正常发育大鼠视皮层神经元特点及突触输入模式对其影响的研究
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摘要
研究目的
研究正常发育大鼠生后视皮层Ⅳ层神经元细胞电学生理特点及不同来源突触的输入及其输入模式的变化对神经元细胞反应的影响。
1.正常发育大鼠视皮层神经元脉冲潜时及膜电导成分动力学变化分析研究;
2.不同来源突触输入及其输入方式对正常发育大鼠视皮层神经元反应特性的影响；
3.年龄因素对大鼠视皮层神经元反应特性的影响。研究方法
1.采用电流钳全细胞记录模式,在电流钳下绘制I-V曲线进行视皮层神经元突触电导成分变化的分析；在电压钳全细胞记录模式下,利用不同的钳制电压用电学分离方式记录兴奋性及抑制性电流的发生,测量两种电流成分的潜时差异。
2.改变刺激模式,观察大鼠视皮层神经元细胞膜脉冲电导的变化。
3.改变白质两个刺激电极间的距离及两个刺激模式及时序,记录大鼠视皮层Ⅳ层的细胞反应变化情况；在电压钳模式下进行全细胞记录,利用计算变异系数的方法对被激发的突触事件进行间接描述。研究结果
1. EPSC的反转电位在-50.39±13.74mV,而IPSC的反转电位在0.47±10.33mV。利用两种电活动的反转电位的差异,将神经元细胞钳制在不同的电位水平,从而分离出EPSC与IPSC。两者分离后,利用潜时测量方法,可以分别测出EPSC与IPSC潜时,EPSC为5.10±0.52ms,IPSC的潜时为11.45±1.84ms。
2.利用突触定量分析公式,得出正常发育的神经元细胞膜电导存在两种成分,即兴奋性成分和抑制性成分。
3.分别利用单纯刺激和联合刺激的模式,在不同的电流水平下记录细胞膜电位的改变,从而计算出生理状态下大鼠视皮层神经元电导成分的改变。可以得出：单纯纯刺激后,兴奋性电导与抑制性电成分均较刺激前发生改变,利用配对t检验,发现这种差异具有统计学意义(P<0.01)。而联合刺激后,两种成分的改变亦具有统计学意义(P<0.01),而对于两种不同的刺激模式,联合刺激后抑制性成分的变化具有统计学意义(P<0.01),而兴奋性成分的变化差异无统计学意义(P>0.01)。
4.观察不同距离白质刺激后视皮层Ⅳ层神经元细胞反应的特点,可以发现在100-120μm差异具有统计学意义(P<0.01)。提示100-120μm可能是大鼠视皮层微功能柱的范围。
5.利用配对t检验,对刺激前后大鼠视皮层神经元反应的幅度大小进行统计学检验,差异具有统计学意义(P<0.01),提示不同的刺激模式对大鼠视皮层神经元细胞的影响不同。
6.根据CV=σ/M=[(1-p)/np]1/2将刺激前后不同组别CV进行标准化,同时将刺激前后反应幅度的变化标准化后得出对于单纯刺激而言,突触效能的改变多依赖于突触前机制,而对于联合刺激而言,突触效能的改变以突触后机制为主
7.不同年龄大鼠白质刺激引起相应视皮层Ⅳ层的细胞反应变化情况：P20大鼠视皮层对于刺激输入,不论单纯刺激或联合刺激,均有可能引发LTP,而P14大鼠视皮层,两种刺激模式诱发的均为LTD改变。
8.改变白质两个刺激模式时序,两个刺激时间差不同,可以得出：对于不同发育时期大鼠,刺激的时序的改变均导致在一特定的时间窗内产生非线性加和结果,即在某一特定时间窗内,联合刺激的模式所产生的神经元细胞反应大小不仅仅是每个刺激单纯刺激时引起的反应的加和。而当超出这个时间窗时,此加和现象不明显。
结论
1.大鼠视皮层Ⅳ层神经元细EPSC的反转电位在-50.39±13.74mV,而IPSC的反转电位在0.47±10.33mV。利用两种电活动的反转电位的差异,将神经元细胞钳制在不同的电位水平,分离出EPSC与IPSC。可以分别测出EPSC与IPSC潜时,EPSC为5.10±0.52ms,IPSC的潜时为11.45±1.84ms,两种电流成分的潜时不同。
2.正常发育的神经元细胞膜电导存在两种成分,即兴奋性成分和抑制性成分，且两种成分发生动态性变化以维持神经元细胞的正常反应。
3.刺激模式的变化对大鼠视皮层神经元细胞膜脉冲的兴奋性电导和抑制性电导均产生影响,且抑制性电导的变化差异具有统计学意义。
4.根据对应于视皮层不同距离白质刺激引起视皮层Ⅳ层的细胞反应变化情况可以得出在正常发育大鼠初级视皮层微小功能柱或类功能柱的存在。
5.对应于视皮层不同距离的两个不同刺激模式的白质刺激引起视皮层Ⅳ层突触效能的变化提示不同的刺激模式对大鼠视皮层神经元细胞的影响不同。且其发生机制可能存在差异：对于单纯刺激而言,突触效能的改变多依赖于突触前机制,而对于联合刺激而言,突触效能的改变以突触后机制为主。
6.不同年龄大鼠白质刺激引起相应视皮层Ⅳ层的细胞反应变化不同,P20大鼠视皮层对于刺激输入,不论单纯刺激或联合刺激,均有可能引发LTP,而P14大鼠视皮层,两种刺激模式诱发的均为LTD改变。
7.对于不同发育时期大鼠,刺激的时序的改变均导致在一特定的时间窗内产生非线性加和结果,即在某一特定时间窗内,联合刺激的模式所产生的神经元细胞反应大小不仅仅是每个刺激单纯刺激时引起的反应的加和。而当超出这个时间窗时,这样的加和现象不明显。对于P14大鼠,其时间窗约为±0.5ms,而对于P20大鼠,时间窗范围则缩短为±0.1ms.
Objective
To observe the electrophysiological characteristics of visual cortex layer IV in normal development rats and the impact of different synapse and the input mode on neuron reaction of visual cortex in rats. Research if or not the time windows of different synapse affect the different afferent and how to regulation.
1. To identify the electrophysiological characteristics and latency of visual cortex layer IV in normal development rats.
