我们描述了一个协议,以监测在模型脊椎动物毛细胞系统运动命令期间的无动于衷的神经元活动的变化。
感官系统收集指导行为必不可少的线索,但动物必须破译哪些信息与生物学相关。运动产生重新获得线索,表明动物必须脱离周围环境的相关感官线索。例如,当鱼游泳时,由构成横向线系统的机械感应神经瘤(包括毛细胞)检测到身体起伏产生的流量。然后,毛细胞通过感官性神经元将流体运动信息从传感器传输到大脑。同时,电机指令的必然放电被传到毛细胞,以防止感官超载。因此,在评估横向线系统的灵敏度时,考虑运动过程中预测运动信号的抑制作用至关重要。我们已经开发出一种体内电生理学方法,可以同时监测斑马鱼幼虫(受精后4-7天)的后横向线轴神经元和腹腔运动根活动,这种活动可以持续几个小时。使用松散的贴片夹紧技术实现对有发声神经元的细胞外记录,该技术可以检测来自单个或多个神经元的活动。用玻璃电极通过皮肤进行心室根记录,以检测运动神经元活动。我们的实验协议提供了在完整、行为灵巧的脊椎动物中监测运动行为的内源性或唤起感官输入变化的潜力。
机械感官系统的神经元在听觉和平衡过程中将信息从毛细胞传输到大脑。电子生理学可以通过直接记录揭示过敏神经元的敏感性。虽然从头发细胞的整个细胞修补可能具有挑战性,从下游的通风神经元记录更容易,并允许评估行动潜力,以响应受控刺激1,2,3。刺激头发细胞导致其偏转,从而改变机械感官结构,从而触发在无动于衷的神经元4,5,6的动作潜力(尖峰)增加。在没有外部刺激的情况下,由于谷氨酸从毛细胞泄漏到发精后7、8的终端,心酸神经元也会自发地尖峰,并被证明有助于保持敏感性9、10。贴片夹记录的通风活动,使观察头发细胞的敏感性和信号动力学,这是不可能使用技术与较低的时间分辨率,如在微音11,12或功能钙成像13,14,15。以下协议将允许记录与运动命令并存的无动于衷的活动,以显示毛细胞敏感性的瞬时变化。
斑马鱼(Danio rerio)使用神经瘤中所含的头发细胞组成横向线系统来检测相对于其身体的水运动,这些水体被转化为导航16、17、18、捕食者躲避、猎物捕获19、20和学校教育21所必需的神经信号。水流也可以由游泳22、23、24、呼吸22、25、26和喂食27的运动自行产生。这些行为包括重复运动,可以疲劳头发细胞和损害传感。因此,横向线系统必须区分外部(外向)和自生成(重现)流量刺激。在斑马鱼的运动过程中,必然放电会减弱自生成的流动信号。这种抑制性预测运动信号通过下降神经元传导到感官受体,以修改输入或中断处理再吸收反馈28,29。开创性的工作有助于我们早期了解这个馈送系统依赖于体外制备,其中神经回路的连接性和内源活动没有保持28,30,31,32,33,34,35。该协议描述了一种保存完整神经回路的方法,其中内源反馈动力学得以保持,从而能够更好地了解体内的必然放电。
此处概述的协议描述了如何同时监测幼虫斑马鱼的后横向线神经元和运动神经元活动。在运动指令之前、期间和之后对发光信号动态进行特征化,可深入了解中枢神经系统实时的内生反馈,这些反馈在运动过程中调节了毛细胞的敏感性。该协议概述了在实验前需要准备哪些材料,然后描述了如何瘫痪和准备斑马鱼幼虫。该协议将描述如何建立一个稳定的松散补丁记录的通风神经元和细胞外腹腔根(VR)记录的运动神经元。使用此协议可以获得的代表性数据来自一个范例个体,并针对实验协议的多个副本进行了分析。使用 MATLAB 中的自定义书面脚本对数据进行预处理。总的来说,这种体内实验范式有望在模型脊椎动物毛细胞系统中更好地了解运动过程中的感官反馈。
描述的实验协议提供了在完整、行为良好的脊椎动物中监测运动行为感官输入的内源性变化的潜力。具体来说,它详细介绍了一种体内方法,用于同时执行横向线轴神经元和幼虫斑马鱼腹腔运动根部的细胞外记录。自发性活动以前在斑马鱼中具有特征,没有考虑到潜在的并发运动活动1、2、39、40、41。</sup…
The authors have nothing to disclose.
