【作者】 王树；

【导师】 阮迪云；

【作者基本信息】 中国科学技术大学 ， 生物物理， 2008， 博士

【摘要】 镉(Cd²⁺)是一种常见的环境污染物，严重危害人类健康。慢性镉暴露会对肾、肺、胃肠道、骨骼以及中枢神经系统造成损伤。在中枢神经系统，特别是在发育期，镉会造成生化和形态学上的改变以及可能由此引起的认知功能障碍。在本论文中，我们运用电生理学的方法，从以下几个方面研究了镉在海马中的神经毒性机制。1)应用全细胞脑片膜片钳技术，我们研究了Cd²⁺对海马雪氏侧枝到CA1区锥体神经元由AMPA受体介导的谷氨酸能突触传递的作用。AMPA受体在中枢神经系统的兴奋性突触传递及突触可塑性方面起着非常重要的作用。中枢神经系统一些正常的生理过程比如学习记忆,以及一些病理状态都可能涉及到AMPA受体所介导的突触传递的改变。Cd²⁺以浓度依赖的方式抑制了诱发的兴奋性突触后电流（eEPSCs），并且增强了双脉冲易化效应和频率易化效应。Cd²⁺降低了自发兴奋性突触后电流（sEPSCs）的频率和幅度，但是没有影响到微小兴奋性突触后电流（mEPSCs）的频率和幅度。这些作用可能是由于Cd²⁺抑制了突触前电压依赖的钙离子流入诱发的谷氨酸释放。此外，Cd²⁺延长了sEPSCs和mEPSCs的衰减相，提示Cd²⁺的突触后作用位点。我们的结果表明Cd²⁺影响了海马雪氏侧枝到CA1区锥体神经元由AMPA受体介导的谷氨酸能突触传递以及短时突触可塑性，这可能是镉神经毒性的一个可能机制。2)我们在急性分离的大鼠海马CA1区神经元上应用全细胞膜片钳技术研究了Cd²⁺对瞬时外向钾电流I_A和延迟整流钾电流I_K的作用。在神经系统，电压门控钾电流对于神经元和神经网络的兴奋性有非常重要的作用，钾通道功能的改变可能会造成神经元发放特性的变化和异常放电。结果表明Cd²⁺以浓度依赖的方式可逆地抑制了I_A和I_K的幅度，半数抑制浓度IC₅₀分别为546±59μM和749±53μM，并且Cd²⁺的抑制作用是电压依赖性的。Cd²⁺使I_A的稳态激活曲线和稳态失活曲线显著右移。与之相比，Cd²⁺使I_K的稳态激活曲线也显著右移但移动程度较小，失活曲线未受到影响。Cd²⁺延缓了I_K的复活，但对I_A的复活时间没影响。这些结果提示Cd²⁺可能是通过与钾通道蛋白上某一特定位点的直接相互作用从而影响I_A和I_K，而不是通过对细胞膜表面电荷的屏蔽作用来产生效应。Cd²⁺对电压门控钾电流的作用可能是其神经毒性机制的一个方面，另外本实验表明Cd²⁺对钾电流的有效浓度与其作用于钙电流的浓度重叠，提示在将其作为钙通道阻断剂时要考虑到其对钾电流的作用是否会对实验结果带来影响。3)我们在急性分离的大鼠海马CA1区神经元上应用全细胞膜片钳技术研究了Cd²⁺对甘氨酸诱导的氯电流(I_Gly)的作用。甘氨酸受体（GlyR）可以调控CA1区海马神经元的发放以及兴奋性突触传递，GlyR功能的改变会影响到海马整个神经环路的信息处理。我们的结果表明Cd²⁺以浓度依赖的方式可逆地抑制了I_Gly,半数抑制浓度IC₅₀和Hill系数分别为1．27mM和0．45。在-60mV到+40mV范围内，Cd²⁺对I_Gly的作用是非电压依赖的，并且其反转电位不受影响。对甘氨酸的剂量效应曲线进行双倒数作图，表明Cd²⁺对I_Gly的抑制是非竞争性抑制。胞内透析3mM的Cd²⁺对I_Gly没有影响表明其作用位点可能在胞外。3mM的Cd²⁺不能影响Zn²⁺对I_Gly的抑制作用，表明Zn²⁺和Cd²⁺在甘氨酸受体上的作用位点可能不同。这些结果表明镉抑制了海马CA1区神经元上甘氨酸受体介导的氯电流，有助于我们对镉神经毒性机制的进一步了解。更多还原

