树突膜片钳记录方法的建立与应用

发布时间：2018-03-31 13:05

本文选题：树突膜片钳记录　切入点：树突特性　出处：《南京医科大学》2012年硕士论文

【摘要】：膜片钳技术是上个世纪90年代初兴起的一种记录细胞膜离子通道电生理特性的技术,它为了解生物膜离子通道的门控动力学特征以及通透性等提供了最直接的手段。在神经科学领域,膜片钳技术多用于探索神经元上离子通道的分布特点和功能特性。大多数的膜片钳记录是在胞体上进行的。神经元的另一重要结构——树突,因其直径较小,一直以来限制树突膜片钳技术的操作。然而,树突接受大多数投射到神经元的突触传入,其上分布着大量不同类型的离子通道,是突触整合、突触可塑性调节的重要位点,具有重要的生理功能。因此在树突上进行直接的膜片钳记录,将为进一步了解树突的兴奋性特点提供新的机会。我们在成功进行胞体膜片钳记录的基础上对树突膜片钳记录进行了摸索。我们的实验以海马脑片为标本,在微分干涉相衬(Differential interference contrast, DIC)显微镜下观察到健康的CA1区锥体神经元的树突后操作的。虽然树突记录的基本操作技术类似于胞体记录,但与后者相比,树突记录需要更高的精确度。经过多次尝试,我们发现,要提高树突记录的成功率,需要注意的主要有以下几个方面：使用最优化的膜片钳设备；提高脑片质量,以看到更多适合封接的健康树突；电极尖端直径的大小要合适,过大过小都会影响树突的封接；电极接触树突的方式要正确,不可过度；破膜时所用负压要适度,也可采用电击破或强超极化的方式破膜。我们在CA1区锥体神经元的胞体和顶树突同时记录,研究树突的被动特性和主动特性。实验中,我们分别在顶树突的不同位点,给予相同大小的兴奋性突触后电流(Excitatory postsynaptic current, EPSC)样注电流,在胞体和顶树突同时记录。结果发现,树突注电流位点距离胞体越远,产生的树突局部兴奋性突触后电位(Excitatory postsynaptic potential, EPSP)幅度越大,而同时在胞体记录的EPSP(?)的幅度则更小,Half-width与Rise time则更长。实验结果说明由于树突本身的特性,同样大小的注电流在树突较远端产生的EPSPi幅度比在较近端产生的大,而且EPSP在向胞体传递的过程中幅度逐渐衰减,持续时间逐渐延长。我们换用玻璃电极刺激海马CA1区的辐射层(Stratum radiatum, SR),分别在不同的树突位点和胞体同时记录,得到一致的结果。因此我们得出结论：EPSP的传递效率依赖突触传入的位点。为观察EPSP由胞体向树突方向的传递情况,我们在胞体给予一定大小的EPSC-样注电流,分别在树突不同位点和胞体同时记录。结果发现,EPSP在向树突传递的过程中,幅度逐渐衰减,Half-width与Rise time逐渐增加。基底树突直径非常小,一方面要求封接基底树突所用的电极尖端直径更精细,同时也需要更精确的封接方法,因此大大增加了封接的难度。我们在多次失败经验的基础上终于在基底树突记录到电流。在获得高质量树突记录的基础上,我们打算对不同树突来源的两个EPSP的整合机制进行探讨。我们用玻璃电极与金属电极同时刺激海马CA1区的SR和始层(Stratum oriens, SO),-10ms的时间窗(SO刺激在SR刺激之前10ms)以10Hz的频率,将顶树突EPSP与基底树突EPSP配对诱导60次,结果发现EPSP的长时程增强(Long-term potentiation, LTP)现象。同样的protocol下,+10ms时获得EPSP的长时程抑制现象(Long-term depression, LTD)。我们计划借助顶树突与胞体、胞体与基底树突同时记录的方法探索顶树突EPSP与基底树突EPSP整合的机制。
[Abstract]:The technique of patch clamp technique is a technique of recording the electrophysiological properties of ionic channel in the cell membrane . It provides the most direct means for understanding the characteristics of the channel and the permeability of the membrane . In the field of neurosciences , the membrane patch clamp technique is used to explore the distribution characteristics and functional characteristics of ion channels in neurons .

We have carried on the study of dendritic patch clamp recordings on the basis of successful cell patch clamp recording . Our experiments show that dendritic cells of healthy CA1 pyramidal neurons are operated under differential interference contrast ( DIC ) microscope . Although the basic operating techniques of dendritic records are similar to those of the latter , the dendritic records require higher accuracy . After many attempts , we have found that to improve the success rate of dendritic records , it is important to pay attention to the following aspects : the use of optimized patch clamp equipment ;
improve the quality of the brain slices so as to see more healthy dendrites suitable for sealing ;
The diameter of the tip of the electrode is suitable , too much too small to affect the sealing of the dendritic ;
the method of electrode contact dendritic is correct and not excessive ;
the negative pressure used in the membrane breaking is moderate , and the membrane can be broken by adopting a mode of electric shock or strong hyperpolarization .

At the same time , we found that the greater the EPSPi amplitude at the distal end of the dendritic cells , the greater the amplitude of the EPSP at the distal end of the dendritic cells , and longer in the EPSP ( ? ) in the cell body . The results show that the transmission efficiency of EPSP depends on the afferent locus of the synaptic transmission .

In order to observe the transmission of EPSP from the cytoplasm to the dendritic direction , we recorded EPSC - like injection currents with a certain size in the cytoplasm at the same time . The results showed that the amplitude of EPSP gradually decreased , half - width and rise time increased gradually in the process of transferring dendritic cells to dendritic cells .

The diameter of the base dendritic is very small , on the one hand , it is required that the diameter of the electrode tip used for sealing the base dendritic is finer , and more accurate sealing method is needed , thus greatly increasing the difficulty of sealing .

On the basis of obtaining high - quality dendritic records , we intend to explore the integration mechanism of two EPSPs from different dendritic sources . We use the glass electrode and the metal electrode to stimulate the SR and the initial layer ( SO ) in the CA1 region of the hippocampus at the same time . The time window of 10 Hz ( 10 ms before SR stimulation ) is used to pair the top dendritic EPSP and the base dendritic EPSP to induce 60 times . The results show that the long - term depression ( LTP ) phenomenon of EPSP is obtained . Under the same protocol , the long - term depression ( LTD ) of EPSP is obtained at + 10 ms . We plan to explore the mechanism of the integration of the top dendritic EPSP with the base dendritic EPSP by means of simultaneous recording of the top dendritic cell and the cell body , the cell body and the base dendritic cell .

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R338

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