十溴联苯醚的神经毒理研究
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摘要
多溴联苯醚(PBDEs)是一种广泛添加于各种生活必须品中的溴代阻燃剂。它的大量使用已造成全球性的环境污染。PBDEs有209种同源物,其中主要的商用五溴和八溴联苯醚已因其严重毒性而被欧盟和美国禁用,但是十溴联苯醚(PBDE209)作为最主要的商用溴系阻燃剂,仍然在大量的使用。已有报道PBDEs能够损伤动物多种器官的功能,尤其是能够损伤中枢神经系统的功能。海马是学习和记忆功能的重要部位,损伤海马的功能,可能是PBDEs损伤中枢神经系统的一条重要途径。本文采用行为和电生理手段,应用morris水迷宫、在位场电位记录和单细胞膜片钳的方法,对PBDE209的神经毒性及其机理进行了研究。
1.PBDE209对空间学习和记忆能力的影响。
学习和记忆是神经系统重要的功能。研究学习记忆功能的机制可采用多种方法,Morris水迷宫是最常用的方法之一,主要应用于啮齿类动物空间学习记忆功能的研究和评估。本研究应用Morris水迷宫对PBDE209染毒的C57BL/6J小鼠的学习和记忆功能进行了测试,结果发现PBDE209在定位航行实验中明显延长了实验动物寻找到隐藏平台所用的总路程和总时间,以及在空间探索实验中明显改变了实验动物在目标象限的游泳路程和时间占总路程和时间的比率。这证实PBDE209既可以影响小鼠定位航行中的学习过程,也能影响小鼠空间搜索中的回忆过程和搜寻策略,表明PBDE209和PBDEs能够损伤神经系统的空间学习记忆功能。
2.PBDE209对大鼠海马DG区突触可塑性的影响。
海马神经元活动依赖的突触可塑性被认为是学习和记忆功能的细胞和分子基础。我们应用在位电生理技术研究了PBDE209对大鼠海马DG区神经元突触可塑性的影响,并进一步探讨了不同发育期的PBDE209暴露对成年大鼠海马DG区突触可塑性影响的差异。结果表明,PBDE209暴露不仅损伤了EPSP和PS的基础突触传递,也损伤了突触后电位和群峰电位的双脉冲反应和长时程增强。并且在不同发育期其对突触可塑性的损害不同,其中哺乳期是大鼠对PBDE209最敏感的时期。我们发现早期对母体暴露PBDE209,其子代海马神经元的突触可塑性的损伤不明显,但是在终身暴露的大鼠上,这种损伤比单纯的早期母体暴露或单纯的断奶后灌胃暴露PBDE209的损伤都大,因此,早期的母体暴露对这种损伤的累积效应起了重要的作用。
3.PBDE209对培养大鼠海马神经元电压门控钠通道的影响。
电压门控钠通道对于神经元动作电位的发放和传导具有重要的意义,钠通道功能特性的变化对于中枢神经系统神经元的功能活动有重要的影响。我们在阻断电压门控型钾通道和钙通道的条件下,在培养的大鼠海马神经元上记录了TTX敏感的电压门控钠通道电流,并研究了PBDE209对钠通道电流以及其动力学特性的作用。结果表明,PBDE209抑制了钠电流的幅度,其最低效应浓度为0.1μM,这种抑制效应具有浓度依赖性并且是不可逆的。PBDE209能使钠通道的激活和失活曲线向超极化方向移动,延长了激活和快速失活的时间,减缓了钠通道复活的时间,减少了钠通道激活的组分。这说明PBDE209可能通过影响钠通道的动力学特性从而损伤中枢神经系统功能。我们进一步施加抗氧化剂,抗坏血酸和维生素E,证实了PBDE209可能通过过氧化途径损伤了钠通道。
Polybrominated diphenyl ethers (PBDEs) is one of the flame-retardant additives, which is largely used in all parts of our lives. Its wide use has caused persistently environmental contamination. There are 209 congeners of PBDEs, and the penta- and octa-brominated diphenyl ethers have been banned by European Union and America for their toxic effects. But the decabrominated diphenyl ether (PBDE209), as the primary commercial product of PBDEs, still persistent and widely use. Previous studies indicated that many organs are targets of PBDEs, especially the central nervous system (CNS). Hippocampus is a region related to learning and memory. Impairing hippocampus may be one of the pathways that PBDEs impair the CNS. In this study, we investigated in depth the effects of PBDE 209 on the CNS, using behavioral and electrophysiological techniques, by morris water maze, field potential recording and patch clamp methods.
