Neuroligin-1在癫痫发作中的作用及机制研究
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摘要
第一部分Neuroligin-1和neurexin-1β在难治性颞叶癫痫患者及动物模型中的表达
目的：脑神经元异常突触传递的同步化能导致癫痫的产生。Neuroligin-1(NL1)是位于兴奋性突触后膜的一种突触粘附分子，它能调节突触传递并能决定中枢神经网络的性质。本研究首先检测了NL1及其突触前膜受体neurexin-1β(NRX1β)在难治性颞叶癫痫（TLE）患者及氯化锂-匹罗卡品（LiCl-PILO）癫痫大鼠模型中的表达，以探讨其在TLE发生及发展中的可能作用。
方法：
1.从课题组所建脑组织标本库中随机抽取22例难治性TLE患者的颞叶皮质和10例对照组颞叶皮质。
2.80只成年雄性SD大鼠随机分为对照组（n=10）和癫痫组（n=70），癫痫组又分为7个亚组（每组n=10），即1d组、2d组、3d组、7d组、14d组、30d组和60d组。癫痫组给予LiCl-PILO进行造模。
3.用免疫组化，免疫荧光和免疫印迹三种方法分别检测NL1和NRX1β在颞叶癫痫患者脑组织和癫痫大鼠脑组织中的表达。
结果：
1.免疫染色显示，NL1蛋白主要在难治性TLE患者及对照颞叶皮质神经元的细胞膜和细胞中表达。NL1在难治性TLE患者脑组织中的免疫组化平均光密度值显著高于对照组(p<0.05)。荧光双标染色结果显示NL1主要在颞叶皮质神经元，且和NMDAR1共表达。免疫印迹结果显示NL1及其受体NRX1β在难治性TLE组中为强阳性，而在对照组中则为弱阳性。难治性TLE患者颞叶皮质的NL1和NRX1β的表达水平显著高于对照组(p<0.05)。
2.在LiCl-PILO癫痫大鼠皮质和海马各区，均可见NL1免疫组化阳性染色，其中在海马齿状回、CA1区、CA3区阳性染色最强。在海马组织中，NL1的免疫组化平均OD值在造模后各个时间点呈动态增高趋势。荧光双标染色结果显示NL1在颞叶皮质和海马神经元表达，并且和NMDAR1共表达。免疫印迹结果显示，与对照组相比，NL1和NRX1β在急性期和慢性期均升高，且表达趋势基本一致。与对照组相比，NL1在海马的表达除了在7d和14d组没有差异（p>0.05）外，在其它各组均有差异（p<0.05）。与对照组相比，NRX1β在海马的表达除了在7d组没有差异（p>0.05）外，在其余各组均有差异（p<0.05）。
结论：NL1及其受体NRX1β在难治性TLE患者和LiCl-PILO癫痫大鼠颞叶组织中表达均增高，提示其可能参与了TLE的发生与形成过程。
第二部分Neuroligin1的knockdown对大鼠癫痫发作的影响
目的：为进一步探讨NL1的knockdown是否会对癫痫发作产生影响，我们用沉默NL1基因的短发卡RNA（shRNA）慢病毒减少大鼠海马内源性NL1表达，并在造模后观察由此产生的对癫痫大鼠行为学的影响。
方法：
1.成年雄性SD大鼠造模前分为三组进行海马立体定位注射：即海马立体定位注射生理盐水假手术组（EP），海马立体定位注射慢病毒空载体组(EP+sh-con.)，以及海马立体定位注射NL1shRNA慢病毒组(EP+sh-NL1)。
2.成年雄性SD大鼠海马立体定位注射NL1shRNA慢病毒，并用免疫印迹实验和免疫荧光实验检测其干扰效率.
