慢性应激对大鼠海马CA3区锥体细胞树突骨架的效应
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摘要
背景与目的：慢性应激可诱发或加重诸如复发性抑郁症、创伤后应激障碍(PTSD)等许多精神神经疾病。尽管这些疾病的发病机制还不清楚，但普遍认为慢性应激引起的海马结构与功能的改变可能参与其中。研究已经表明，慢性应激可引起大鼠海马CA3区锥体细胞项树突萎缩，且应激大鼠的行为与复发性抑郁症和PTSD患者的临床表现极为相似。海马是司职学习、记忆、情绪反应及认知功能的重要脑区，临床发现复发性抑郁症及PTSD患者的海马功能受损，慢性应激引起的海马萎缩可能与上述疾病的发生发展密切相关。神经元的形态结构需要完整的细胞骨架来维持，海马萎缩可能与其细胞骨架的改变有关。微管是树突骨架的重要成分，故微管破坏可能参与了海马CA3区锥体细胞顶树突萎缩的机制。本研究观察了慢性应激大鼠海马CA3区锥体细胞树突内微管的形态结构和微管相关蛋白2(MAP2)表达水平及[Ca~(2+)]_i浓度变化，旨在探讨慢性应激引起海马神经元树突萎缩的可能机制。
材料和方法：实验对象为Sprague-Dawley (SD)雄性大鼠(2月龄)，体重180-250g。随机分为对照组、应激组。
Background and objective: Exposure to chronic stress is known to precipitate or exacerbate many neuropsychiatric disorders such as depression and PTSD. Although the responsible brain sites in the pathogenesis of these disorders have been obscure, the hippocampus is thought to be involved. Indeed, chronic stress in rats induces atrophy of apical dendrites of the pyramidal neurons in the hippocampal CA3 subfield. In addition, these stressed rats show depression- or PTSD-like behavior. Since the hippocampal formation is the important structure in brain involving memory, emotion and cognitive function, and these functions are impaired in patients with depression or PTSD, the stress-induced hippocampal atrophy may be implicated in the pathogenesis of these disorders. Several morphological studies suggest that this atrophy is caused by the damage of cytoskeleton such as microtubule, microfilament and intermediate filament. Among them, microtubule is the most important segment of cytoskeleton in dendrites of neuron
s. Therefore, the damage of it may be involved in atrophy of apical dendrites of the pyramidal neurons. The purpose of this study is to clarify
the mechanism of the chronic stress-induced atrophy of the hippocampal neurons. To this point, we investigated the effects of chronic stress on the morphology of microtubules, the level of phosphorylated MAP2 in dendrites and the concentration of [Ca2+]i in the hippocampal CA3 pyramidal neurons.
Materials and methods: Male SD rats were divided into two groups: control and stress. Rats in the stress group were subjected to forced-swimming for a 20-minute a day for four weeks, the diameter and length of dendrite shaft of the rat hippocampal CA3 pyramidal neurons were measured by using Golgi staining. The ultrastructure of dendrite, especially, microtubules of rat hippocampal CA3 pyramidal neurons were observed by using an electron-microscope. The level of phosphorylated MAP2 of rat hippocampal CA3 pyramidal neurons was measured quantitatively by using the immunohistochemistry method and the computerized image technique. LSCM was used to measure the concentration of [Ca2+]i in the CA3 pyramidal neurons.
Result: The diameter of dendritic shaft of the hippocampal CA3 pyramidal neurons was significantly thicker in the stress group (6.40um ±0.94um) than that in the control group (5.65um±0.85um) (p<0.05); the length of them (125.35um±18.69um) was shorter than that in the control group (98.51um ± 17.48um) (p<0.01). The following ultrastructural changes of dendrites were observed in the hippocampal
CA3 pyramidal neurons of the stress group: breaking into fragmentation of microtubule, widening of spacing between parallel microtubules in longitudinal-section, irregularity of annular and maldistribution of monome in cross-section, crista clouding or degeneration as vacuoles of mitochondrion.