1. The effects of PBDE209 on spatial learning and memory.
Learning and memory is one of the most important functions of the CNS. There are many methods to study learning and memory. Morris water maze is one of the most frequently used methods, which is used to study and evaluate the spatial learning and memory of the rodents. In our study, we detected the effects of PBDE209 on the learning and memory of C57BL/6J mice through Morris water maze. The results showed PBDE209 could prolong the time and distance of mice finding the hide plate in the place navigation, and also changed the rate of distance and time between mice in target quadrant and all the quadrants in the spatial probe. Our founding indicated that PBDE209 could affect the learning process in the place navigation, and also affect the memory recall process and search strategy in the spatial probe. These confirm that PBDE209 can impair the spatial learning and memory of the CNS.
2. The influence of PBDE 209 on synaptic plasticity in the rat dentate gyrus (DG) in vivo.
The activity-dependent synaptic plasticity of hippocampus is regarded as the electrophysioloical substrate of learning and memory. We studied the effects of PBDE209 on synaptic plasticity in the DG in vivo, and discussed different effects of PBDE209 in the different developmental periods. The results demonstrated that PBDE 209 could impair the basic synaptic transmission, the paired-pulse reactions (PPR) and the long-term potentiation (LTP) of the field excitatory postsynaptic potential (fEPSP) slope and the population spike (PS) amplitude, and the lactation period is the most sensitive time of development towards PBDE 209. It was also found that PBDE 209 received from mother in early developmental periods play an important role to the toxic effects of PBDE 209 in whole range exposure.
3. The effects of PBDEs on voltage-gated sodium channels (VGSCs) in rat primary cultured hippocampal neurons.
VGSCs are responsible for both initiation and propagation of action potentials of the neurons in the hippocampus and throughout the central nervous system. Therefore, modulation of the functional properties of VGSCs would be expected to alter the activity and function of CNS neurons. We recorded TTX-sensitive voltage-gated sodium channels currents (I_(Na)) in rat primary cultured hippocampal neurons and studied the effects of PBDE 209 on I_(Na) and their kinetic properties, when the voltage-gated potassium channels and calcium channels were blocked. The results showed that PBDE 209 could irreversibly decrease I_(Na) in a concentration-dependent manner and the lowest assayed concentration was 0.1μM, PBDE 209 could shift the activation and inactivation of I_(Na) toward hyperpolarizing direction, prolong the time course of activation and fast inactivation, slow down the recovery from inactivation of I_(Na), and decrease the fraction of activated sodium channels. These results suggested that PBDE 209 could modulate voltage-gated sodium channels (VGSCs), which may lead to change in electrical activity and contribute to worsen the neurotoxicological damage. Furthermore, ascorbic acid, as an antioxidant, was found capable of partially reversing those effects, which indicates that PBDE 209 might inhibit I_(Na) through peroxidation.
1.PBDE209对空间学习和记忆能力的影响。
学习和记忆是神经系统重要的功能。研究学习记忆功能的机制可采用多种方法,Morris水迷宫是最常用的方法之一,主要应用于啮齿类动物空间学习记忆功能的研究和评估。本研究应用Morris水迷宫对PBDE209染毒的C57BL/6J小鼠的学习和记忆功能进行了测试,结果发现PBDE209在定位航行实验中明显延长了实验动物寻找到隐藏平台所用的总路程和总时间,以及在空间探索实验中明显改变了实验动物在目标象限的游泳路程和时间占总路程和时间的比率。这证实PBDE209既可以影响小鼠定位航行中的学习过程,也能影响小鼠空间搜索中的回忆过程和搜寻策略,表明PBDE209和PBDEs能够损伤神经系统的空间学习记忆功能。
2.PBDE209对大鼠海马DG区突触可塑性的影响。
海马神经元活动依赖的突触可塑性被认为是学习和记忆功能的细胞和分子基础。我们应用在位电生理技术研究了PBDE209对大鼠海马DG区神经元突触可塑性的影响,并进一步探讨了不同发育期的PBDE209暴露对成年大鼠海马DG区突触可塑性影响的差异。结果表明,PBDE209暴露不仅损伤了EPSP和PS的基础突触传递,也损伤了突触后电位和群峰电位的双脉冲反应和长时程增强。并且在不同发育期其对突触可塑性的损害不同,其中哺乳期是大鼠对PBDE209最敏感的时期。我们发现早期对母体暴露PBDE209,其子代海马神经元的突触可塑性的损伤不明显,但是在终身暴露的大鼠上,这种损伤比单纯的早期母体暴露或单纯的断奶后灌胃暴露PBDE209的损伤都大,因此,早期的母体暴露对这种损伤的累积效应起了重要的作用。
3.PBDE209对培养大鼠海马神经元电压门控钠通道的影响。
电压门控钠通道对于神经元动作电位的发放和传导具有重要的意义,钠通道功能特性的变化对于中枢神经系统神经元的功能活动有重要的影响。我们在阻断电压门控型钾通道和钙通道的条件下,在培养的大鼠海马神经元上记录了TTX敏感的电压门控钠通道电流,并研究了PBDE209对钠通道电流以及其动力学特性的作用。结果表明,PBDE209抑制了钠电流的幅度,其最低效应浓度为0.1μM,这种抑制效应具有浓度依赖性并且是不可逆的。PBDE209能使钠通道的激活和失活曲线向超极化方向移动,延长了激活和快速失活的时间,减缓了钠通道复活的时间,减少了钠通道激活的组分。这说明PBDE209可能通过影响钠通道的动力学特性从而损伤中枢神经系统功能。我们进一步施加抗氧化剂,抗坏血酸和维生素E,证实了PBDE209可能通过过氧化途径损伤了钠通道。
