【作者】 牟海燕；

【导师】 赵堪兴；

【作者基本信息】 天津医科大学 ， 眼科学， 2010， 博士

【摘要】 目的探讨发育期大鼠高级视皮层活动对初级视皮层Ⅱ/Ⅲ层锥体神经元突触传递效能的影响及相关机制。方法1.通过对睁眼前后关键期内的大鼠的离体视皮层脑片白质和LM区细胞外刺激,V1区Ⅱ/Ⅲ层锥体神经元全细胞膜片钳记录,分别钳制在-70mv和-50mv,刺激间隔时间ISIs=0,±0.1,±0.2,±0.3,±0.4,±0.5,±1,±2ms,观察随着白质和LM区刺激的时间隔及钳制电位的不同,V1区Ⅱ/Ⅲ层锥体神经元兴奋性突触后电流(EPSCs)的变化,分析计算加和指数(SI)。2.睁眼初期(P14-16)大鼠离体视皮层脑片在单纯白质刺激、白质联合LM区间隔2ms刺激、白质联合LM区间隔0.1ms刺激,V1区Ⅱ/Ⅲ层锥体神经元EPSCs的变化,比较LTP/LTD诱发率异同。3.采用脑片膜片钳全细胞记录技术观察视皮层Ⅱ/Ⅲ层锥体神经元对Ⅳ层双极电极刺激的反应。将全细胞膜电位钳制在0mv从而电学分离抑制性突触后电流(IPSCs),比较不同天龄组双脉冲刺激(刺激间隔40ms)诱导的IPSCs短时程突触可塑性的变化。将全细胞膜电位钳制在Cl-平衡电位以上不同水平获得IPSCs电流-电压曲线,并计算得到抑制性电导(gi),观察gi的发育性变化及其与IPSCs双脉冲易化间的关系。通过计算各天龄组双脉冲刺激诱发IPSCs的变异系数(CV)及不同钳制电位水平IPSCs的双脉冲系数(PPR)结合各天龄阶段双脉冲刺激诱导的△gi的变化,研究生后早期大鼠视皮层短时程突触可塑性的可能机制。结果1.在静息电位水平,白质(WM)和LM区同时刺激,睁眼前、睁眼早期、睁眼后期SI分别为4.65±1.28；2.90±0.44;2.54±0.33。钳制在Cl-反转电位(-50mv),SI分别为2.82±0.63；1.99±0.42；1.34±0.30。SI随着天龄增大而降低,随着钳制电压而降低。2.单纯白质刺激、白质联合LM区间隔2ms刺激、白质联合LM区间隔0.1ms刺激,V1区Ⅱ/Ⅲ层锥体神经元EPSCs幅值改变分别为29.59±26.28;22.93±17.05；67.28±44.94。1组和3组,2组和3组有统计学差异(P<0.05)；1组和2组无统计学差异(P>0.05)。LTP/LTD诱发率各组相比,1组(55.0%)和3组(88.2.0%),2组(50.0%)和3组有统计学差异(P<0.05)；1组和2组无统计学差异(P>0.05)。3.(1)在所有3组中IPSCs对双脉冲刺激均表现出双脉冲易化(PPF)现象,Omv水平各组PPR值分别为2.41±0.72(A组)、1.62±0.83(B组)、0.19±0.79(C组)。(2)gi的水平随着天龄的增加而不断升高,各组分别为：1.48±0.78nS(A组)、2.05±0.84nS(B组)、5.67±2.22nS(C组)。(3)双脉冲刺激使各天龄组IPSCs的CV值均出现降低,各组CV降低指数分别为：-2.09±0.46(A组)、-1.90±0.58(B组)、-0.29±0.18(C组)。各组IPSCs的双脉冲系数(PPR)均表现出电压依赖性,即随着钳制电位的正向升高而降低,双脉冲刺激诱导的△gi分别为-0.31±0.21nS(A组)、1.13±0.53nS(B组)、1.43±0.38nS(C组)。结论1.睁眼前后关键期内的大鼠的离体视皮层脑片白质和LM区细胞外刺激,V1区Ⅱ/Ⅲ层锥体神经全细胞膜片钳记录当ISIs=0,±0.1ms时,加和指数(SI)表现为明显的非线性加和,随着天龄的增大而降低。随着刺激间隔时间(ISIs)的延长,SI在1上下波动,基本表现为线性加和。整个曲线呈现典型的“钟形”。2.白质联合LM区刺激在小于0.1ms时可提高视皮质Ⅱ/Ⅲ层锥体神经元LTP/LTD的诱发率提示高级视皮层对初级视皮层的可塑性可产生影响。3.大鼠出生早期视皮层Ⅱ/Ⅲ层锥体神经元抑制性突触联系随着发育不断成熟并得到加强,同时其短时程可塑性能力逐渐降低。生后早期尤其是睁眼后最初几天抑制性突触联系的大量建立促发了经验依赖的视皮层可塑性关键期的开始。抑制性突触后电流PPF的电压依赖性提示视皮层抑制性回路的成熟存在突触后机制,而PPF的突触前机制在睁眼及睁眼后最初几天表现更为突出。睁眼前PPR值与△gi的分离提示突触后GABA受体可能存在较睁眼后更为显着地双脉冲诱导的失敏现象。IPSCs双脉冲易化机制可能参与了大鼠生后早期经验依赖的视皮层发育过程。4.睁眼初期大鼠初级视皮层Ⅱ/Ⅲ层锥体神经元的IPSCs的短时程易化可能是LTD诱发率高的原因。更多还原