3. LiCl-PILO造模之后60min之内对大鼠进行行为学观察，分别记录每20min时间间隔内的Racine评分，60min之内最大Racine评分，各组在60min之内达到Ⅳ级以上发作的大鼠所占比例，以及大鼠从PILO注射到首次出现Ⅳ级以上发作的时间（癫痫发作潜伏期）。
结果：
1. NL1shRNA慢病毒海马立体定位注射后，内源性NL1表达逐渐下降。与相应时间点对照组相比，NL1的平均OD比值在慢病毒注射后3d组和7d组中显著下降（p<0.05）。3d组和7d组之间没有显著差异（p>0.05）。在慢病毒注射后的第3d和7d，NL1的总量分别降低了36.3±4.3%和44.2±5.3%。荧光结果显示EGFP阳性表达位于海马，尤其是CA1区。
2.在PILO注射后，大鼠癫痫发作的平均Racine评分随着时间的延长逐渐增高。在各20min间隔内，EP+sh-NL1组的癫痫发作评分均比其它组低（p<0.05），组间有差异（p<0.05）而时间与组间交互作用无差异（p>0.05）。EP+sh-NL1组的最大Racine评分比其它组低（p<0.05）,而另外两组对照无差异（p>0.05）。在EP+sh-NL1组中只有33.3%的大鼠出现了Ⅳ级以上的发作，而在其它两组对照组均有83.3%的大鼠出现了Ⅳ级以上的发作。NL1的knockdown能显著降低Ⅳ级以上癫痫的发生率（p<0.05）。与两组对照组相比，EP+sh-NL1组癫痫发作潜伏期显著延长，而两组对照之间无显著差异（p>0.05）。
结论：
1.对NL1进行的体内shRNA慢病毒干扰能有效抑制内源性NL1的表达，其干扰效率在干扰后第7d达到稳定水平。
2.海马内NL1shRNA慢病毒干扰能减轻癫痫发作程度，降低癫痫发作的易感性。
第三部分Neuroligin1在大鼠癫痫发作中的作用机制
目的：为了研究NL1在自发性癫痫发作（SRS）中的可能作用机制，我们在癫痫大鼠造模后第30d检测NL1shRNA慢病毒干扰效率并进行电生理研究，同时检测与NL1关系密切的NMDAR1在NL1knockdown之后的表达变化。
方法：
1.癫痫大鼠按海马内立体定位注射试剂不同分为三组：生理盐水组（EP）、空载体组(EP+sh-con.)、NL1shRNA组(EP+sh-NL1)；正常大鼠海马内注射生理盐水作为正常对照组（control）。
2.用免疫印迹和免疫荧光实验检测造模后30d大鼠海马NL1shRNA慢病毒干扰效率。
3.用全细胞膜片钳技术记录造模后30d各组大鼠脑片海马CA1区细胞的动作电位（AP）、微小兴奋性突触后电流（mEPSC）、诱发兴奋性突触后电流（evoked EPSC）。
4.用免疫印迹实验检测造模后30d大鼠海马NMDAR1的总蛋白和膜蛋白。
结果：
1.免疫印迹实验结果表明，大鼠造模后30d，NL1在海马的表达在EP+sh-NL1组与EP+sh-con.组或EP组之间有显著差异（p<0.05）,而在EP+sh-con.组与EP组之间以及EP+sh-NL1组与control组之间没有显著差异（p>0.05）。 EGFP免疫荧光结果提示慢病毒已成功转染至海马CA1区神经元。
2.自发性AP的频率在EP+sh-NL1组显著低于EP+sh-con.组（p<0.05）；与control组相比，AP频率在EP+sh-con.组以及EP+sh-NL1组均显著增高（p<0.05）。mEPSC的幅度在EP+sh-con.组与EP+sh-NL1组之间无差别（p>0.05）；与control组相比，mEPSC的幅度在EP+sh-con.组以及EP+sh-NL1组均显著增高（p<0.05）。mEPSC的频率在EP+sh-NL1组比EP+sh-con.组.显著降低（p<0.05）；与control组相比，mEPSC的频率在EP+sh-con.组以及EP+sh-NL1组均显著增高（p<0.05）；mEPSC的频率在control组和EP+sh-NL1组有显著差异（p>0.05）。
3.与control组和EP+sh-con.组相比，EP+sh-NL1组的NMDAR/AMPAR比值显著降低（p<0.05）。与EP+sh-con.组相比，EP+sh-NL1组的NMDAR依赖性EPSCs幅度的显著降低（p<0.05）而AMPAR依赖性EPSCs幅度没有改变（p>0.05）。
4.与control组比，EP组和EP+sh-con.组的总蛋白、表面蛋白/总蛋白比值均显著降低（p<0.05）。而EP+sh-NL1组的总蛋白、表面蛋白/总蛋白比值明显低于EP组和EP+sh-con.组（p<0.05）。
结论：
1.对NL1进行的体内shRNA慢病毒干扰在30d后仍然能有效抑制内源性NL1的表达。
2.癫痫大鼠海马NL1的knockdown能抑制海马CA1区神经元的过度兴奋。
3.癫痫大鼠海马NL1的knockdown能通过突触后NMDAR1的作用选择性抑制海马脑片NMDAR依赖性突触电流。
Part one: The expression of neuroligin-1and neurexin-1β in temporallobe epileptic foci in patients and experimental animals
Objective: Abnormally synchronized synaptic transmission in the brainleads to epilepsy. Neuroligin-1(NL1) is an synaptic cell adhesion moleculelocated at excitatory synapse, which modulates synaptic transmission anddetermines the properties of neuronal networks in the mammalian centralnervous system. Here we investigated the expression of NL1and its bindingpartner neurexin-1β (NRX1β) in temporal lobe epileptic foci in patients andexperimental animals in order to explore the probable relationship betweentheir expression and temporal lobe epilepsy (TLE).
