【作者】 陈珂；

【导师】 戴躍；

【作者基本信息】 华东师范大学 ， 运动人体科学， 2017， 硕士

【摘要】 研究表明,耐力训练可使得大鼠脊髓α-运动神经元静息膜电位超极化和产生动作电位的电压阈值降低,而运动剥夺后大鼠α-脊髓运动神经元则表现出动作电位的电流阈值和电压阈值同时增加。这一研究结果提示我们运动干预可对脊髓神经系统电生理特性产生影响:增加运动量可使得脊髓运动神经元兴奋性提高,剥夺运动则降低脊髓运动神经元的兴奋性。我们知道,脊椎动物的肢体运动是通过脊髓网络对运动神经元的控制来完成的。在运动训练过程中,除运动神经元以外还有大量的脊髓中间神经元参与运动协调与控制,然而运动干预是否对这些中间神经元的兴奋性也产生影响我们却不得而知。此外,现有的运动训练对脊髓神经元兴奋性影响的研究均选择在动物成年后进行运动干预,未见在动物成年之前的青少年时期进行运动训练的研究。鉴此我们选择在小鼠3周龄时对其进行3周的跑台训练,在小鼠6周龄时通过全细胞膜片钳技术对T12-L6脊髓切片中的中间神经元的电生理指标进行测量。观测6周龄时其脊髓中间神经元的电生理指标的变化。我们的研究表明,运动训练可提高脊髓中间神经元的兴奋性。目的:本研究以青少年小鼠为研究对象,以跑台训练作为运动干预的手段,使用离体脊髓切片的方式对脊髓神经元进行全细胞膜片钳测量,对比运动组和对照组脊髓神经元电生理指标的差异,评价运动干预对青少年小鼠脊髓神经元的调节作用。方法:实验动物:本实验使用B6品系小鼠,购自美国Jackson Lab实验室。实验过程严格按照华东师范大学动物中心的伦理要求进行,伦理编号:20141003。使用B6野生型小鼠繁殖交配,小鼠出生后3周随机分为运动组和对照组。运动组小鼠进行为期3周的跑台训练,对照组不运动。两组小鼠均在其6周龄时进行电生理实验。(1)运动干预方案:运动前对小鼠进行适应性训练,使其适应动物跑台。首次训练,跑台速度为13米/分钟,时间30分钟。适应性训练为期3天,之后开始20米/分钟(运动强度相当于85%VO2max),60min/次,1次/天,6天/周的正式训练,持续训练3周。(2)电生理实验记录两组小鼠脊髓神经元基础膜特性:本实验所测量反应细胞模特性的指标包括电流阈值(current threshold:Ith)、电压阈值(voltage threshold:Vth)、静息膜电位(resting membrane potential:Em)、输入电阻(input resistance:Rin)、细胞膜时间常数(membrane time constant:Tm)、全细胞电容(whole cell capacitance:Cm)、动作电位(action potential)高度及宽度、后超极化(after hyperpolarization:AHP)深度和半衰时间。结果:6周龄小鼠脊髓神经元基础膜特性描述1.电生理指标1.1对照组小鼠脊髓神经元基础膜特性(n=27):Em(-64.2±5.7mV)、Ith(11.9±6.9pA)、Rin(1483.3±682.3MΩ)、Tm(41.0±16.6ms)、Cm(27.6±24.3pF)、Vth(-36.9±5.1mV)、APheight(49.9±10.9mV)、AP width(1.3±0.4ms)、AHP depth(21.2±4.5mV)、AHP1/2 decay time(122.0±95.1ms);1.2训练组小鼠脊髓神经元基础膜特性(n=47):Em(-62.2±4.9mV)、Ith(11.7±9.6pA)、Rm(1608.2±586.4MΩ)、Tm(41.1 ±23.3ms)、Cm(28.1 ±24.9pF)、Vth(-40.4±3.2mV)、AP height(52.7±11.9mV)、AP width(1.8±0.6ms)、AHP depth(18.7±5.2mV)、AHP1/2 decay time(117.4±83.0ms)。2.板层分布特性2.1对照组小鼠脊髓背侧角(dorsalhorn,1-6板层)神经元(n=10)和脊髓腹侧(ventrol horn,7/8/10板层)神经元(n=17)仅在电流阈值这一指标间存在差异:脊髓背侧角神经元电流阈值(16.3±4.8pA)显著高于脊髓腹侧中间神经元(9.1±6.4pA),(P<0.05)。2.2训练组小鼠脊髓背侧角神经元(n=20)和脊髓腹侧中间神经元(n=27)各项电生理指标均无显著差异。3.放电模式按照脊髓神经元在受到去极化电流时的放电情况,可将其划分为:单峰放电、相位放电和持续性放电三种类型,这三种类型的神经元在对照组和训练组小鼠中均有发现。3.1对照组单峰放电型神经元后超极化电位半衰时间(25.9± 14.5ms)明显低于相位放电型神经元(159.3±22.1ms)和持续放电型神经元(180.1±58.0ms)(P<0.05),电压阈值(-31.0±5.7mV)显著高于相位放电型神经元(-39.4±7.4mV)和持续放电型(-39.3±5.4mV)(P<0.05);3.2训练组小鼠持续放电型神经元电压阈值(-40.3±3.3mV,n=21)极显著低于相位放电型(-35.4±4.5mV,n=18)(P<0.01)。特殊放电现象1.六周龄小鼠脊髓神经元具有超极化电流激活的膜电位凹,这一现象在对照组(57%)和训练组(47.6%)小鼠脊髓神经元中出现的概率近似。2.本文在电生理实验过程中发现训练组小鼠和对照组小鼠脊髓神经元在接受去极化步进电流刺激时,均发现呈阵发式放电模式(burst)的神经元。其中训练组呈现阵发式放电的两个神经元分别位于脊髓背侧角第三和第五板层,对照组中一个呈现阵发式放电的神经元分布于第七板层。运动干预对脊髓神经元兴奋性的调节作用1.三周跑台训练后,训练组小鼠腹侧中间神经元与对照组在动作电位的阈电压、动作电位宽度和后超极化深度等指标间存在差异:训练组小鼠脊髓腹侧中间神经元动作电位的阈电压(-40.2±3.2mV,n=27)与对照组相(-34.9±4.4mV,n=17)相比更加超极化(P<0.05);训练组小鼠脊髓腹侧中间神经元动作电位宽度(2.1±0.6ms,n=27)与对照组(1.3±0.4ms,n=17)相比显著增加(P<0.01);同时与对照组(24.1±3.9mV)相比,训练组小鼠脊髓神经元后超极化深度(16.6±4.3mV)极显著减小(P<0.01)。2.对照组小鼠脊髓背侧(1-6板层)神经元(n=10)各项电生理指标与训练组脊髓背侧(1-6板层)神经元(n=20)相比均无显著性差异。3.三周跑台训练结束后,训练组小鼠所有的持续性放电神经元(n=22)平均内向电阻(1834.0±580.6MΩ)与对照组(n=13)(1332.1±419.6MΩ)相比显著增加(P<0.05);训练组小鼠所有的相位放电型神经元(n=17)平均动作电位宽度(1.9±0.4ms)与对照组(n=9)(1.5±0.5ms)相比显著增大(P<0.05)。结论:1.运动干预对生长发育敏感期的小鼠脊髓中间神经元的兴奋性具有明显的调节作用,但这种作用与神经元在脊髓板层中的分布有关;2.运动训练可显著降低脊髓腹侧(7/8/10板层)中间神经元产生动作电位的电压阈值,降低动作电位后超级电位,以及增加动作电位的宽度,这些结果表明:运动训练可提高腹侧中间神经元的兴奋性;3.运动训练对脊髓背角(1-6板层)中间神经元各项电生理指标均未产生显著差异,说明运动干预对传递感觉信号的中间神经元的兴奋性没有显著影响。相比之下,与运动相关的腹侧中间神经元对运动训练的反应更为敏感;4.运动训练对持续放电型神经元和相位放电型神经元兴奋性均有明显提高,表明这两类神经元比单峰放电型神经元对运动干预的调节作用更为敏感。更多还原