Polybrominated diphenyl ethers (PBDEs) is one of the flame-retardant additives, which is largely used in all parts of our lives. Its wide use has caused persistently environmental contamination. There are 209 congeners of PBDEs, and the penta- and octa-brominated diphenyl ethers have been banned by European Union and America for their toxic effects. But the decabrominated diphenyl ether (PBDE209), as the primary commercial product of PBDEs, still persistent and widely use. Previous studies indicated that many organs are targets of PBDEs, especially the central nervous system (CNS). Hippocampus is a region related to learning and memory. Impairing hippocampus may be one of the pathways that PBDEs impair the CNS. In this study, we investigated in depth the effects of PBDE 209 on the CNS, using behavioral and electrophysiological techniques, by morris water maze, field potential recording and patch clamp methods.
1. The effects of PBDE209 on spatial learning and memory.
Learning and memory is one of the most important functions of the CNS. There are many methods to study learning and memory. Morris water maze is one of the most frequently used methods, which is used to study and evaluate the spatial learning and memory of the rodents. In our study, we detected the effects of PBDE209 on the learning and memory of C57BL/6J mice through Morris water maze. The results showed PBDE209 could prolong the time and distance of mice finding the hide plate in the place navigation, and also changed the rate of distance and time between mice in target quadrant and all the quadrants in the spatial probe. Our founding indicated that PBDE209 could affect the learning process in the place navigation, and also affect the memory recall process and search strategy in the spatial probe. These confirm that PBDE209 can impair the spatial learning and memory of the CNS.
2. The influence of PBDE 209 on synaptic plasticity in the rat dentate gyrus (DG) in vivo.
The activity-dependent synaptic plasticity of hippocampus is regarded as the electrophysioloical substrate of learning and memory. We studied the effects of PBDE209 on synaptic plasticity in the DG in vivo, and discussed different effects of PBDE209 in the different developmental periods. The results demonstrated that PBDE 209 could impair the basic synaptic transmission, the paired-pulse reactions (PPR) and the long-term potentiation (LTP) of the field excitatory postsynaptic potential (fEPSP) slope and the population spike (PS) amplitude, and the lactation period is the most sensitive time of development towards PBDE 209. It was also found that PBDE 209 received from mother in early developmental periods play an important role to the toxic effects of PBDE 209 in whole range exposure.
3. The effects of PBDEs on voltage-gated sodium channels (VGSCs) in rat primary cultured hippocampal neurons.
VGSCs are responsible for both initiation and propagation of action potentials of the neurons in the hippocampus and throughout the central nervous system. Therefore, modulation of the functional properties of VGSCs would be expected to alter the activity and function of CNS neurons. We recorded TTX-sensitive voltage-gated sodium channels currents (I_(Na)) in rat primary cultured hippocampal neurons and studied the effects of PBDE 209 on I_(Na) and their kinetic properties, when the voltage-gated potassium channels and calcium channels were blocked. The results showed that PBDE 209 could irreversibly decrease I_(Na) in a concentration-dependent manner and the lowest assayed concentration was 0.1μM, PBDE 209 could shift the activation and inactivation of I_(Na) toward hyperpolarizing direction, prolong the time course of activation and fast inactivation, slow down the recovery from inactivation of I_(Na), and decrease the fraction of activated sodium channels. These results suggested that PBDE 209 could modulate voltage-gated sodium channels (VGSCs), which may lead to change in electrical activity and contribute to worsen the neurotoxicological damage. Furthermore, ascorbic acid, as an antioxidant, was found capable of partially reversing those effects, which indicates that PBDE 209 might inhibit I_(Na) through peroxidation.
引文
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