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Neurosciences

体外楔形切片准备模拟在Vivvo神经元电路连接

Published: August 18, 2020
doi:
10.3791/61664
,
1Section on Neuronal Circuitry, National Institute on Deafness and Other Communication Disorders,NIH

Summary

将不同的突触输入与神经元的整合最好用一种制备来测量，这种制备保留了所有突触前核，以便自然计时和电路可塑性，但大脑切片通常切断许多连接。我们开发了一个经过修改的大脑切片来模拟体内回路活动，同时保持体外实验能力。

Abstract

体外切片电生理学技术以精确的电和时间分辨率测量单细胞活动。脑片必须相对薄，以正确可视化和访问神经元进行贴片夹紧或成像，并且对大脑电路的体外检查仅限于急性切片中物理存在的。为了保持体外切片实验的好处，同时保留大部分前突触核，我们开发了一种新型切片制备。这种”楔形切片”设计用于贴片式电生理学记录，以描述脑干中中层胆汁（MOC）神经元的多种单声道、声驱动输入。这些神经元从由反边耳和相应的耳蜗核（CN）的刺激激活的神经元中接收其主要兴奋和抑制性输入。设计了一个不对称的大脑切片，在一个半球的横向边缘的玫瑰花外域中最厚，然后向相反半球的横向边缘变薄。在厚的一面，该片包含听觉神经根向大脑传递有关听觉刺激的信息，内在的CN电路，以及融合在反向MOC神经元上的脱联兴奋和三联突抑制的通路。记录从片片薄侧的MOC神经元进行，在该实验中，使用DIC光学元件进行典型贴片夹实验。当听觉神经进入听觉脑干时，直接刺激它，允许内在CN电路活性和突触可塑性发生在MOC神经元上游的突触。使用这种技术，可以尽可能密切地模拟切片内的体内电路激活。这种楔形切片制备适用于其他脑电路，其中电路分析将受益于保持上游连接和远程输入，结合体外切片生理学的技术优势。

Introduction

神经回路活动的观察是理想地用原生感官输入和反馈进行的，以及大脑区域之间在体内的完整连接。然而，进行神经回路功能单细胞分辨率的实验仍然受到完整大脑中技术挑战的受限。虽然在体内细胞外电生理学或多光子成像方法可用于调查完整神经系统的活动，但解释不同输入如何集成或测量亚星体突触输入仍然很困难。体内全细胞记录克服了这些限制，但很难执行，即使在大脑区域，很容易访问。单细胞分辨率实验的技术挑战在位于大脑深处的某些神经元群体或空间扩散群体中进一步放大，这些群体需要基因工具来定位体内细胞（例如，与视光记录配对的通道霍多辛的遗传表达）或记录站点标记后（例如具有神经传递特异性标记）后组织化学识别。位于脑干的腹外表面附近，中层胆道（MOC）神经元受到上述限制1，使得它们极难进入体内实验。

脑片（±100-500μm厚度）长期以来一直用于研究脑电路，包括听觉脑干电路，因为同一切片2、3、4、5、6、7、8、9中所含的神经元的物理分离。其他实验室都使用更厚的切片（>1 mm）进行实验，以了解双边输入如何整合在高级橄榄复合物（SOC）区域，包括中层高级橄榄10，11。这些切片的准备，使听觉神经（AN）的轴突保持完好无损的切片内，并受电刺激，启动突触神经递质释放在CN，模仿一阶听觉神经元的活动，因为他们会响应声音。这些厚切片的一个主要缺点是神经元的可见性，用于贴片电生理记录（”修补”）。修补变得越来越困难，因为在这个区域的许多轴突成为骨髓与年龄12，13，14，15，使组织光学密集和模糊神经元，即使在一个典型的，薄的大脑切片。我们的目标是创造体外准备，更类似于体内记录电路连接，但与高通量和高分辨率记录能力视觉引导补丁夹电生理学在脑片。

我们的实验室研究听觉发泡系统神经元的生理学，包括MOC神经元。这些胆碱神经元通过调节外发细胞（OHCs）16、17、18、19、20的活性，为耳蜗提供发向反馈。先前的研究显示，这种调制在耳蜗21、22、23、24、25、26和隔音创伤27、28、29、30、31、32、33等的增益控制中起着一定的作用。在小鼠中，MOC神经元弥漫地位于听觉脑干1中梯形体（VNTB）的腹核。我们组利用与 tdTomato 记者鼠标线交叉的 ChAT-IRES-Cre 鼠标线，在显光照明下瞄准脑干切片中的 MOC 神经元。我们表明，MOC神经元从梯形体（MNTB）的吸管内核接收一个抑制性输入，而梯形体（MNTB）则由反边合体核（CN）34、35、36、37、38中的球状浓密细胞（GBC）的轴突激发。此外，MOC神经元可能从反面CN39、40、41中的T-斯特拉特细胞接收其兴奋性输入。综合起来，这些研究表明，MOC神经元接收来自同一（反向）耳朵的兴奋和抑制性输入。然而，前突触神经元，及其轴突聚集在MOC神经元上，在典型的日冕切片制备中，彼此不够接近，无法完全完好无损。为了研究突触输入与MOC神经元的整合如何影响其作用潜在的射击模式，我们开发了一种制备，我们可以以最生理上现实的方式刺激从一只耳朵对MOC神经元的不同影响，但具有体外脑切片实验的技术益处。