The average gray degree of phosphorylated MAP2 in hippocampal CA3 pyramidal neurons was significantly lower in the stress group (145 ±4.40) than that in the control group (149±1.81) (p<0.05). As the average gray degree is correlated negatively with expression of intensity, the decrease of phosphorylated MAP2 average gray degree in stress group indicates that the chronic stress enhances the phosphorylation of MAP2. Moreover, the number of phosphorylated MAP2 positive pyramidal neurons was significantly decreased in the stress group (40.36 ±1.35) compared to the control group (42.73 ± 1.56) (p<0.05).
The fluorescent intensity of [Ca2+]i in hippocampal CA3 pyramidal neurons was significantly stronger in the stress group (1548.83 ± 118.53 ) than that in the control group (598.71 ±48.02) (p<0.01), which indicated
that chronic stress increase the concentration of [Ca2+ ]i in the pyramidal neurons.
Conclusions: Chronic stress could damage the microtubules of dendrites leading to dendritic shaft retraction in rat hippocampal CA3 pyramidal neurons. In addition, chronic stress enhances phosphorylation
of MAP2 and increases the concentration of [Ca2+]i in the CA
材料和方法：实验对象为Sprague-Dawley (SD)雄性大鼠(2月龄)，体重180-250g。随机分为对照组、应激组。
Background and objective: Exposure to chronic stress is known to precipitate or exacerbate many neuropsychiatric disorders such as depression and PTSD. Although the responsible brain sites in the pathogenesis of these disorders have been obscure, the hippocampus is thought to be involved. Indeed, chronic stress in rats induces atrophy of apical dendrites of the pyramidal neurons in the hippocampal CA3 subfield. In addition, these stressed rats show depression- or PTSD-like behavior. Since the hippocampal formation is the important structure in brain involving memory, emotion and cognitive function, and these functions are impaired in patients with depression or PTSD, the stress-induced hippocampal atrophy may be implicated in the pathogenesis of these disorders. Several morphological studies suggest that this atrophy is caused by the damage of cytoskeleton such as microtubule, microfilament and intermediate filament. Among them, microtubule is the most important segment of cytoskeleton in dendrites of neuron
s. Therefore, the damage of it may be involved in atrophy of apical dendrites of the pyramidal neurons. The purpose of this study is to clarify
the mechanism of the chronic stress-induced atrophy of the hippocampal neurons. To this point, we investigated the effects of chronic stress on the morphology of microtubules, the level of phosphorylated MAP2 in dendrites and the concentration of [Ca2+]i in the hippocampal CA3 pyramidal neurons.
Materials and methods: Male SD rats were divided into two groups: control and stress. Rats in the stress group were subjected to forced-swimming for a 20-minute a day for four weeks, the diameter and length of dendrite shaft of the rat hippocampal CA3 pyramidal neurons were measured by using Golgi staining. The ultrastructure of dendrite, especially, microtubules of rat hippocampal CA3 pyramidal neurons were observed by using an electron-microscope. The level of phosphorylated MAP2 of rat hippocampal CA3 pyramidal neurons was measured quantitatively by using the immunohistochemistry method and the computerized image technique. LSCM was used to measure the concentration of [Ca2+]i in the CA3 pyramidal neurons.
Result: The diameter of dendritic shaft of the hippocampal CA3 pyramidal neurons was significantly thicker in the stress group (6.40um ±0.94um) than that in the control group (5.65um±0.85um) (p<0.05); the length of them (125.35um±18.69um) was shorter than that in the control group (98.51um ± 17.48um) (p<0.01). The following ultrastructural changes of dendrites were observed in the hippocampal
CA3 pyramidal neurons of the stress group: breaking into fragmentation of microtubule, widening of spacing between parallel microtubules in longitudinal-section, irregularity of annular and maldistribution of monome in cross-section, crista clouding or degeneration as vacuoles of mitochondrion.
