通过电生理场记录研究跨体海马CA1的长期突触可塑性
Summary
我们在纵向海马脑切片中使用记录和刺激电极,并在体内背海马区中纵向定位记录和刺激电极,以唤起细胞外的显微电位并演示沿纵向间层 CA1 的长期突触可塑性。
Abstract
海马突触可塑性的研究主要集中在CA3-CA1层状网络的使用上。对纵向间层CA1-CA1网络的关注较少。然而,最近,CA1-CA1金字塔神经元之间的关联连接被显示出来。因此,有必要研究海马的纵向间层CA1-CA1网络是否支持突触可塑性。
我们设计了一个协议,利用体内和体外电生理场记录,研究海马间CA1网络中是否存在长期突触可塑性。对于体内细胞外场记录,记录和刺激电极被放置在背海马的隔音-时间轴以纵向的角度,以唤起场内兴奋的后分感电位。对于体外细胞外场记录,海马纵向切片被切割平行于隔膜-时间平面。记录和刺激电极被放置在沿纵轴的海马层或地层(S.R)和地层辐射(S.R) 中。这使我们能够研究诱发兴奋性脱发潜能的方向和层特异性。已经建立的协议用于诱导体内和体外的长期强效(LTP)和长期抑郁症(LTD)。我们的结果表明,纵向间层CA1网络支持N-甲基-D-阿斯巴酸酯(NMDA)受体依赖性长期强效(LTP),没有方向或层特异性。然而,与横向层状层网络相比,间层网络没有出现任何显著的长期抑郁症(LTD)。
Introduction
海马在认知研究1,2,3中被广泛应用。横轴中的海马层状网络形成由倒角陀螺、CA3 和 CA1 区域组成的三突触电路。层网络被认为是一个平行和独立的单元4,5。这种层状的观点影响了海马体体内和体外电生理学研究的横向方向和横片的使用。根据新兴的研究,正在重新评估层状假说,并关注海马的间层网络。关于海马间海马网络,CA3区域早已被调查7,8,9,10,但纵向CA1海马区域已收到直到最近,人们很少关注。关于CA1间间层网络,大鼠的多索文拉纵向海马CA1轴线的短期突触特性已被证明变化11。此外,发现对相和地点作出反应的海马细胞簇被系统地排列在大鼠的海马的纵轴上,完成短期记忆任务12。此外,癫痫发作活动被发现沿整个海马沿着纵轴13同步。
然而,大多数对纵向CA1海马区域的研究,都利用了从CA3到CA1区域11、14、15的输入。使用独特的协议来制作纵向脑切片,我们以前的工作证明了CA1金字塔神经元沿纵轴的关联连通性,并牵连到其有效处理神经元信号的能力16。然而,需要确定没有横向输入的沿纵轴的CA1金字塔神经元是否能支持长期突触可塑性。这一发现可以为调查与海马有关的神经问题增加另一个角度。
神经元适应信息传递功效的能力称为突触可塑性。突触可塑性被牵连为认知过程的基础机制,如学习和记忆17,18,19,20。长期突触可塑性表现为长期强效(LTP),代表神经元反应的增强,或长期抑郁(LTD),代表神经元反应的减弱。在海马的横轴上研究了长期突触可塑性。然而,这是第一次研究证明CA1金字塔神经元的海马纵向轴的长期突触可塑性。
根据杨等人16日使用的一个协议,我们设计了该协议,以在CA1金字塔神经元的海马纵轴中演示LTP和LTD。我们使用年龄介于5-9周之间的C57BL6雄性小鼠进行体外实验,使用6-12周大的雄性小鼠进行体内实验。本详细文章展示了如何从小鼠获得纵向海马脑切片进行体外记录,以及如何在纵轴中记录体内记录。对于体外记录,我们研究了纵向CA1突触可塑性的方向特异性,通过瞄准海马的隔膜和时间端。我们还通过记录海马的地层或层和地层辐射,研究了纵向CA1突触可塑性的层特异性。对于体内记录,我们研究了与海马的纵向方向最对应的角度。
使用体内和体外细胞外场外记录,我们观察到纵向连接的CA1金字塔神经元呈现LTP,而不是LTD。然而,涉及CA3和CA1神经元的横向方向同时支持LTP和LTD。海马的横向和纵向之间的突触能力区别可能推测表明其功能连通性的差异。需要进一步的实验来破译其突触能力的差异。
Protocol
Representative Results
Discussion
该方案演示了在体外海马的纵向CA1-CA1轴中诱导体内和脑切片的长期突触可塑性的方法。概述的步骤为实验者提供了足够的细节,以便他们研究纵向海马CA1-CA1连接中的LTP和LTD。需要练习来磨练成功记录现场兴奋潜力所需的技能。
除了需要练习外,还有几个关键步骤对于取得良好结果至关重要。首先,它之前显示,大脑切片的角度可以截截或保留金字塔神经元的纵向投影在CA1-CA1区域?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了仁川国立大学(国际合作)研究资助。我们要感谢崔女士协助收集一些数据。
Materials
| Atropine Sulphate salt monohydrate, ≥97% (TLC), crystalline | Sigma-Aldrich | 5908-99-6 | Stored in Dessicator |
| Axon Digidata 1550B | |||
| Calcium chloride | Sigma-Aldrich | 10035-04-8 | |
| Clampex 10.7 | |||
| D-(+)-Glucose ≥ 99.5% (GC) | Sigma-Aldrich | 50-99-7 | |
| Eyegel | Dechra | ||
| Isoflurane | RWD Life Sciences | R510-22 | |
| Magnesium chloride hexahydrate, BioXtra, ≥99.0% | Sigma-Aldrich | 7791-18-6 | |
| Matrix electrodes, Tungsten | FHC | 18305 | |
| Multiclamp 700B Amplifier | |||
| Potassium chloride, BioXtra, ≥99.0% | Sigma-Aldrich | 7447-40-7 | |
| Potassium phosphate monobasic anhydrous ≥99% | Sigma-Aldrich | 7778-77-0 | Stored in Dessicator |
| Pump | Longer precision pump Co., Ltd | T-S113&JY10-14 | |
| Silicone oil | Sigma-Aldrich | 63148-62-9 | |
| Sodium Bicarbonate, BioXtra, 99.5-100.5% | Sigma-Aldrich | 144-55-8 | |
| Sodium Chloride, BioXtra, ≥99.5% (AT) | Sigma-Aldrich | 7647-14-5 | |
| Sodium phosphate monobasic, powder | Sigma-Aldrich | 7558-80-7 | |
| Sucrose, ≥ 99.5% (GC) | Sigma-Aldrich | 57-50-1 | |
| Temperature controller | Warner Instruments | TC-324C | |
| Tungsten microelectrodes | FHC | 20843 | |
| Urethane, ≥99% | Sigma-Aldrich | 51-79-6 | |
| Vibratome | Leica | VT-1200S | |
| Water bath | Grant Instruments | SAP12 |
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