【作者】 张卫鹏；

【导师】 赵堪兴；

【作者基本信息】 天津医科大学 ， 眼科学， 2009， 博士

【摘要】 目的本文通过建立可视法脑片膜片钳技术，研究发育期大鼠视皮层突触功能活动以及发育期大鼠视皮层脉冲时间依赖的突触可塑性(spike timing-dependentplasticity，STDP)，了解视皮层经验依赖的突触可塑性，进一步探索视觉经验在视皮层神经元突触发育成熟过程中的作用，以及在异常视觉经验状态下视皮层突触功能状态的改变，从功能的角度来探索弱视发生机制，为寻求合理有效的预防与治疗弱视的方法建立基础。方法1．应用红外微分干涉相差显微镜(IR-DIC)结合电耦合式摄像机(CCD-Camera)可视法膜片钳全细胞记录生后2～7 d、8～14 d、15～21 d、22～28d各组自发兴奋性突触后电流(spontaneous excitatory postsynaptic currents，sEPSCs)变化，同时于电极内液中加入0．3％荧光黄(luciferyellow)对所记录细胞进行染色观察形态学改变，分别记录17 d(正常饲养组及双眼剥夺组)大鼠脑片的sEPSCs并行统计学分析。2．在较低频率细胞内刺激下应用双膜片钳全细胞记录技术，观察在不同的配对刺激方案下，大鼠脑片视皮质2／3层内两锥体神经元突触传递效能的长时程变化情况。同时通过电极内液加入荧光染料观察细胞与突触形态。3．采用STDP模式，应用双膜片钳技术，对双眼剥夺组和对照组的兴奋性突触后电位基线值(control EPSP)进行测量，统计平均EPSP幅值，观察突触传递效能变化。在精确控制突触前和突触后神经元刺激时间下，采用不同的突触前后配对刺激方案对EPSP变化进行测量，观察双眼剥夺组和对照组的区别。同时采用传统的长时程增强(LTP)诱导方式(仅给予突触前脉冲刺激)，采取频率由低到高，并逐渐接近近似θ波刺激频率(theta-burst stimulation，TBS)，逐一观察不同频率下EPSP变化。结果1．可视法脑片膜片钳技术可对神经元直接进行准确定位及健康细胞的筛选。视皮层神经元sEPSCs幅值与频率均随发育逐渐升高，(单因素方差分析，频率F=87．46，幅值F=20．69，P＜0．01详见第一部分结果)。但2～7 d组与8～14 d组差异无统计学意义(P＞0．05)。视皮层2／3层神经元胞体及突起以及生物电学特性随发育逐渐成熟。双眼剥夺组和健康对照组的sEPSCs比较，幅值下降明显P＜0．05，频率比较差异无显著意义P＞0．05。2．精确控制突触前和突触后神经元刺激时间下，突触前后配对刺激后测试突触后动作电位(test EPSP)平均增幅在120％以上，LTP诱导成功。而只有突触前或只有突触后刺激，test EPSP平均增幅在110％以下，LTP未诱导成功。3．双眼剥夺组视皮层2／3层内两相邻锥体神经元之间的突触传递效能下降。剥夺组与对照组平均EPSP幅值(基线值)比较差异具有显著统计学意义(P＜0．01)。配对诱导刺激50分后，双眼剥夺组EPSP幅值上升幅度较对照组高，两组差异具有统计学意义P＜0．05，两组均可诱导成功LTP。而采用传统的LTP诱导方法低频刺激，24例均未诱导出LTP。逐渐增加频率，EPSP增幅也随之略增加，但即使频率增加到100Hz左右近似TBS刺激频率时，EPSP平均增幅也不能达到110％以上。结论1．可视法脑片膜片钳技术操作更直观方便，对神经元的分辨率及封接细胞的成功率都大大提高。生后早期视皮层2／3层神经元及突触发育处于相对不成熟状态，随着发育逐渐成熟，视觉经验在这一过程中起了关键性作用。研究显示发育早期突触并非完全处于静息状态，具有一定的早期自发的突触功能活动。2．突触传递效能的变化依赖于突触前后的联合脉冲刺激，脉冲时间前与后的顺序设定决定了突触传递效能变化的方向。突触前后脉冲刺激对突触传递效能的变化存在一个时间窗关系，STDP突触修饰法则可以解释经验活动改变皮层神经元功能的一些现象。3．外界视觉经验异常或受到干扰时，影响了视皮质神经元信息呈递，下降的信息传递可能间接影响了高级中枢的信息整合。但发育期双眼剥夺的视皮层神经元突触可塑性并不比健康组降低反而提高。更多还原