Method:
1. The NL1and NRX1β expression were assessed in twenty two humanbrain tissues derived from patients undergoing operation for intractable TLEand was also detected in ten temporal lobes from controls.
2. Adult male Sprague–Dawley (SD) rats were used in this study,including lithium–pilocarpine-treated rats (n=70) on days1,2,3,7,14,30,and60post-seizure, and control rats (n=10).
3. Expression of NL1and NRX1β were assessed byimmunohistochemistry, immunofluorescence, and Western blot analysis.
Result:
1. NL1protein was mainly expressed in the membrane and cytoplasmof neurons in the temporal neocortex from both the control and intractableTLE groups by immumohistochemical staining. the mean optical density(OD) value of NL1was significantly higher in the temporal neocortex of theTLE group compared with the control group (p<0.05) Double-labelimmunofluorescent staining showed that NL1and GFAP were seldomcoexpressed in astrocytes, but NL1-positive cells coexpressed withNMDAR1. Western blot analysis showed the expression of NL1and NRX1βwas strong in the subjects with intractable TLE, whereas it was relativelyweak in the control subjects.
2. The NL1immumohistochemical staining was extensively observedin neocortex and all regions of the hippocampus, and the intense staining inhippocampus was in the dentate gyrus, CA1and CA3regions. Comparedwith the controls, the mean OD values of NL1expression were elevateddynamically. Double-labeling immunofluoresence showed NL1-positivecells did not co-express with GFAP in astrocytes but co-expressed withNMDAR1in neurons. Western blot analysis showed compared with thecontrols, both NL1and NRX1β expression elevated in the acute period andthe chronic period, and their expression profile presented nearly the same. The difference of the mean OD ratio in the NL1expression level inhippocampus between the control and every treated group was significant(p<0.05), except the7-d and14-d groups (p>0.05). There was a significantdifference of the mean OD ratio in the NRX1β expression between thecontrol and every treated group (p<0.05), except the7-d group (p>0.05).
Conclusion: The expression of NL1and its binding partner NRX1βwere increased in temporal lobe epileptic foci in patients andlithium–pilocarpine-treated epileptic rats. Our results suggest that NL1andNRX1β may play an important role in the development of TLE.Part two: The effect of knockdown of neuroligin1on epileptic seizures
Objective: To further study whether supression of NL1could preventseizures, we investigated the behavioral changes of epileptic rats performedby lentivirally mediated knockdown of NL1in the hippocampus.
Method:
1. The following three groups with hippocampal injection wereincluded, namely sham operation group with hippocampal injection of saline(EP), group with hippocampal injection of vehicle virus (EP+sh-con.), andgroup with hippocampal injection of NL1shRNA (EP+sh-NL1).
2. Western blot analysis of NL1expression in hippocampus was used toverify that in vivo RNA interference for NL1was effective and selective. Onthe other hand, EGFP antibody was used to identify and amplify the virus-infected neurons in the hippocampus by immunofluoresence.
3. We perform rats behavioral investigation7days after virus infusion.We observed the behavioral changes of the rats within60-min afterpilocarpine injection. Scoring in rats for each20-min interval, the maximumRacine score during the60-min trial,the percentage of the rats reached above5score, and the seizure latency in the three groups were recorded.
Result:
1. Western blot analysis showed that the mean OD ratio of NL1significantly decreased in the epileptic group at days3,7respectively afterinfusion (p<0.05). There was no significantly difference between the3-d and7-d groups (p>0.05). The results revealed that at days3and7after infusion,the amount of NL1was respectively decreased by36.3±4.3%, and44.2±5.3%of control levels. The immunfluresene showed that EGFP waslocalized in the hippocampus, especially in CA1area.