【Abstract】 It was shown in previous studies that endurance training could hyperpolarized the resting membrane potential and lowered the voltage threshold for action potential generation in-motoneurons of rat spinal cord.In contrary,however,increase in both current and voltage thresholds for action potential generation was reported in limb-unweighting rats.These results suggest that electrophysiological properties of spinal motoneurons can be changed by exercise intervention.While endurance training increases the excitability of spinal motoneurons,the limb-unweighting reduces their excitability.It is well known that locomotion in vertebrates is produced through the control of motoneurons by the networks of spinal cord.In addition to the motoneurons,spinal interneurons are required to participate in generating and coordinating locomotion.However,it remains unknown if exercise intervention could modulate the intrinsic membrane properties of the spinal interneurons.Furthermore,previous studies of the exercise intervention were all carried on in adult rats and a few of adolescent mice were used as the experimental models.For the above reasons we used adolescent mice of three weeks in this study and induced three-week treadmill exercise in these mice.And then the electrophysiological properties of the spinal interneurons were measured from the spinal cord slices of T12-L6 with whole-cell patch clamp technique.Results from this study show that the excitability of spinal interneurons can be increased by the exercise training.Objective:In the current study our purpose was to determine if the adolescent mouse allowed access to treadmill training for 3 weeks would possess interneurons with different electrophysiological properties than their control counterparts.And we use the whole cell patch recording in the slice of the spinal cord to test the electrophysiological properties of the interneurons.Method:Experiments were conducted on the B6 mouse obtained from Jackson Lab,The experiment was carried out in strict accordance with the ethical requirements of the animal center of East China Normal University,Ethical code:20141003.The 3 weeks postnatal animals were assigned to either a control group or a treadmill-training group randomly.The training is last for 3 weeks,we test the electrophysiological properties of the interneurons from both the two groups when they were 6 weeks old.(1)Treadmill training:Following the adjustment period,exercise animals were acclimated to treadmill running(13m/min,30min).The acute exercise training protocol:20m/min(85%VO2),lh/day,6 days/week on a motor-driven treadmill for 3weeks.(2)Electrophysiology:Membrane properties observed in the current study include rest membrane potential(Em),input resistance(Rin),voltage threshold(Vth),rheobase,membrane time constant(Tm),whole cell capacitance(Cm),amplitude and width of the action potential,amplitude and half decay time after hyperpolarization(AHP)in the whole cell patch recording.Results:Membrane properties of the 6 weeks old mouse.1.Electrophysiology:1.1 Control group(n=27):Em(-64.2±5.7 mV)、Ith(11.9±6.9pA)、Rin(1483.3±682.3MΩ2)、Tm(41.0±16.6ms)、Cm(27.6±24.3pF)、Vth(-36.9±5.1mV)、AP height(49.9±10.9mV)、AP width(1.3±0.4ms)、AHP depth(21.2±4.5mV)、AHP1/2 decay time(122.0±95.1ms);1.2 Training