【Abstract】 ObjectiveTo explore the influences of higher cortical areas activity to primary visual cortex on synaptic plasticity in developing rats.Methods1. To examine the spatiotemporal interactions between the WM inputs and LM inputs under clamp voltages at-70mv and-50mv respectively, the responses to concurrent activation at various interstimulus intervals (ISIs) of any two inputs were recorded. The ISIs used were0,±0.1,±0.2,±0.3,±0.4,±0.5,±1,±2ms and ISIs were analysised.2. Compare the LTP/LTD induction rates between stimulate wm alone,wm and LM with ISIs=±2ms, wm and LM with ISIs=0,±0.1ms in cortex brain slices of P14-16rats.3. A short-term (inter-stimuli interval of40ms) paired-pulse stimuli was delivered to the layer IV of slices to evoke postsynaptic currents of pyramidal neurons of layer Ⅱ/Ⅲ. Inhibitory postsynaptic currents (IPSCs) were recorded under different clamp voltages though whole-cell patch clamp technique. Inhibitory input conductance (gi) calculated from their current-voltage fitting curves were used for analysis of their developmental changes and effect of paired-pulse stimuli. The changes of coefficient of variance (CV) between the postsynaptic currents induced by paired-pulse stimuli in different groups were compared.Results1. At the resting membrane potential, when two inputs were driven simultaneously, the SI of the three groups were4.65±1.28;2.90±0.44;2.54±0.33. At the-50mv membrane potential,the SI were2.82±0.63;1.99±0.42;1.34±0.30respectively.The SI decreased with the growth.2. The changes of amplitude of EPSCs were29.59±26.28;22.93±17.05;67.28±44.94respectively when stimulate wm alone,wm and LM with ISIs=±2ms, win and LM with ISIs=0,±0.1ms in cortex brain slices of P14-16rats.The average amplitude of the wm and LM with ISIs=0,±0.1ms was significantly larger than that of the other groups(p<0.05),and the LTP/LTD induction rates was significantly larger than that of the other groups(p<0.05).3. The IPSCs showed that PPF decreased with ages2.41±0.72(A group),1.62±0.83(B group),0.19±0.79(C group); while the gi level increased ages1.48±0.78nS(A group),2.05±0.84nS(B group),5.67±2.22nS(C group).The CV value was reduced significantly with ages-2.09±0.46(A group),-1.90±0.58(B group),-0.29±0.18(C group).Conclusions1.When WM inputs and LM input were driven simultaneously on the rat slices around critical period, the evoked EPSCs were larger than the mathematical summation of the two individual EPSCs. The SI decreased with the growth and the positive changes of clamp voltage.2.Higher visual areas activity can affect Primary visual cortex on synaptic plasticity in developing rats,it can improve the LTP/LTD induction rates.3.In the postnatal early period of rat, the inhibitory synaptic circuitry was strengthened and matured with the visual cortex development, but its short-term plasticity decreased with the time. In the first several days after open eyes, the presynaptic mechanism of IPSCs-PPF is in the dominant status. The short-term plasticity reflected by PPF may participates in the experience dependent visual cortical development of rats.4. LTD induction maybe connected with the PPF of IPSCs during the postnatal early period.更多还原