楔形切片是一种经过修改的厚切片制备，用于调查MOC神经元中的电路集成（图1A中所示）。在切片的厚侧，楔子包含听觉神经的被切断轴突（以下简称”听觉神经根”），因为它们从边缘进入脑干，并在CN中突触。听觉神经根可以电刺激，唤起神经递质释放和突触激活完全完整的CN 42，43，44，45，46细胞。这种刺激格式对电路分析具有若干优点。首先，我们刺激的不是直接刺激向MOC神经元提供输入的T-斯特拉特和GBC轴突，而是刺激性，以允许激活CN中丰富的内在电路。这些电路调节CN神经元的输出到他们在整个大脑的目标，包括MOC神经元46，47，48，49，50，51。其次，从安到MOC神经元的CN上游的多合成激活允许更自然的激活时间，使可塑性在这些突触中发生，因为它们在听觉刺激期间在体内发生。第三，我们可以改变我们的刺激模式来模仿一个活动。最后，对MOC神经元的兴奋和抑制单声投影在楔片中完好无损，它们在MOC神经元中可以测量其整合，并精确测量贴片-夹触电生理学。总的来说，与典型的脑切片制备相比，这种激活方案为MOC神经元提供了一个更完整的电路。这种脑干楔形切片也可用于研究其他听觉区域，接受从ipilatermNTB的抑制性输入，包括横向优越的橄榄，优越的橄榄核和中高级橄榄10，11，52，53，54，55，56。除了我们的具体准备，这种切片方法可以使用或修改来评估其他系统，其好处是保持远程输入的连接，并改善神经元的可视化，用于各种单细胞分辨率电生理学或成像技术。

该协议要求使用可倾斜约 15° 的振动台或平台。在这里，我们使用一个市售的两件式磁舞台，”舞台”是一个金属盘，底部弯曲，放置在凹式磁性”舞台基座”中。然后可以移动舞台以调整切片角度。舞台基座上的同心圆用于可重复估计角度。舞台和舞台底座放置在切片室中，其中磁级底座也可以旋转。

Protocol

所有实验程序都得到国家神经疾病和中风研究所/国家耳聋和其他沟通障碍动物护理和使用委员会的批准。 1. 实验准备 注：有关切片制备的详细信息，包括切片溶液、切片温度、切片孵化温度和装置（等）是本实验中进行脑干制备的特明。切片孵育细节可以根据实验室经验进行更改。 准备用于修补的内部解决方案。 准备含有（以 mM）76 Cs-甲…

Representative Results

楔形切片的组织学研究为了研究听觉脑干神经元功能，楔形切片制剂被设计为包含听觉神经根和CN反面的MOC神经元的目标记录（例如切片如图1B所示）。初步组织学检查的准备是重要的，以确认切片包含电路激活所必需的核，并且与弓投影完好无损。CN 中的两种细胞类型为 MOC 神经元提供声音信息。T-斯特拉特细胞被假设为MOC神经元39，40，41，58提供<sup…

Discussion

这里描述的楔形切片切片过程对于保持完整的前突触神经元电路是强大的，但脑切片实验的可访问性，用于分析神经元功能。必须注意几个初始步骤，以最大限度地提高电路分析准备的效用。楔形的尺寸应使用组织学检查进行确认，这是确认前突触核及其囊突投影都包含在准备好的楔形切片中不可或缺的。如果投影被切断或几个轴子到达目标核，则切片几何可能需要修改。更一般来说，楔形切片?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项研究得到了国家卫生和宫内研究计划的支持，NIDCD，Z01 DC00091（CJCW）。