The average gray degree of phosphorylated MAP2 in hippocampal CA3 pyramidal neurons was significantly lower in the stress group (145 ±4.40) than that in the control group (149±1.81) (p<0.05). As the average gray degree is correlated negatively with expression of intensity, the decrease of phosphorylated MAP2 average gray degree in stress group indicates that the chronic stress enhances the phosphorylation of MAP2. Moreover, the number of phosphorylated MAP2 positive pyramidal neurons was significantly decreased in the stress group (40.36 ±1.35) compared to the control group (42.73 ± 1.56) (p<0.05).
The fluorescent intensity of [Ca2+]i in hippocampal CA3 pyramidal neurons was significantly stronger in the stress group (1548.83 ± 118.53 ) than that in the control group (598.71 ±48.02) (p<0.01), which indicated
that chronic stress increase the concentration of [Ca2+ ]i in the pyramidal neurons.
Conclusions: Chronic stress could damage the microtubules of dendrites leading to dendritic shaft retraction in rat hippocampal CA3 pyramidal neurons. In addition, chronic stress enhances phosphorylation
of MAP2 and increases the concentration of [Ca2+]i in the CA
引文
1、McEwen BS. Stress and hippocampal plasticity. Ann Rev Neurosci 1999; 22: 105-22
2、张艳美，杨权，许崇涛等．慢性应激对大鼠海马CA3区锥体细胞形态结构的效应。中华精神科杂志 2002；35(1)：22-24
3、Porter KR. The cytomatrix: a short history of its study. J Cell Biol 1984 suppl: 3s-12s
4、Stein-Behrens B, Mattson MP, Chang I, et al. Stress exacerbates neuron loss and cytoskeletal pathology in the hippocampus. J Neurosci 1994 Sep;14(9): 5373-5380
5、McEwen BS, Magarinos AM. Stress effects on morphology and function of the hippocampus. Ann N Y Acad Sci. 1997 Jun 21; 821:271-84
6、Kaech S, Ludin B, Matus A. Cytoskeletal plasticity in cells expressing neuronal microtubule-associated proteins. Neuron 1996 Dec; 17(6): 1189-99
7、Wilson MT, Keith CH. Glutamate modulation of dendrite outgrowth: alterations in the distribution of dendritic MTs. J Neurosci Res 1998 Jun 1; 52(5): 599-611
8、Chan J, Jensen CG, Jensen LC, et al. The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules. PNAS 1999 Dec 21; 96(26): 14931-6
9、Weisshaar B, Doll T, Matus A. Reorganisation of the microtubular cytoskeleton by embryonic microtubule-associated protein 2 (MAP2c). Development 1992 Dec; 116(4): 1151-61
10、Matus A. Microtubule-associated proteins and the determination of neuronal form. J Physiol(Paris) 1990; 84(1): 134-7
11、Maccioni RB, Cambiazo V. Role of microtubule-associated proteins in the control of microtubule assembly. Physiol Rev 1995 Oct; 75(4): 835-64
12、Desai A, Mitchison TJ. Microtubule polymerization dynamics. Annu Rev Cell Dev Biol 1997; 13:83-117
13、Kossel AH, Williams CV, Schweizer M, et al. Afferent innervation influences the
development of dendritic branches and spines via both activity-dependent and non-activity-dependent mechanisms. J Neurosci 1997 Aug 15; 17(16): 6314-24
14、Malefic-Savatic M, Malinow R, Svoboda K. Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 1999 Mar 19; 283(5409): 1923-7
15、Wilson MT, Kisaalita WS, Keith CH. Glutamate-induced changes in the pattern of hippocampal dendrite outgrowth: A role for calcium-dependent pathways and the microtubule cytoskeleton. J Neurobiol 2000 May; 43(2): 159-72
16、Quinlan EM, Halpain S. Postsynaptic mechanisms for bi-directional control of MAP2 phosphorylation by glutamate receptors. Neuron 1996 Feb; 16(2): 357-68
17、Quinlan E M, Halpain S. Emergence of activity-dependent, bi-directional control of microtubule-associated protein MAP2 phosphorylation during postnatal development. J Neurosci 1996 Dec; 16(23):7627-7637
18、Fukunaga K, Soderling TR, Miyamoto E. Activation of Ca~(2+)/calmodulin-dependent protein kinase Ⅱ and protein kinade C by glutamate in cultured rat hippocampal neurons. J Neurochem 1992 Nov 5; 267(31): 22527-33
19、Huang Xin P, Hampson DR. Inhibition of Microtubule Formation by Metabotropic Glutamate Receptors. J Neurochem 2000 Jan; 74(1): 104-13.
