【作者】 王烨；

【导师】 李朝义；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 神经生物学， 2003， 博士

【摘要】 本文用在体全细胞记录技术，结合细胞内染色的方法，研究了猫纹状皮层神经元不同类型感受野和整合野的突触特性，以及相关神经元的形态学特征。在本文第一部分，讨论了我们实验室建立的一种新的全细胞记录方法。此方法的关键是不打开硬脑膜，先用胶原酶（collagenase）将其软化，使玻璃微电极能够穿过。此方法能有效地减弱由心跳和呼吸引起的脑波动，同时又能减少皮层表面的细胞直接受外界环境的影像，从而获得长时间稳定的胞内记录和胞内注射，并极大地简化了通常胞内记录所必须的繁杂手术准备。从细胞内记录和胞内染色的结果可以看出，用胶原酶软化硬脑膜对皮层神经元的功能和形态均无影响。第二部分用上述在体胞内记录技术研究了视皮层内兴奋性和抑制性回路在方位调谐加工中的作用。在全细胞记录状态下，多数皮层细胞的静息膜电位可以达到－30到－60mV,并可清晰地观察到被记录细胞的兴奋性突触后电位（EPSPs）、抑制性突触后电位（IPSPs）和动作电位（AP）。在用不同方位的正弦调制光栅刺激感受野时，膜电位也呈现与刺激频率相同的正弦形波动。表现为膜电位去极化，主要由复合的EPSP构成，也包含少数IPSP。根据此波动的振幅与刺激方位的关系，可以测出视皮层神经元输入端（PSPs）的方位调谐曲线。同样，根据AP频率与刺激方位的关系，可以测出视皮层神经元输出端的方位调谐曲线。将同一神经元的输入和输出曲线进行比较可以看出，经过视皮层内神经回路加工以后，方位调谐曲线的宽度变窄，调谐特性被明显地锐化。为了阐明这种锐化作用的机制，我们进一步分析了视皮层神经元的调制传递函数，观察到在输入和输出信号之间，存在着一种指数函数关系。这说明方位调谐的锐化取决于皮层神经元的非线性传递特性。在对比度调谐和空间频率调谐的加工过程中，皮层神经元的这种非线性机制也同样起到了锐化作用。第三部分在在体全细胞记录的过程中，通过给细胞施加去极化或超极化电流，可以改变细胞的发放活动。据此我设计实验让细胞在去极化，超极化和正常状态下接受长时间的非最佳方位光栅刺激，考查细胞的方位和方向选择特性是否会发生可塑性变化。结果发现细胞的方位方向调谐特性确实会发生一些变化，但细胞的个体差异很大。<WP=7>第四部分用在体全细胞记录方法研究了整合野与感受野之间相互作用的突触机制。分别用正弦光栅刺激感受野和/或整合野，用一种统计学方法定量地测量被记录细胞的EPSP和IPSP的变化。在用最佳光栅同时刺激感受野易化型整合野时，记录到的EPSP增加，细胞的膜平均电位水平被去极化，但主要改变的成分因细胞而异；在用最佳光栅同时刺激感受抑制型型整合野时，记录到的细胞平均膜电位趋向超极化，同样被改变的成分因细胞各异，有的EPSP减少，有的IPSP成分加强，有的膜电位波动幅度减小，有的电位分布整体向超极化移动。提示整合野的作用可能涉及多种机制。第五部分用细胞内注射生物胞素（biocytin）染色的方法，研究了不同类型整合野神经元的细胞形态学及其与功能的关系。易化型整合野神经元多数为多棘锥体细胞，胞体和树突野较大；树突棘密度高，多为粗短型；轴突分枝广泛，分布范围包括多个皮层层次，在II、III层可见到大范围的水平分支和间隔400-700?m的簇状分布。抑制型整合野神经元为少棘锥体细胞或非锥体细胞；胞体和树突野较小，树突棘少而稀疏，多为纤细型；轴突分枝极少，有的是以单一纤维形式直接投射到白质内。更多还原

【Abstract】 The synaptic properties of classical receptive field (CRF) and integration field (IF) of cat’s striate cortical neurons and their morphological features were investigated using the whole cell recording and intracellular staining techniques in vivo. In part I of this thesis, a new approach of the whole cell recording method developed by our laboratory is discussed. The core of this method is to remain the dura intact. To ensure the penetration of glass microelectrode, dura mater was softened by collagenase. A continuous positive pressure was applied during penetration to keep electrode clean of tissue debris. When the tip of the electrode was finally resting on the cell membrane, the positive pressure was released and a small negative pressure was applied to the electrode for the formation of a seal. This method can effectively minimize cardiopulmonary pulsations of the brain and remain the intracellular recordings and injections stable for an hour or two without performing a pneumothorax preparation. Cortical neurons had well-preserved morphological state and normal electrophysiological properties after dura mater was treated by the enzyme.In part II, the effects of intracortical excitatory and inhibitory circuitries on the orientation selectivity were studied using the in vivo whole cell recording technique. Under this condition, the resting membrane potentials of most neurons were between -30 and -60 mV, and the EPSPs, IPSPs and action potentials (AP) were clearly observed. When a sinusoidal luminance grating moving across the simple cell’s receptive field, all the cells tested responded with periodic fluctuations in their membrane potentials at the temporal frequency of the visual stimulus. The<WP=9>fluctuations were mostly due to periodic depolarization of membrane potential resulted from the summed EPSPs, which were occasionally interrupted by individual IPSPs. Orientation tuning of the cells can be deduced from the input signals, i.e., the amplitude of EPSPs and from the output signals, the firing frequency of AP, respectively. By comparing the tuning width of the input and output tuning curves, it is shown that the orientation selectivity is sharpened after being processed by the intracortical connection. To explore the mechanisms of this sharpening process, we analysed the transfer function of cortical neurons. An exponential transfer characteristic was observed between the synaptic input and the cell’s output. This non-linear transformation of cortical neurons may underlie the sharpening of orientation tuning properties. This mechanism was also involved in the sharpening of the other selectivities of visual attributes. On the other hand, the amplitude of summed IPSPs was also modulated by the visual stimulation. In most cases, the IPSPs reached maximum at the optimal orientation, implying an effect of enhancing temporal and spatial precision.In part III, the synaptic plasticity of orientation and direction selectivity of simple cells in primary visual cortex was studied. We use hyperpolarize and depolarize current to change the membrane potential and discharge activity of the cells, paired with selected orientation drifting grating stimulation. The subsequent orientation tuning properties were examined. We found the optimal orientation and direction shifted not only related to the polarity of the current paired with training orientation. In part IV, synaptic mechanisms of the interaction between the CRF and IF were investigated using the whole cell recording technique in vivo. The probability of depolarization and hyperpolarization of membrane potential was counted when CRF and/or IF were stimulated by the drifting sinusoidal gratings. When an optimal grating moved across the facilitatory both the CRF and IF, the amplitude and frequency of EPSPs increased and the membrane potential depolarized. The mechanisms and components of the changes in membrane potential may differ in different cells. Similarly, stimulating the inhibitory IF induced increase of the amplitude and frequency of IPSPs or supp更多还原