2. With time, all the rats developed more severe seizures. The seizuresin sh-NL1group are less severe than that in the other groups in each20-minintervals (P<0.05). There was a significant main effect of time (P<0.05) butno significant of time×group interaction (P>0.05). The maximum racinescore in sh-NL1group was less severe than that in the other groups (P<0.05), but there was no significant difference between the two control groups(P>0.05). Finally, only33.3%rats in sh-NL1group showed generalizedclonic or tonic seizures (seizure score4or5) compared with83.3%rats in both other control groups. NL1knockdown significantly reduced theincidence of generalized clonic or tonic seizures (P<0.05). NL1knockdownsignificantly delayed seizure latency compared with the control goups (P<0.05), but there was no significant difference between the two control groups(P>0.05).
Conclusion:
1. The expression of endogenous NL1in the hippocampus wassuccessfully suppressed in vivo by sh-NL1, and the efficiency of RNAinterference in vivo for NL1got to a stable level at day7.
2. Knockdown of NL1in epileptic rats could reduce seizure severityand prolonged seizure latency.Part three: The protential mechanism of neuroligin1involved inepileptigenesis
Objective: To further explore the probable underlying mechanisms ofof NL1involved in spontaneous recurrent seizure(SRS), we investigated theelectrophysiological changes of epileptic rats performed by lentivirallymediated knockdown of NL1in the hippocampus.
Method:
1. Three epileptic groups with hippocampal injection of saline (EP),vehicle virus (EP+sh-con.), and NL1shRNA (EP+sh-NL1) respectivly, aswell as one normal control group with hippocampal injection of saline (control) were included.
2. Western blot analysis and immunfluresene were used for analysis ofthe efficiency of RNA interference in the epileptic rats at day30.
3. Whole-cell recordings of CA1pyramidal neurons in brain slices wereperformed, and the action potential (AP), miniature excitatory postsynapticcurrents (mEPSCs) and evoked excitatory postsynaptic currents (mEPSCs)were recorded.
4. Western blot analysis was used for analysis of the total or the surface/total ratio of NMDAR1expression in the epileptic rats at day30.
Result:
1. Western blot analysis showed the difference in the NL1expressionlevel in hippocampus between the sh-NL1epileptic group and sh-con.epileptic group or epileptic group was significant (p<0.05), but there was nosignificant difference between the sh-con. epileptic group and epilepticgroup as well as the the sh-NL1epileptic group and normal control group(p>0.05). The immunfluresene of EGFP indicated that the lentivirus wassuccessfully transfected into hippocampal neurons.
2. the frequency of spontaneous AP in sh-NL1epileptic group waslower than that in the sh-con. epileptic group (P <0.05). Compared with thenormal control group, the spontaneous AP was increased in both sh-con.epileptic and sh-NL1epileptic groups (P <0.05). There was no significantlydifference of the mean mEPSC amplitude between the sh-NL1epileptic group and the sh-con. epileptic group (P>0.05). The mean mEPSCamplitude averaged from the sh-con. epileptic group and the sh-NL1epileptic group were significantly increased respectively compared with thenormal control group (P <0.05). the mEPSC frequency averaged from thesh-NL1epileptic group was lower than that in the sh-con. epileptic group(P <0.05). Compared with the normal control group, the mean mEPSCfrequency in both sh-con. epileptic group and sh-NL1epileptic weredramaticlly increased (P <0.05). There was a significant difference betweenthe normal control group and the sh-NL1epileptic group (P <0.05).
3. A clear reduction in NMDAR/AMPAR ratio was observed in thesh-NL1epileptic group comparing with the normal control group or sh-con.epileptic group(P <0.05). Compared with the sh-con. epileptic group, thesh-NL1epileptic group showed a significant decrease in the averageamplitude of NMDAR-dependent EPSCs (P<0.05), but not AMPAR-dependent EPSCs (P>0.05).
4. The total or the surface/total ratio of NMDAR1expression in bothepileptic group and sh-con. epileptic group were significantly higher thanthat in the control group (P <0.05). The total or the surface/total ratio ofNMDAR1protein expression in the sh-NL1epileptic group wassignificantly lower than that in the epileptic group and sh-con. epilepticgroup (P <0.05).
Conclusion:
1. The expression of endogenous NL1in the hippocampus remainssuppressed in vivo by shNL1in the rats at day30.
2. Knockdown of NL1in epileptic rats inhibits hyperexcitability inepileptic hippocampal slices.
3. NL1knockdown in epileptic rats could reduces NMDAR-mediatedsynaptic currents in hippocampal slices via postsynaptic NMDAR1.