2. To search the impact of different synapse and the input mode on neuron reaction of visual cortex in rats.
3. To study the age impact on the neuron reaction.
Methods1. Analyze the synaptic conductance according to the mode with current patch clamp; and dissociated the EPSC and IPSC under different voltage condition, which is used to calculate the latency of the two different current.
2. Change the stimulation mode to observe the dynamics of synaptic conductance in visual cortex layer IV of rats.
3. Change the distance between two stimulation pipettes to record the synaptic response and calculate the CV of before and after stimulation to describe the synaptic events under the voltage patch clamp mode.
Result
1. The reversal potential of EPSC is at-50.39±13.74mV, but IPSC's is at0.47±10.33mV. The latency of EPSC is5.10±0.52ms, and the latency of IPSC is11.45±1.84ms.
2. There have two different composition of synaptic conductance:excitation and inhibition.
3. Different stimulation mode can result in distinct change, in which the combined stimulation with S1and S2may displays much more distinction; and the synaptic conductance has altered before and after different synaptic input.
4.100-120μm may be the mini-column in visual cortex of rats.
5. Different synaptic input may alter the synaptic response in visual cortex of rats.
6. The classical coefficient of variation method for "quantal" analysis of synaptic response allows unambiguous identification of the before and after stimulation synaptic plasticity mechanisms.
7. LTP is absent in layer IV of visual cortex in postnatal14rats, but can be induced in postnatal20rats, whatever the stimulation mode.
8. There has an nonlinear reconciliation between S1and S2during an'"time window" and the “time window”of P14is±0.5ms and P20is±0.1ms
Conclusions
1. The reversal potential and latency of EPSC and IPSC is different.
2. Dynamics of excitation and inhibition underlying different current injection in rat visual cortex layer IV.
3. The different synaptic inputs have impact on the synaptic conductance, respectively, including the excitation and inhibition conductance.
4. mini-column or alike-column may exist in rats visual cortex.
5. The synaptic mechanism may differ underlying different synaptic inputs, including pre-or/and postsynaptic mechanism.
6. Age may have an role in synaptic response:LTP is absent in layer IV of visual cortex in postnatal14rats, but can be induced in postnatal20rats, whatever the stimulation mode.
7. It displays an “time window” in P14and P20rats, and during this time, there has an nonlinear reconciliation between S1and S2.