20、Whitford KL, Dijkhuizen P, Polleux F, et al. Molecular control of cortical dendrite development. Annu. Rev. Neurosci. 2002; 25:127-49
21、McEwen BS, Albeck D, Cameron H, Stress and the brain: a paradoxical role for adrenal steroids. Vitam Horm 1995; 51:371-402
22、Audesirk G, Cabell L, Kern M. Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons. Dev Brain Res 1997 Sep 20; 102(2): 247-60
23、Shea TB, Beermann ML. Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization. Mol Biol Cell 1994 Aug; 5(8): 863-75
24、Duman RS, Malberg J, Thome J. Neural plasticity to stress and antidepressant
treatment. Biol Psychiatry 1999 Nov 1; 46(9): 1181-91
25、Leza JC, Sales E, Sawicki G, et al. The effects of stress on homeostasis in JCR-la-cp rats: the role of nitric oxide. J Phamacol Exp Ther 1998 Sep; 286(3): 1397-403
26、Magarinos AM, McEwen BS. Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: involvement of glucocorticoid secretion and excitatory amino acid receptors. Neurosci 1995;69(1):89-98
27、马光瑜，樊小力，徐小虎等．用激光扫描共聚焦显微镜观察吗啡成瘾对大鼠海马神经细胞内钙离子的影响．中国神经精神疾病杂志 2000；26(2)：109-111
28、韩济生《神经科学原理》第二版 北京医科大学出版社 1999；145-148
29、Diez-Guerra FJ, Avila J. MAP2 phosphorylation parallels dendrite arborization in hippocampal neurons in culture. NeuroReport 1993 Apr; 4(4): 419-22
30、Friedrich P, Aszodi A. MAP2: a sensitive cross-linker and adjustable in dendritic architecture. FEBS Lett 1991 Dec 16; 295(1-3):5-9
31、Walaas SI, Nairn AC. Multisite phosphorylation of mierotubule-assoeiated protein2(MAP-2)in rat brain: peptide mapping distinguishes between cyclic-AMP-, calcium/calmodulin-, and calcium/phosphorylation mechanisms. J Mol Neurosci 1989; 1(2): 117-27
32、Berling B, Wille H, Roll B, et al. Phosphorylation of microtubule-associated proteins MAP2a, b and MAP2c at Ser 136 by proline-directed kinases in vivo and in vitro. Eur J Cell Biol 1994 Jun; 64(1): 120-30
33、Yamamoto H, Saitoh Y, Fukunaga K, et al. Dephosphorylation of microtubule proteins by brain protein phosphatases 1 and 2A, and its effect on microtubule assembly. J Neurochem 1988 May; 50(5): 1614-23
34、Checkley S. The neuroendocrinology of depression and chronic stress. Br Med Bull. 1996 Jul; 52(3): 597-617
35、Sapolsky RM. Why stress bad for your brain. Science 1996 Aug 9; 273(5276): 749-50
36、Herman JP, Cullinan WE. Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis. Trends Neurosci 1997 Feb; 20(2):
78-84
37、Blumcke I, Becker AJ, Klein C, et al. Temporal lobe epilepsy associated up-regulation of metabotropic glutamate receptors: correlated changes in mGluR1 mRNA and protein expression in experimental animals and human patients. J Neuropathol Exp Neurol 2000 Jan; 59(1): 1-10
38、Schwendt M, Jezova D. Gene expression of two glutamate receptor subunits in response to repeated stress exposure in rat hippocampus. Cell Mol Neurobiol 2000 Jun; 20(3): 319-29
39、Woolf NJ, Zinnerman MD, Johnson GV. Hippocampal microtubule-associated protein-2 alterations with contextual memory. Brain Res 1999 Mar 6; 821(1): 241-9