group(n=47):Em(-62.2±4.9mV)、Ith(11.7±9.6pA)、Rin(1608.2±586.4 MΩ)、Tm(41.1±23.3ms)、Cm(28.1 ±24.9pF)、Vth(-40.4±3.2mV)、AP height(52.7±11.9mV)、AP width(1.8±0.6ms)、AHP depth(18.7±5.2mV)、AHP1/2 decay time(117.4±83.0ms).2.Membrane properties in the different lamina.2.1 In the control group,significant differences were found between the dorsal(lamina Ⅰ-Ⅵ)and ventral horn(lamina Ⅶ,Ⅷ and Ⅹ)interneurons in the rheobase(16.3 ±4.8pA and 9.1 ±6.4 pA for the dorsal and ventral respectively).2.2 In the training group,there was no significant difference between the neurons distributed in the dorsal and ventral horn.3.Three types of interneurons.All the cells recorded in both groups in this study were classified into three types basing on their response to the injected depolarizing current:type I(single-spike firing),type II(phasic firing)and type III(tonic firing).3.1 For the control group,type Ⅰ has significant smaller AHP half decay time and depolarized voltage threshold.3.2 For the training group,type Ⅲ has significant hyperpolarized voltage threshold.Membrane currents and special firing pattern.1.Hyperpolarization-activated inward current(Ih)was measured in this study.Ih was observed in 57%of the total neurons in the control group and 47.6%in the training group.2.In current study,we found 3 burst neurons.Two of them were from training group and they were distributed in lamia Ⅲ and lamina Ⅴ,and the rest one was from the control and was distributed in the lamia VII.Effects of activity.The treadmill training did change the properties of the spinal cord interneurons significantly,and the treatment effect depended on the interneuron type and distribution.1.The ventral horn interneuron changed their properties after 3 week treadmill training.The interneuron from the training group had significant hyperpolarized voltage threshold(-40.2±3.2 mV and-34.9±4.4 mV for training and control group respectively),increased AP width(2.1 ±0.6ms and 1.3±0.4ms for training and control group respectively)and reduced the AHP depth(16.6±4.3mV and 24.1±3.9mV for training and control group respectively)compare with control group.2.After 3 weeks treadmill training type Ⅲ neuron had significant increased input resistance(1834.0±580.6MΩ and 1332.1±419.6MQ for training and control group respectively);and type Ⅱ neuron has significant larger AP width(1.9±0.4ms and 1.5± 0.5ms for training and control group respectively).Conclusion:1.The treadmill training do change the properties of the spinal cord interneuron significantly,and the treatment effect depends on the interneuron type and distribution.2.After exercise training the ventral horn interneuron has significant hyperpolarized voltage threshold,larger AP width and smaller AHP depth;These results suggest that the exercise training can improve the excitability of the ventral horn interneurons.3.No significant difference was found in the sensory interneurons located in the dorsal horn between the control and the training group;This result indicates that the interneurons distributed in the ventral horn are more responsible to the exercise intervention than those in dorsal horn.4.Exercise training echanced the excitability of the type Ⅱ and type Ⅲ neuron,and suggesting that these two types are more responsible to the exercise intervention than type Ⅰ.更多还原