Summary

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

Published: January 19, 2012
doi:

Summary

在这里,我们描述了一个协议,准备琼脂嵌入式适合电和Ca2 +成像视网膜切片。这种方法允许一个研究使用录音单突触前神经末梢的直接膜片钳视网膜微型带状型突触。

Abstract

发现视觉刺激,并转达了专门的神经元在脊椎动物视网膜的光线强度和频率的变化在很宽的动态范围。两个班的视网膜神经细胞,感光细胞和双极细胞,使用色带类型的活动区,从而使长的时间周期,持续和高吞吐量的神经递质的释放。型混合双极细胞终端(MB)在金鱼视网膜,从而去极化光刺激和接收混合杆和锥感光输入,适合的带状型突触都因他们的大尺寸(〜10-12的研究微米直径)和他们的许多横向和互惠的无长突细胞树突的突触联系。 MB双极细胞在金鱼视网膜切片膜片钳技术终端直接访问允许测量突触前的Ca 2 +电流,膜电容的变化,和互惠的反馈抑制GABA介导的突触<分> A和GABA 彗星受体表达上的终端。突触前膜电容测量胞吐允许一个短期的可塑性研究的兴奋性神经递质释放14,15。此外,短期和长期的无长突细胞的抑制性神经递质释放的可塑性也可以抵达的21 MB终端的相互反馈抑制录音进行调查。 GABA能无长突细胞的相互反馈抑制经过短时间的时间(例如〜10秒),通过GABA的小泡池枯竭 11双脉冲抑郁症。在视网膜内丛状层视网膜微型突触动力学因此可以直接研究。

大脑切片技术引入超过40年前,但仍然是非常有用的神经元的电性能,无论是在单细胞胞体,树突或轴突单,调查,和Microcircuit突触水平19。组织太小,无法直接粘到切片室往往是先嵌入在琼脂(或放置到一个滤纸),然后切片20,23,18,9。在这段视频中,我们采用预嵌入琼脂技术,使用金鱼视网膜。在金鱼视网膜片的巨型双极细胞终端axotomized(轴突切割)在切片过程中。这允许我们分离单个突触前神经末梢输入,录音排除,因为从axotomized终端的信号从胞体树突状车厢。另外,也可以从完整的MB双极细胞记录,从没有减少在切片过程中的轴突连接到终端记录。总体而言,这个实验性的协议的使用将有助于在视网膜突触生理学,微电路功能分析,带状突触的突触传递的研究。

Protocol

1。外部和内部的解决方案准备从10倍原液切片溶液(低钙)和每天添加2,氯化钙 ,氯化镁和D -葡萄糖。最后1X解决方案,包括(毫米):119氯化钠,氯化钾2.5,3.2,0.25 MgCl 2的氯化钙,12个D -葡萄糖,0.2 L -抗坏血酸,12 HEPES 。设置pH值至7.4(用NaOH),并调节渗透压260 mOsm(使用H 2 O和10倍原液) 。 称取3%的低胶凝温度琼脂(琼脂糖类型VII – A,A0701,Sigma公司…

Discussion

一个关键和艰难的一步,在我们的协议转让的视网膜上成的琼脂溶液(3.4协议) 。这是必要的,小心地取出玻璃体和视网膜片残余片解决方案,传输不失真或弯曲。为了做到这一点,我们使用了一个尖弯曲90 °角,沿直角尖镊子(11251-35,精细科学的工具),位置视网膜的小抹刀(21 – 401 – 25B,Fisherbrand)片解决方案的整个传输过程中的一块。仔细涂抹表面与一个折叠或卷起Kimwipes(金…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢他的弗雷德雷基博士琼脂包埋的视网膜切片制备的一种解释,当我们开始在我们的实验室中使用的协议。我们也感谢洛瑞Vaskalis的示意图和DRS的插图。 Veeramuthu维文和Soyoun赵上的文字和视频的有益的意见。这项工作是支持由聂NIH RO1的授予,由韩国政府资助的韩国研究基金会格兰特也部分支持KRF – 2008 – 357 – E00032]。

Materials

Name of the reagent Company Catalogue number Comments
Low gelling-temperature agar Sigma A0701 Agarose type VII-A
Patch pipette World Precision Instruments 1B150F-4 Thick-walled (1.5 mm outer diameter) borosilicate glass
Vertical puller Narishige PP830  
Dental wax Cavex    
Spring scissors Fine Science Tools 15003-08  
45° angled fine tip forceps Fine Science Tools 11251-35  
Razor blade Personna   Double-edged, cleaned with 70% ethanol and H2O
Cylindrical tube Fisherbrand 03-338-1B Polyethylene sample vials 2.5 ml
Hyaluronidase Sigma H6254  
Vibratome slicer Leica VT1000S or VT1200S  
Upright microscope Olympus BX51WI  
60x water-immersion objective Olympus LUMPlanFl NA 0.90
CCD camera Sony XC-75  
Camera controller Hamamatsu C2400  
Monitor Sony   13” black and white monitor
Syringe filter Nalgene   0.2 μm
Micromanipulator Sutter Instrument MPC-200  
Lock-in amplifier HEKA   EPC-9/10 amplifiers have software emulation
Spinning disk laser confocal microscope Yokogawa CSU-X1 Live cell imaging after patch clamp whole cell recording
Slidebook software Intelligent Imaging Instruments (3i)   Imaging data acquisition and analysis
Paraformaldehyde Sigma P6148  
Phosphate buffer solution GIBCO 70013  
Superfrost slide Fisher Scientific   Slide glass
Anti-fading agents Biomeda corp.    
Confocal laser-scanning microscope Carl Zeiss LSM 710 Imaging of fixed tissue
Spatula Fisherbrand 21-401-25B  
Manuel vertical slicer Narishige ST-20  
Oregon Green 488 BAPTA-1 Invitrogen O-6806 Ca2+ sensitive fluorescent dye
Alexa Fluor 555 Hydrazide Invitrogen A-20501MP Fluorescent dye

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Kim, M., Vickers, E., von Gersdorff, H. Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices. J. Vis. Exp. (59), e3345, doi:10.3791/3345 (2012).

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