【Abstract】 ObjectivesIntroduce the visual patch clamp whole-cell recording technique and investigatefunctional synaptic activities and spike-timing dependent plasticity of rats visualcortex during development stage.So as to account for aspects of experience-dependentvisual-cortical plasticity.To explore the role of visual experience when aging.And tounderstand the mechanism of amplyopia from functional aspects,through studyingthe changes of synaptic function status.Thus to find a reasonable and effectiveprevention methods and treatment of amblyopia.Methods1.By combining infrared differential interference contrast(IR-DIC)technique andCCD-Camera system with visual patch clamp whole-cell recording technique,spontaneous EPSCs of P2～7,P8～14,P15～21,P22～28 groups were oberved andanalyzed,simultaneously the neurons were stained by adding 0.3% luciferyellow tothe pipette solultion.Including the sEPSCs of P17 coronal slices of normal andabnormal visual experience group.2.Dual whole-cell voltage recording technique was used to stimulate in the cell atlow-frquency.To observe the long-lasting changed efficency of transmission betweentwo neurons in layer 2/3 of visual cortex under deferent stimulating protocols.At thesame time to observe the morphology of neurons and dendrites through addingluciferyellow to the pipette solution.3.Using dual whole-cell recording technique with STDP mode,control EPSP wasobtained from binocular deprived and control group respectively Then theeffectiveness of changes in synaptic transmission was compared.Observe the changeof test EPSP in the two groups after different pairing of individual postsynaptic APswith EPSPs precisely.And with the conventional LTP evoking protocol that onlyconsists of presynaptic stimulation,enhancing the frequency from lower to higher tilla theta-burst like stimulation,test EPSP was measured in sequence. Results1.Visual patch clamp technique can be used to make direct localization and healthscreening exactly.The amplitude and frequency of sEPSCs were all enhanced whenaging (One way ANOV,P＜0.01).But there was no significant difference betweenP2～7 group and P8～14 group(P＞0.05).The electrophysiology and dendrite of neuronbecame mature whilst development.The amplitude of sEPSCs in binocular deprivedgroup was significantly lower than that of the controled group(P＜0.05).Yet thediference of frequency between the two groups was not significant(P＞0.05)2.Depending on the precise control of timing of spike evoked in the pre/post neuron,after paring stimulating average test EPSP increased beyond 120%,LTP was evokedsucessfully.Yet when stimulation only applied to the pre-or postsynaptic neuron,testEPSP increased below 110%,LTP was not evoked sucessfully.3.The effectiveness of synaptic transmission decreased in binocular deprived group.The difference between binocular deprived and control groups was significant atP＜0.01 50 minutes after paring stimulation,it produced stable LTP in slices frombinocular deprived anmimals,but little potentiation in controls.Yet with theconventional LTP evoking protocol at lower frequency,no LTP was producedsuccessfully in 24 slices.Potentiation did not arrive at 110% even when the frequencywas near 100 Hz a theta-burst like stimulation.Conclusions1.Visual patch clamp technique,better than blind patch clamp technique,can improvethe success of sealing process.During the early postnatal days neurons wererelatively immature,and became mature during development.Visual experienceplayed an important role in this process.The results suggested that early AMPAreceptor-mediated functional activities existed in layer 2/3 pyramidal neurons,andsynapses were not completely silent,but had early spontaneous activities.2.The varing transmissional efficency of synapse was determined by paring pre-andpostsynaptic spike and the pre/post spike orders.There was an STDP timing windowin the layer 2/3.Spike-timing dependent plasticity may explain some experience-dependentmodifications in cortex.3.Abnormal visual experience during early postnatal days can decrease theeffectiveness of synaptic transmission,thus affecting the visual cortex neurons in presenting information.During developmental stages,visual-cortical plasticity inbinocular deprived group was not lower than the control group.更多还原