Materials

Experimental Preparations
Agar, powder Fisher Scientific BP1423500 4% agar block used to stabilize brain tissue during vibratome sectioning
AlexaFluor Hydrazide 488 Invitrogen A10436 Fluorophore used in internal solution to confirm successful MOC neuron patch
Analytical Balance Geneses Scientific (Intramalls) AV114 Weighing chemicals
Double edged razor blade Ted Pella 121-6 Vibratome cutting blade
Kynurenic acid (5g) Sigma Aldrich K3375-5G Slicing ACSF additive used to reduce neuron activity during dissection and slicing in order to improve tissue health for patch clamping
pH Meter Fisher Scientific (Intramalls) 13-620-451 Solution pH tester
Plastic petri dishes 100mm dia X 20mm Fisher Scientific (Intramalls) 12-556-002 4% Agar Prep
Stirring Hotplate Fisher Scientific (Intramalls) 11-500-150 Heating for 4% Agar preparation
Dissection and Slicing
Biocytin Sigma Aldrich B4261-250MG Chemical used for axonal tracing (conjugated to Streptavidin 488)
Dissecting Microscope Amscope SM-1BN For precision dissection during brain removal
Dumont #5 Forceps Fine Science Tools 11252-20 Fine forceps dissection tool
Economy tweezers #3 WPI 501976 Forceps dissection tool
Glass Petri Dish 150mm dia x 15mm H Fisher Scientific (Intramalls) 08-747E Dissection dish
Interface paper (203 X 254mm PCTE Membrane 10um) Thomas Scientific 1220823 Slice incubation/biocytin application
Leica VT1200S Vibratome Leica 1491200S001 Vibratome for wedge slice sectioning
Mayo scissors Roboz RS-6872 Dissection tool
Single-edged carbon steel blades Fisher Scientific (Intramalls) 12-640 Razor blade for dissection
Specimen disc, orienting Leica 14048142068 Specialized vibratome stage for reproducible tilting
Spoonula FisherSci 14-375-10 Dissection tool
Super Glue Newegg 15187 Used for glueing tissue to vibratome stage
Vannas Spring Scissors Fine Science Tools 91500-09 Dissection tool
Electrophysiology
A1R Upright Confocal Microscope Nikon Instruments Electrophysiology and imaging microscope, can be any microscope compatible with electrophysiology
Electrode Borosilicate glass w/ Filament OD 1.5mm, ID 1.1mm, 10 cm long Sutter Instrument BF150-110-10 Patch clamping pipette glass
Electrode Filler MicroFil WPI CMF20G Patch electrode pipette filler
In-line solution heater Warner Instruments (GSAdvantage) SH-27B Slice perfusion system heater
Multi-Micromanipulator Systems Sutter Intruments MPC-200 with MP285 Micromanipulators for patch clamp and stimulation electrode placement
P-1000 horizontal pipette puller for glass micropipettes Sutter instruments FG-P1000 Patch clamp pipetter puller
Patch-clamp amplifier and Software HEKA EPC-10 / Patchmaster Next Can be any amplifier/software
Recording Chamber Warner Instruments RC26G Slice "bath" during recording
Recording Chamber Harp Warner Instruments 640253 Stablizes slice during electrophysiology recording
Slice Incubation Chamber Custom Build Heated, oxygenated holding chamber for slices during recovery after slicing
Stimulus isolation unit A.M.P.I. Iso-Flex Stimulus isolation unit for electrophysiology
Syringe 60CC Fischer Scientific (Intramalls) 14-820-11 Electrophysiology perfusion fluid handling
Temperature controller Warner Instruments (GSAdvantage) TC-324C Slice perfusion system temperature controller
Tubing 1/8 OD 1/16 ID Fischer Scientific (Intramalls) 14-171-129 Electrophysiology perfusion fluid handling
Tugsten concentric bipolar microelectrode WPI TM33CCINS Stimulating electrode for electrophysiology
Histology
24 well Plate Fisher Scientific (Intramalls) 12-556006 Histology slice collection and immunostaining
Alexa Fluor 488 Streptavidin Jackson Immuno labs 016-540-084 Secondary antibody for biocytin visualization
Corning Orbital Shaker Sigma CLS6780FP Shaker for immunohistochemistry agitation
Cresyl Violet Acetate Sigma Aldrich (Intramalls) C5042-10G Cellular stain for histology
Disposable Microtome Blades Fisher Scientific 22-210-052 Sliding microtome blade
Filter-syringe Nalgene 4mm Cellulose Acetate 0.2um Fisher Scientific (Intramalls) 09-740-34A Syringe filter for filling recording pipettes with internal solution
Fluoromount-G Slide Mounting Medium Fisher Scientific OB100-01 Immunohistochemistry fluorescence mounting medium
glass slide staining dish with rack Fisher Scientific (Intramalls) 08-812 Cresyl Violet staining chamber
Microm HM450 Sliding Microtome ThermoFisher 910020 Freezing microtome for histology
Microscope Cover Glasses: Rectangles 50mm X 24mm Fisher Scientific (Intramalls) 12-543D Histochemistry slide cover glass
Permount mounting medium Fisher Scientific SP15-100 Cresyl violet section mounting medium
Superfrost Slides Fisher Scientific 22-034980 Histology slides
DOWNLOAD MATERIALS LIST

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In VitroWedge SliceNeuronal Circuit ConnectivityBrain SlicePresynaptic CircuitryPatch Clamp RecordingsPharmacologyActivity ImagingHistologySkullBrain DissectionCranial NervesSpinal CordBrain Fixation
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Fischl, M. J., Weisz, C. J. C. In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity. J. Vis. Exp. (162), e61664, doi:10.3791/61664 (2020).
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