目的：脑神经元异常突触传递的同步化能导致癫痫的产生。Neuroligin-1(NL1)是位于兴奋性突触后膜的一种突触粘附分子，它能调节突触传递并能决定中枢神经网络的性质。本研究首先检测了NL1及其突触前膜受体neurexin-1β(NRX1β)在难治性颞叶癫痫（TLE）患者及氯化锂-匹罗卡品（LiCl-PILO）癫痫大鼠模型中的表达，以探讨其在TLE发生及发展中的可能作用。
方法：
1.从课题组所建脑组织标本库中随机抽取22例难治性TLE患者的颞叶皮质和10例对照组颞叶皮质。
2.80只成年雄性SD大鼠随机分为对照组（n=10）和癫痫组（n=70），癫痫组又分为7个亚组（每组n=10），即1d组、2d组、3d组、7d组、14d组、30d组和60d组。癫痫组给予LiCl-PILO进行造模。
3.用免疫组化，免疫荧光和免疫印迹三种方法分别检测NL1和NRX1β在颞叶癫痫患者脑组织和癫痫大鼠脑组织中的表达。
结果：
1.免疫染色显示，NL1蛋白主要在难治性TLE患者及对照颞叶皮质神经元的细胞膜和细胞中表达。NL1在难治性TLE患者脑组织中的免疫组化平均光密度值显著高于对照组(p<0.05)。荧光双标染色结果显示NL1主要在颞叶皮质神经元，且和NMDAR1共表达。免疫印迹结果显示NL1及其受体NRX1β在难治性TLE组中为强阳性，而在对照组中则为弱阳性。难治性TLE患者颞叶皮质的NL1和NRX1β的表达水平显著高于对照组(p<0.05)。
2.在LiCl-PILO癫痫大鼠皮质和海马各区，均可见NL1免疫组化阳性染色，其中在海马齿状回、CA1区、CA3区阳性染色最强。在海马组织中，NL1的免疫组化平均OD值在造模后各个时间点呈动态增高趋势。荧光双标染色结果显示NL1在颞叶皮质和海马神经元表达，并且和NMDAR1共表达。免疫印迹结果显示，与对照组相比，NL1和NRX1β在急性期和慢性期均升高，且表达趋势基本一致。与对照组相比，NL1在海马的表达除了在7d和14d组没有差异（p>0.05）外，在其它各组均有差异（p<0.05）。与对照组相比，NRX1β在海马的表达除了在7d组没有差异（p>0.05）外，在其余各组均有差异（p<0.05）。
结论：NL1及其受体NRX1β在难治性TLE患者和LiCl-PILO癫痫大鼠颞叶组织中表达均增高，提示其可能参与了TLE的发生与形成过程。
第二部分Neuroligin1的knockdown对大鼠癫痫发作的影响
目的：为进一步探讨NL1的knockdown是否会对癫痫发作产生影响，我们用沉默NL1基因的短发卡RNA（shRNA）慢病毒减少大鼠海马内源性NL1表达，并在造模后观察由此产生的对癫痫大鼠行为学的影响。
方法：
1.成年雄性SD大鼠造模前分为三组进行海马立体定位注射：即海马立体定位注射生理盐水假手术组（EP），海马立体定位注射慢病毒空载体组(EP+sh-con.)，以及海马立体定位注射NL1shRNA慢病毒组(EP+sh-NL1)。
2.成年雄性SD大鼠海马立体定位注射NL1shRNA慢病毒，并用免疫印迹实验和免疫荧光实验检测其干扰效率.