【Abstract】 Cadmium (Cd²⁺), a common environmental pollutant, is a nonphysiological metal potentially toxic to human. Cadmium exposure causes a variety of impairments to kidney, lung, gastrointestinal tract, bone and the central nervous system （CNS）. In the CNS, especially during development, chronic cadmium exposure causes biochemical and morphological changes of the brain and results in cognitive function disabilities. In this study, using electrophysiological methods, we investigated the neurotoxic mechanisms of Cd²⁺ in hippocampus in the following directions.1) With whole-cell patch-clamp recording in rat hippocampal slices, we examined the effects of Cd²⁺ on AMPA （α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid） receptor-mediated synaptic transmission and short-term synaptic plasticity in hippocampal CA1 area.The subtype excitatory amino acid AMPA receptors play a central role in fast synaptic transmission and plasticity at most CNS excitatory synapses. Changes to the strength of AMPA receptor-mediated synaptic transmission are involved with normal physiological brain functions, including learning and memory, as well as with many neuropathological disorders. Cd²⁺ significantly inhibited the peak amplitude of evoked EPSCs （eEPSCs） in a concentration-dependent manner and enhanced the short-term synaptic plasticity including paired-pulse facilitation and frequency facilitation. Cd²⁺ also decreased the frequency and amplitude of spontaneous EPSCs （sEPSCs） but had no effect on those of miniature EPSCs （mEPSCs）. These effects of Cd²⁺ may involve a presynaptic mechanism of blockade of action potential-sensitive, calcium-dependent release of glutamate. In addition, Cd²⁺ prolonged the decay time of both sEPSCs and mEPSCs, which suggested a postsynaptic action site of Cd²⁺. This study demonstrates that Cd²⁺ impairs the Schaffer collateral-commissural-CA1 glutamatergic synaptic transmission and short-term plasticity in rat hippocampal slices, which may be a possible mechanism underlying the Cd²⁺-induced neurotoxic effects.2) The effects of Cd²⁺ on the transient outward potassium current （I_A） and delayed rectifier potassium current （I_K） were investigated in acutely dissociated rat hippocampal CA1 neurons using the whole-cell patch-clamp technique.In nervous system, Voltage-gated potassium currents play crucial roles in modifying neuronal cellular and network excitability, the regulation of potassium channels would make neurons display aberrant firing properties and abnormal neuronal discharge. The results showed that Cd²⁺ inhibited the amplitudes of I_A and I_Kin a reversible and concentration-dependent manner, with IC₅₀ values of 546±59 and 749±53μM, and the inhibitory effect of Cd²⁺ was voltage-dependent. Cd²⁺ significantly shifted the steady-state activation and inactivation curve of I_A to more positive potentials. In contrast, Cd²⁺ caused a relatively less but still significant positive shift in the activation of I_K without effect on the inactivation curve. Cd²⁺ significantly slowed the recovery from inactivation of I_K but had no effect on the recovery time course of I_A. The results suggested that the modulation of I_A and I_K was most likely mediated by the interaction of Cd²⁺ with a specific site on the potassium channel protein, rather than by screening of bulk surface negative charge. The effects of Cd²⁺ on the voltage-gated potassium currents may be a possible contributing mechanism for the Cd²⁺-induced neurotoxic damage. In addition, the effects of Cd²⁺ on the potassium currents at concentrations that overlap with its effects on calcium currents raise the concerns about its use in pharmacological or physiological studies.3) The effects of Cd²⁺ on glycine-induced Cl^- current (I_Gly) were investigated in acutely dissociated rat hippocampal CA1 neurons using the whole-cell patch-clamp technique in this study.In hippocampus, GlyR may participate in an inhibitory mechanism by affecting somatical firing and synaptic transmission, the modulation of GlyR may affect hippocampal information processing. We found that Cd²⁺ reversibly and concentration-dependently reduced the amplitudes of I_Gly, with an IC₅₀ of 1.27 mM and Hill coefficient of 0.45. The depression of I_Gly by Cd²⁺ was independent of membrane voltage between -60mV and +40mV and didn’t involve a shift in the reversal potential of the current. A noncompetitive inhibition was suggested by a double reciprocal plot of the effects of Cd²⁺ on the concentration-response curve of the I_Gly. Since intracellular dialysis with 3 mM Cd²⁺ failed to modify I_Gly, it was suggested that the site of action of Cd²⁺ is extracellular. The suppression of I_Gly by Zn²⁺ was unaffected by 3 mM Cd²⁺, which indicated that Zn²⁺ and Cd²⁺ bind to independent sites on glycine receptor. The results show that Cd²⁺ decreases I_Gly in acutely dissociated rat hippocampal neurons and the present study may be helpful to understand the mechanisms of cadmium-induced neurotoxicity.更多还原