研究正常发育大鼠生后视皮层Ⅳ层神经元细胞电学生理特点及不同来源突触的输入及其输入模式的变化对神经元细胞反应的影响。
1.正常发育大鼠视皮层神经元脉冲潜时及膜电导成分动力学变化分析研究;
2.不同来源突触输入及其输入方式对正常发育大鼠视皮层神经元反应特性的影响；
3.年龄因素对大鼠视皮层神经元反应特性的影响。研究方法
1.采用电流钳全细胞记录模式,在电流钳下绘制I-V曲线进行视皮层神经元突触电导成分变化的分析；在电压钳全细胞记录模式下,利用不同的钳制电压用电学分离方式记录兴奋性及抑制性电流的发生,测量两种电流成分的潜时差异。
2.改变刺激模式,观察大鼠视皮层神经元细胞膜脉冲电导的变化。
3.改变白质两个刺激电极间的距离及两个刺激模式及时序,记录大鼠视皮层Ⅳ层的细胞反应变化情况；在电压钳模式下进行全细胞记录,利用计算变异系数的方法对被激发的突触事件进行间接描述。研究结果
1. EPSC的反转电位在-50.39±13.74mV,而IPSC的反转电位在0.47±10.33mV。利用两种电活动的反转电位的差异,将神经元细胞钳制在不同的电位水平,从而分离出EPSC与IPSC。两者分离后,利用潜时测量方法,可以分别测出EPSC与IPSC潜时,EPSC为5.10±0.52ms,IPSC的潜时为11.45±1.84ms。
2.利用突触定量分析公式,得出正常发育的神经元细胞膜电导存在两种成分,即兴奋性成分和抑制性成分。
3.分别利用单纯刺激和联合刺激的模式,在不同的电流水平下记录细胞膜电位的改变,从而计算出生理状态下大鼠视皮层神经元电导成分的改变。可以得出：单纯纯刺激后,兴奋性电导与抑制性电成分均较刺激前发生改变,利用配对t检验,发现这种差异具有统计学意义(P<0.01)。而联合刺激后,两种成分的改变亦具有统计学意义(P<0.01),而对于两种不同的刺激模式,联合刺激后抑制性成分的变化具有统计学意义(P<0.01),而兴奋性成分的变化差异无统计学意义(P>0.01)。
4.观察不同距离白质刺激后视皮层Ⅳ层神经元细胞反应的特点,可以发现在100-120μm差异具有统计学意义(P<0.01)。提示100-120μm可能是大鼠视皮层微功能柱的范围。
5.利用配对t检验,对刺激前后大鼠视皮层神经元反应的幅度大小进行统计学检验,差异具有统计学意义(P<0.01),提示不同的刺激模式对大鼠视皮层神经元细胞的影响不同。
6.根据CV=σ/M=[(1-p)/np]1/2将刺激前后不同组别CV进行标准化,同时将刺激前后反应幅度的变化标准化后得出对于单纯刺激而言,突触效能的改变多依赖于突触前机制,而对于联合刺激而言,突触效能的改变以突触后机制为主
7.不同年龄大鼠白质刺激引起相应视皮层Ⅳ层的细胞反应变化情况：P20大鼠视皮层对于刺激输入,不论单纯刺激或联合刺激,均有可能引发LTP,而P14大鼠视皮层,两种刺激模式诱发的均为LTD改变。
8.改变白质两个刺激模式时序,两个刺激时间差不同,可以得出：对于不同发育时期大鼠,刺激的时序的改变均导致在一特定的时间窗内产生非线性加和结果,即在某一特定时间窗内,联合刺激的模式所产生的神经元细胞反应大小不仅仅是每个刺激单纯刺激时引起的反应的加和。而当超出这个时间窗时,此加和现象不明显。
结论
1.大鼠视皮层Ⅳ层神经元细EPSC的反转电位在-50.39±13.74mV,而IPSC的反转电位在0.47±10.33mV。利用两种电活动的反转电位的差异,将神经元细胞钳制在不同的电位水平,分离出EPSC与IPSC。可以分别测出EPSC与IPSC潜时,EPSC为5.10±0.52ms,IPSC的潜时为11.45±1.84ms,两种电流成分的潜时不同。
2.正常发育的神经元细胞膜电导存在两种成分,即兴奋性成分和抑制性成分，且两种成分发生动态性变化以维持神经元细胞的正常反应。
3.刺激模式的变化对大鼠视皮层神经元细胞膜脉冲的兴奋性电导和抑制性电导均产生影响,且抑制性电导的变化差异具有统计学意义。
4.根据对应于视皮层不同距离白质刺激引起视皮层Ⅳ层的细胞反应变化情况可以得出在正常发育大鼠初级视皮层微小功能柱或类功能柱的存在。
5.对应于视皮层不同距离的两个不同刺激模式的白质刺激引起视皮层Ⅳ层突触效能的变化提示不同的刺激模式对大鼠视皮层神经元细胞的影响不同。且其发生机制可能存在差异：对于单纯刺激而言,突触效能的改变多依赖于突触前机制,而对于联合刺激而言,突触效能的改变以突触后机制为主。
6.不同年龄大鼠白质刺激引起相应视皮层Ⅳ层的细胞反应变化不同,P20大鼠视皮层对于刺激输入,不论单纯刺激或联合刺激,均有可能引发LTP,而P14大鼠视皮层,两种刺激模式诱发的均为LTD改变。
7.对于不同发育时期大鼠,刺激的时序的改变均导致在一特定的时间窗内产生非线性加和结果,即在某一特定时间窗内,联合刺激的模式所产生的神经元细胞反应大小不仅仅是每个刺激单纯刺激时引起的反应的加和。而当超出这个时间窗时,这样的加和现象不明显。对于P14大鼠,其时间窗约为±0.5ms,而对于P20大鼠,时间窗范围则缩短为±0.1ms.
Objective
To observe the electrophysiological characteristics of visual cortex layer IV in normal development rats and the impact of different synapse and the input mode on neuron reaction of visual cortex in rats. Research if or not the time windows of different synapse affect the different afferent and how to regulation.
1. To identify the electrophysiological characteristics and latency of visual cortex layer IV in normal development rats.
2. To search the impact of different synapse and the input mode on neuron reaction of visual cortex in rats.