40、韩济生《神经科学原理》第二版 北京医科大学出版社1999；223-225
41、Neely MD, Nicholls JG. Electrical activity, growth cone motility and the cytoskeleton. J Exp Biol 1995 Jul; 198(Pt 7): 1433-46
42、Wu GY, Cline HT. Stabilization of dendritic arbor structure in vivo by CaMK Ⅱ. Science 1998 Jan 9; 279(5348): 222-6
2、张艳美，杨权，许崇涛等．慢性应激对大鼠海马CA3区锥体细胞形态结构的效应。中华精神科杂志 2002；35(1)：22-24
3、Porter KR. The cytomatrix: a short history of its study. J Cell Biol 1984 suppl: 3s-12s
4、Stein-Behrens B, Mattson MP, Chang I, et al. Stress exacerbates neuron loss and cytoskeletal pathology in the hippocampus. J Neurosci 1994 Sep;14(9): 5373-5380
5、McEwen BS, Magarinos AM. Stress effects on morphology and function of the hippocampus. Ann N Y Acad Sci. 1997 Jun 21; 821:271-84
6、Kaech S, Ludin B, Matus A. Cytoskeletal plasticity in cells expressing neuronal microtubule-associated proteins. Neuron 1996 Dec; 17(6): 1189-99
7、Wilson MT, Keith CH. Glutamate modulation of dendrite outgrowth: alterations in the distribution of dendritic MTs. J Neurosci Res 1998 Jun 1; 52(5): 599-611
8、Chan J, Jensen CG, Jensen LC, et al. The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules. PNAS 1999 Dec 21; 96(26): 14931-6
9、Weisshaar B, Doll T, Matus A. Reorganisation of the microtubular cytoskeleton by embryonic microtubule-associated protein 2 (MAP2c). Development 1992 Dec; 116(4): 1151-61
10、Matus A. Microtubule-associated proteins and the determination of neuronal form. J Physiol(Paris) 1990; 84(1): 134-7
11、Maccioni RB, Cambiazo V. Role of microtubule-associated proteins in the control of microtubule assembly. Physiol Rev 1995 Oct; 75(4): 835-64
12、Desai A, Mitchison TJ. Microtubule polymerization dynamics. Annu Rev Cell Dev Biol 1997; 13:83-117
13、Kossel AH, Williams CV, Schweizer M, et al. Afferent innervation influences the
development of dendritic branches and spines via both activity-dependent and non-activity-dependent mechanisms. J Neurosci 1997 Aug 15; 17(16): 6314-24
14、Malefic-Savatic M, Malinow R, Svoboda K. Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 1999 Mar 19; 283(5409): 1923-7
15、Wilson MT, Kisaalita WS, Keith CH. Glutamate-induced changes in the pattern of hippocampal dendrite outgrowth: A role for calcium-dependent pathways and the microtubule cytoskeleton. J Neurobiol 2000 May; 43(2): 159-72
16、Quinlan EM, Halpain S. Postsynaptic mechanisms for bi-directional control of MAP2 phosphorylation by glutamate receptors. Neuron 1996 Feb; 16(2): 357-68
17、Quinlan E M, Halpain S. Emergence of activity-dependent, bi-directional control of microtubule-associated protein MAP2 phosphorylation during postnatal development. J Neurosci 1996 Dec; 16(23):7627-7637
18、Fukunaga K, Soderling TR, Miyamoto E. Activation of Ca~(2+)/calmodulin-dependent protein kinase Ⅱ and protein kinade C by glutamate in cultured rat hippocampal neurons. J Neurochem 1992 Nov 5; 267(31): 22527-33
19、Huang Xin P, Hampson DR. Inhibition of Microtubule Formation by Metabotropic Glutamate Receptors. J Neurochem 2000 Jan; 74(1): 104-13.