3. LiCl-PILO造模之后60min之内对大鼠进行行为学观察，分别记录每20min时间间隔内的Racine评分，60min之内最大Racine评分，各组在60min之内达到Ⅳ级以上发作的大鼠所占比例，以及大鼠从PILO注射到首次出现Ⅳ级以上发作的时间（癫痫发作潜伏期）。
结果：
1. NL1shRNA慢病毒海马立体定位注射后，内源性NL1表达逐渐下降。与相应时间点对照组相比，NL1的平均OD比值在慢病毒注射后3d组和7d组中显著下降（p<0.05）。3d组和7d组之间没有显著差异（p>0.05）。在慢病毒注射后的第3d和7d，NL1的总量分别降低了36.3±4.3%和44.2±5.3%。荧光结果显示EGFP阳性表达位于海马，尤其是CA1区。
2.在PILO注射后，大鼠癫痫发作的平均Racine评分随着时间的延长逐渐增高。在各20min间隔内，EP+sh-NL1组的癫痫发作评分均比其它组低（p<0.05），组间有差异（p<0.05）而时间与组间交互作用无差异（p>0.05）。EP+sh-NL1组的最大Racine评分比其它组低（p<0.05）,而另外两组对照无差异（p>0.05）。在EP+sh-NL1组中只有33.3%的大鼠出现了Ⅳ级以上的发作，而在其它两组对照组均有83.3%的大鼠出现了Ⅳ级以上的发作。NL1的knockdown能显著降低Ⅳ级以上癫痫的发生率（p<0.05）。与两组对照组相比，EP+sh-NL1组癫痫发作潜伏期显著延长，而两组对照之间无显著差异（p>0.05）。
结论：
1.对NL1进行的体内shRNA慢病毒干扰能有效抑制内源性NL1的表达，其干扰效率在干扰后第7d达到稳定水平。
2.海马内NL1shRNA慢病毒干扰能减轻癫痫发作程度，降低癫痫发作的易感性。
第三部分Neuroligin1在大鼠癫痫发作中的作用机制
目的：为了研究NL1在自发性癫痫发作（SRS）中的可能作用机制，我们在癫痫大鼠造模后第30d检测NL1shRNA慢病毒干扰效率并进行电生理研究，同时检测与NL1关系密切的NMDAR1在NL1knockdown之后的表达变化。
方法：
1.癫痫大鼠按海马内立体定位注射试剂不同分为三组：生理盐水组（EP）、空载体组(EP+sh-con.)、NL1shRNA组(EP+sh-NL1)；正常大鼠海马内注射生理盐水作为正常对照组（control）。
2.用免疫印迹和免疫荧光实验检测造模后30d大鼠海马NL1shRNA慢病毒干扰效率。
3.用全细胞膜片钳技术记录造模后30d各组大鼠脑片海马CA1区细胞的动作电位（AP）、微小兴奋性突触后电流（mEPSC）、诱发兴奋性突触后电流（evoked EPSC）。
4.用免疫印迹实验检测造模后30d大鼠海马NMDAR1的总蛋白和膜蛋白。
结果：
1.免疫印迹实验结果表明，大鼠造模后30d，NL1在海马的表达在EP+sh-NL1组与EP+sh-con.组或EP组之间有显著差异（p<0.05）,而在EP+sh-con.组与EP组之间以及EP+sh-NL1组与control组之间没有显著差异（p>0.05）。 EGFP免疫荧光结果提示慢病毒已成功转染至海马CA1区神经元。
2.自发性AP的频率在EP+sh-NL1组显著低于EP+sh-con.组（p<0.05）；与control组相比，AP频率在EP+sh-con.组以及EP+sh-NL1组均显著增高（p<0.05）。mEPSC的幅度在EP+sh-con.组与EP+sh-NL1组之间无差别（p>0.05）；与control组相比，mEPSC的幅度在EP+sh-con.组以及EP+sh-NL1组均显著增高（p<0.05）。mEPSC的频率在EP+sh-NL1组比EP+sh-con.组.显著降低（p<0.05）；与control组相比，mEPSC的频率在EP+sh-con.组以及EP+sh-NL1组均显著增高（p<0.05）；mEPSC的频率在control组和EP+sh-NL1组有显著差异（p>0.05）。
3.与control组和EP+sh-con.组相比，EP+sh-NL1组的NMDAR/AMPAR比值显著降低（p<0.05）。与EP+sh-con.组相比，EP+sh-NL1组的NMDAR依赖性EPSCs幅度的显著降低（p<0.05）而AMPAR依赖性EPSCs幅度没有改变（p>0.05）。
4.与control组比，EP组和EP+sh-con.组的总蛋白、表面蛋白/总蛋白比值均显著降低（p<0.05）。而EP+sh-NL1组的总蛋白、表面蛋白/总蛋白比值明显低于EP组和EP+sh-con.组（p<0.05）。
结论：
1.对NL1进行的体内shRNA慢病毒干扰在30d后仍然能有效抑制内源性NL1的表达。
2.癫痫大鼠海马NL1的knockdown能抑制海马CA1区神经元的过度兴奋。
3.癫痫大鼠海马NL1的knockdown能通过突触后NMDAR1的作用选择性抑制海马脑片NMDAR依赖性突触电流。
Part one: The expression of neuroligin-1and neurexin-1β in temporallobe epileptic foci in patients and experimental animals
Objective: Abnormally synchronized synaptic transmission in the brainleads to epilepsy. Neuroligin-1(NL1) is an synaptic cell adhesion moleculelocated at excitatory synapse, which modulates synaptic transmission anddetermines the properties of neuronal networks in the mammalian centralnervous system. Here we investigated the expression of NL1and its bindingpartner neurexin-1β (NRX1β) in temporal lobe epileptic foci in patients andexperimental animals in order to explore the probable relationship betweentheir expression and temporal lobe epilepsy (TLE).