3. To study the age impact on the neuron reaction.
Methods1. Analyze the synaptic conductance according to the mode with current patch clamp; and dissociated the EPSC and IPSC under different voltage condition, which is used to calculate the latency of the two different current.
2. Change the stimulation mode to observe the dynamics of synaptic conductance in visual cortex layer IV of rats.
3. Change the distance between two stimulation pipettes to record the synaptic response and calculate the CV of before and after stimulation to describe the synaptic events under the voltage patch clamp mode.
Result
1. The reversal potential of EPSC is at-50.39±13.74mV, but IPSC's is at0.47±10.33mV. The latency of EPSC is5.10±0.52ms, and the latency of IPSC is11.45±1.84ms.
2. There have two different composition of synaptic conductance:excitation and inhibition.
3. Different stimulation mode can result in distinct change, in which the combined stimulation with S1and S2may displays much more distinction; and the synaptic conductance has altered before and after different synaptic input.
4.100-120μm may be the mini-column in visual cortex of rats.
5. Different synaptic input may alter the synaptic response in visual cortex of rats.
6. The classical coefficient of variation method for "quantal" analysis of synaptic response allows unambiguous identification of the before and after stimulation synaptic plasticity mechanisms.
7. LTP is absent in layer IV of visual cortex in postnatal14rats, but can be induced in postnatal20rats, whatever the stimulation mode.
8. There has an nonlinear reconciliation between S1and S2during an'"time window" and the “time window”of P14is±0.5ms and P20is±0.1ms
Conclusions
1. The reversal potential and latency of EPSC and IPSC is different.
2. Dynamics of excitation and inhibition underlying different current injection in rat visual cortex layer IV.
3. The different synaptic inputs have impact on the synaptic conductance, respectively, including the excitation and inhibition conductance.
4. mini-column or alike-column may exist in rats visual cortex.
5. The synaptic mechanism may differ underlying different synaptic inputs, including pre-or/and postsynaptic mechanism.
6. Age may have an role in synaptic response:LTP is absent in layer IV of visual cortex in postnatal14rats, but can be induced in postnatal20rats, whatever the stimulation mode.
7. It displays an “time window” in P14and P20rats, and during this time, there has an nonlinear reconciliation between S1and S2.
引文
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