules have different binding properties. Nature.1983; 304(5924):347-349.
130 Chuong CM, Edelman GM. Alterations in neural cell adhesion molecules during development of different regions of the nervous system. Neurosci.1984; 4(9): 2354-2368.
131 Seki T. Expression patterns of immature neuronal markers PSA-NCAM, CRMP-4 and NeuroD in the hippocampus of young adult and aged rodents. Neurosci Res.2002; 70(3):327-34.
132 Hu QS, Fu H, Ren T, et al. Maternal low-level lead exposure reduces the expression of PSA-NCAM and the activity of sialyltraansferase in the hippocampi of neonatal rat pups. NeuroToxicology.2008; 29(4):675-681.
133 Hu QS, Ren TL, Xue Y, et al. Impact of low-level lead exposure on neural cell adhesion molecule expression of primarily cultured hippocampal neurons. Zhonghua Yu Fang Yi Xue Za Zhi.2004; 38(6):379-382.
134 Larry IB, Aryeh R. A membrane phosphoprotein associated with neural development, axonal regenration, phospholipid metabolim,and synaptic plasticity. TINS.1987; 10 (12):527-537.
135 Tolner EA, Van Vliet EA, Holtmaast AJ, et al. GAP-43mRNA and protein expression in the hippocampal and parahippocampal region during the course of epileptogenesis in rats. Eur J Neuresei.2003;17(11):2369-2380.
136Hassiontis M, Ashwell KW, Marotte LR, et al. GAP-43 immunoreacivity in the brain of the brain of the developing and adult wallaby. Anat Embryol Berl.2002; 206(1): 97-118.
137 Akulinin VA,Belichenko PV,Dahlstrom A. The cellular distribution of GAP-43 immunoreactivity in human neocortical areas using im munofluorescence and confocal microscopy:post-ischemic influence. Brain Res.1998; 784(1/2): 341-346.
138 Routtenberg A,Cantallops 1, Zafuto S, et al. Enhanced learning after genetic overexpression of a brain growth protein. Proc Natl A cad Sci USA.2000; 97(13): 7657-7662.
139 Schmitt TJ, Zawia N, Harry GJ, et al. GAP-43 mRNA expression in the developing rat brain:alterations following lead-acetate exposure. Neurotoxicology.1996; 17(2):407-414.