Method:
1. The NL1and NRX1β expression were assessed in twenty two humanbrain tissues derived from patients undergoing operation for intractable TLEand was also detected in ten temporal lobes from controls.
2. Adult male Sprague–Dawley (SD) rats were used in this study,including lithium–pilocarpine-treated rats (n=70) on days1,2,3,7,14,30,and60post-seizure, and control rats (n=10).
3. Expression of NL1and NRX1β were assessed byimmunohistochemistry, immunofluorescence, and Western blot analysis.
Result:
1. NL1protein was mainly expressed in the membrane and cytoplasmof neurons in the temporal neocortex from both the control and intractableTLE groups by immumohistochemical staining. the mean optical density(OD) value of NL1was significantly higher in the temporal neocortex of theTLE group compared with the control group (p<0.05) Double-labelimmunofluorescent staining showed that NL1and GFAP were seldomcoexpressed in astrocytes, but NL1-positive cells coexpressed withNMDAR1. Western blot analysis showed the expression of NL1and NRX1βwas strong in the subjects with intractable TLE, whereas it was relativelyweak in the control subjects.
2. The NL1immumohistochemical staining was extensively observedin neocortex and all regions of the hippocampus, and the intense staining inhippocampus was in the dentate gyrus, CA1and CA3regions. Comparedwith the controls, the mean OD values of NL1expression were elevateddynamically. Double-labeling immunofluoresence showed NL1-positivecells did not co-express with GFAP in astrocytes but co-expressed withNMDAR1in neurons. Western blot analysis showed compared with thecontrols, both NL1and NRX1β expression elevated in the acute period andthe chronic period, and their expression profile presented nearly the same. The difference of the mean OD ratio in the NL1expression level inhippocampus between the control and every treated group was significant(p<0.05), except the7-d and14-d groups (p>0.05). There was a significantdifference of the mean OD ratio in the NRX1β expression between thecontrol and every treated group (p<0.05), except the7-d group (p>0.05).
Conclusion: The expression of NL1and its binding partner NRX1βwere increased in temporal lobe epileptic foci in patients andlithium–pilocarpine-treated epileptic rats. Our results suggest that NL1andNRX1β may play an important role in the development of TLE.Part two: The effect of knockdown of neuroligin1on epileptic seizures
Objective: To further study whether supression of NL1could preventseizures, we investigated the behavioral changes of epileptic rats performedby lentivirally mediated knockdown of NL1in the hippocampus.
Method:
1. The following three groups with hippocampal injection wereincluded, namely sham operation group with hippocampal injection of saline(EP), group with hippocampal injection of vehicle virus (EP+sh-con.), andgroup with hippocampal injection of NL1shRNA (EP+sh-NL1).
2. Western blot analysis of NL1expression in hippocampus was used toverify that in vivo RNA interference for NL1was effective and selective. Onthe other hand, EGFP antibody was used to identify and amplify the virus-infected neurons in the hippocampus by immunofluoresence.
3. We perform rats behavioral investigation7days after virus infusion.We observed the behavioral changes of the rats within60-min afterpilocarpine injection. Scoring in rats for each20-min interval, the maximumRacine score during the60-min trial,the percentage of the rats reached above5score, and the seizure latency in the three groups were recorded.
Result:
1. Western blot analysis showed that the mean OD ratio of NL1significantly decreased in the epileptic group at days3,7respectively afterinfusion (p<0.05). There was no significantly difference between the3-d and7-d groups (p>0.05). The results revealed that at days3and7after infusion,the amount of NL1was respectively decreased by36.3±4.3%, and44.2±5.3%of control levels. The immunfluresene showed that EGFP waslocalized in the hippocampus, especially in CA1area.