20、Whitford KL, Dijkhuizen P, Polleux F, et al. Molecular control of cortical dendrite development. Annu. Rev. Neurosci. 2002; 25:127-49
21、McEwen BS, Albeck D, Cameron H, Stress and the brain: a paradoxical role for adrenal steroids. Vitam Horm 1995; 51:371-402
22、Audesirk G, Cabell L, Kern M. Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons. Dev Brain Res 1997 Sep 20; 102(2): 247-60
23、Shea TB, Beermann ML. Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization. Mol Biol Cell 1994 Aug; 5(8): 863-75
24、Duman RS, Malberg J, Thome J. Neural plasticity to stress and antidepressant
treatment. Biol Psychiatry 1999 Nov 1; 46(9): 1181-91
25、Leza JC, Sales E, Sawicki G, et al. The effects of stress on homeostasis in JCR-la-cp rats: the role of nitric oxide. J Phamacol Exp Ther 1998 Sep; 286(3): 1397-403
26、Magarinos AM, McEwen BS. Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: involvement of glucocorticoid secretion and excitatory amino acid receptors. Neurosci 1995;69(1):89-98
27、马光瑜，樊小力，徐小虎等．用激光扫描共聚焦显微镜观察吗啡成瘾对大鼠海马神经细胞内钙离子的影响．中国神经精神疾病杂志 2000；26(2)：109-111
28、韩济生《神经科学原理》第二版 北京医科大学出版社 1999；145-148
29、Diez-Guerra FJ, Avila J. MAP2 phosphorylation parallels dendrite arborization in hippocampal neurons in culture. NeuroReport 1993 Apr; 4(4): 419-22
30、Friedrich P, Aszodi A. MAP2: a sensitive cross-linker and adjustable in dendritic architecture. FEBS Lett 1991 Dec 16; 295(1-3):5-9
31、Walaas SI, Nairn AC. Multisite phosphorylation of mierotubule-assoeiated protein2(MAP-2)in rat brain: peptide mapping distinguishes between cyclic-AMP-, calcium/calmodulin-, and calcium/phosphorylation mechanisms. J Mol Neurosci 1989; 1(2): 117-27
32、Berling B, Wille H, Roll B, et al. Phosphorylation of microtubule-associated proteins MAP2a, b and MAP2c at Ser 136 by proline-directed kinases in vivo and in vitro. Eur J Cell Biol 1994 Jun; 64(1): 120-30
33、Yamamoto H, Saitoh Y, Fukunaga K, et al. Dephosphorylation of microtubule proteins by brain protein phosphatases 1 and 2A, and its effect on microtubule assembly. J Neurochem 1988 May; 50(5): 1614-23
34、Checkley S. The neuroendocrinology of depression and chronic stress. Br Med Bull. 1996 Jul; 52(3): 597-617
35、Sapolsky RM. Why stress bad for your brain. Science 1996 Aug 9; 273(5276): 749-50
36、Herman JP, Cullinan WE. Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis. Trends Neurosci 1997 Feb; 20(2):
78-84
37、Blumcke I, Becker AJ, Klein C, et al. Temporal lobe epilepsy associated up-regulation of metabotropic glutamate receptors: correlated changes in mGluR1 mRNA and protein expression in experimental animals and human patients. J Neuropathol Exp Neurol 2000 Jan; 59(1): 1-10
38、Schwendt M, Jezova D. Gene expression of two glutamate receptor subunits in response to repeated stress exposure in rat hippocampus. Cell Mol Neurobiol 2000 Jun; 20(3): 319-29
39、Woolf NJ, Zinnerman MD, Johnson GV. Hippocampal microtubule-associated protein-2 alterations with contextual memory. Brain Res 1999 Mar 6; 821(1): 241-9
40、韩济生《神经科学原理》第二版 北京医科大学出版社1999；223-225
41、Neely MD, Nicholls JG. Electrical activity, growth cone motility and the cytoskeleton. J Exp Biol 1995 Jul; 198(Pt 7): 1433-46
42、Wu GY, Cline HT. Stabilization of dendritic arbor structure in vivo by CaMK Ⅱ. Science 1998 Jan 9; 279(5348): 222-6