2. With time, all the rats developed more severe seizures. The seizuresin sh-NL1group are less severe than that in the other groups in each20-minintervals (P<0.05). There was a significant main effect of time (P<0.05) butno significant of time×group interaction (P>0.05). The maximum racinescore in sh-NL1group was less severe than that in the other groups (P<0.05), but there was no significant difference between the two control groups(P>0.05). Finally, only33.3%rats in sh-NL1group showed generalizedclonic or tonic seizures (seizure score4or5) compared with83.3%rats in both other control groups. NL1knockdown significantly reduced theincidence of generalized clonic or tonic seizures (P<0.05). NL1knockdownsignificantly delayed seizure latency compared with the control goups (P<0.05), but there was no significant difference between the two control groups(P>0.05).
Conclusion:
1. The expression of endogenous NL1in the hippocampus wassuccessfully suppressed in vivo by sh-NL1, and the efficiency of RNAinterference in vivo for NL1got to a stable level at day7.
2. Knockdown of NL1in epileptic rats could reduce seizure severityand prolonged seizure latency.Part three: The protential mechanism of neuroligin1involved inepileptigenesis
Objective: To further explore the probable underlying mechanisms ofof NL1involved in spontaneous recurrent seizure(SRS), we investigated theelectrophysiological changes of epileptic rats performed by lentivirallymediated knockdown of NL1in the hippocampus.
Method:
1. Three epileptic groups with hippocampal injection of saline (EP),vehicle virus (EP+sh-con.), and NL1shRNA (EP+sh-NL1) respectivly, aswell as one normal control group with hippocampal injection of saline (control) were included.
2. Western blot analysis and immunfluresene were used for analysis ofthe efficiency of RNA interference in the epileptic rats at day30.
3. Whole-cell recordings of CA1pyramidal neurons in brain slices wereperformed, and the action potential (AP), miniature excitatory postsynapticcurrents (mEPSCs) and evoked excitatory postsynaptic currents (mEPSCs)were recorded.
4. Western blot analysis was used for analysis of the total or the surface/total ratio of NMDAR1expression in the epileptic rats at day30.
Result:
1. Western blot analysis showed the difference in the NL1expressionlevel in hippocampus between the sh-NL1epileptic group and sh-con.epileptic group or epileptic group was significant (p<0.05), but there was nosignificant difference between the sh-con. epileptic group and epilepticgroup as well as the the sh-NL1epileptic group and normal control group(p>0.05). The immunfluresene of EGFP indicated that the lentivirus wassuccessfully transfected into hippocampal neurons.
2. the frequency of spontaneous AP in sh-NL1epileptic group waslower than that in the sh-con. epileptic group (P <0.05). Compared with thenormal control group, the spontaneous AP was increased in both sh-con.epileptic and sh-NL1epileptic groups (P <0.05). There was no significantlydifference of the mean mEPSC amplitude between the sh-NL1epileptic group and the sh-con. epileptic group (P>0.05). The mean mEPSCamplitude averaged from the sh-con. epileptic group and the sh-NL1epileptic group were significantly increased respectively compared with thenormal control group (P <0.05). the mEPSC frequency averaged from thesh-NL1epileptic group was lower than that in the sh-con. epileptic group(P <0.05). Compared with the normal control group, the mean mEPSCfrequency in both sh-con. epileptic group and sh-NL1epileptic weredramaticlly increased (P <0.05). There was a significant difference betweenthe normal control group and the sh-NL1epileptic group (P <0.05).
3. A clear reduction in NMDAR/AMPAR ratio was observed in thesh-NL1epileptic group comparing with the normal control group or sh-con.epileptic group(P <0.05). Compared with the sh-con. epileptic group, thesh-NL1epileptic group showed a significant decrease in the averageamplitude of NMDAR-dependent EPSCs (P<0.05), but not AMPAR-dependent EPSCs (P>0.05).
4. The total or the surface/total ratio of NMDAR1expression in bothepileptic group and sh-con. epileptic group were significantly higher thanthat in the control group (P <0.05). The total or the surface/total ratio ofNMDAR1protein expression in the sh-NL1epileptic group wassignificantly lower than that in the epileptic group and sh-con. epilepticgroup (P <0.05).
Conclusion:
1. The expression of endogenous NL1in the hippocampus remainssuppressed in vivo by shNL1in the rats at day30.
2. Knockdown of NL1in epileptic rats inhibits hyperexcitability inepileptic hippocampal slices.
3. NL1knockdown in epileptic rats could reduces NMDAR-mediatedsynaptic currents in hippocampal slices via postsynaptic NMDAR1.
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