我们感谢国家卫生研究院(DC010809)、国家科学基金会(IOS1257150,1856237)和惠特尼海洋生物科学实验室对J.C.L的支持。我们要感谢廖实验室过去和现在的成员鼓励讨论。
100 mL beaker | PYREX | 1000 | resceptacle for etchant |
10x water immersion objective | Olympus | UMPLFLN10xW | low magnification for positioning larvae and recording electrode |
40x water immersion objective | Olympus | LUMPLFLN40XW | higher magnification for position electrode tip and establishing patch-clamp |
abfload.m | supplemental coding file | custom written MATLAB script for converting raw electrophysiology recordings to .mat files | |
AffVR_preprocess.m | supplemental coding file | custom written MATLAB script for preprocessing recording data | |
BNC coaxial cables | ThorLabs | 2249-C-12 | connecting amplifier and digitizer channels; require 4 |
borosilicate glass capillaries w/ filament | Warner Instruments | G150F-3 | inner diameter: 0.86, outer diameter: 1.50; capillary glass used to form recording electrodes |
burst_detect | supplemental coding file | custom written MATLAB function necessary to run AffVR_preprocess.m | |
computer | N/A | N/A | any computer should work |
DC Power Supply | Tenma | 72-420 | used for electrically etching dissection pins |
electrophysiology digitizer | Axon Instruments, Molecular Devices | Axon DigiData 1440A | enables acquisition of patch-clamp data |
filament | Sutter Instrument Company | FB255B | 2.5 mm box filament used in micropipette puller |
fine forceps | Fine Science Tools | Dumont #5 (0.05 x 0.02 mm) Item No. 11295-10 | used to manipulate larvae and insert pins |
fixed stage DIC microscope | Olympus | BX51WI | microscope used to visualize and establish patch-clamp recordings |
flexible, tapered pipette tip | Fisher Scientific | 02-707-169 | flexible tips enable insertion into recording electrode to dispense extracellular solution at the tip |
FluoroDish | World Precision Instruments Inc. | FD3510-100 | cover glass bottomed dish recording dish |
KimWipe | KimTech | 34155 | task wipe used for wicking away excess fluid from larvae |
Kwik-Gard | World Precision Instruments Inc. | 710172 | self-mixing sylgard elastomer |
MATLAB | MathWorks | R2020b | command line software for preprocessing data |
microelectrode amplifier | Axon Instruments, Molecular Devices | MultiClamp 700B | patch clamp amplifier for dual channel recordings |
microforge | Narishige | MF-830 microforge | to polish recording electrode |
micromanipulator control unit | Siskiyou | MC1000-eR/T | 4-axis dial coordinator for controlling micromanipulator |
micropipette puller | Sutter Instrument Company | Flaming/Brown P-97 | for pulling capillary glass into recording electrodes |
microscope control unit | Siskiyou | MC1000e | positions the microscope around the fixed stage and preparation |
motorized micromanipulator | Siskiyou | MX7600 | positions the headstage and attached recording electrode for patch-clamp recording |
MultiClamp Commander | Molecular Devices | 2.2.2 | downloadable from Axon MultiClamp 700B Commander download page |
optical air table | Newport Corporation | VH3036W-OPT | breadboard isolation table to float microscope and minimize vibrations during recordings |
pCLAMP | Molecular Devices | 10.7.0 | downloadable from Axon pCLAMP 10 Electrophysiology Data Acquisition & Analysis Software Download page |
permanent ink marker | Sharpie | order from amazon.com | for marking the leading edge side of the VR electrode to ensure proper orientation when inserting into pipette holder |
petri-dish | Falcon | 35-3001 | used to immerse larvae in paralytic |
pipette holder | Molecular Devices | 1-HL-U | hold recording electrode and connect to the headstage |
pneumatic transducer | Fluke Biomedical Instruments | DPM1B | for controlling recording electrode internal pressure |
potassium hydroxide | Sigma-Aldrich | 221473-25G | etchant for etching dissection pins |
silicone tubing | Tygon | 14-169-1A | tubing to connect pneumatic transducer to pipette holder |
spike_detect | supplemental coding file | custom written MATLAB function necessary to run AffVR_preprocess.m | |
stereomicroscope | Carl Zeiss | Stemi 2000-C | used to visualize pin tips and during preparation of larvae |
straight edge razor blade | Canopus | order from amazon.com | cuts the tungsten wire while making dissection pins |
swimbout_detect | supplemental coding file | custom written MATLAB function necessary to run AffVR_preprocess.m | |
syringe | Becton Dickinson Compoany | 309602 | filled with extracellular solution to inject into recording electrodes |
transfer pipette | Sigma-Aldrich | Z135003-500EA | single use, non-sterile pipette for transfering larvae |
tricaine methanesulfonate | Syndel | 12854 | pharmaceutical aneasthetic used to euthanize larvae with high dosage. |
tungsten wire | World Precision Instruments Inc. | 715500 | 0.002 inch, 50.8 μm diameter; used to make dissection pins |
vacuum filtration unit | Sigma-Aldrich | SCGVU11RE | single use, sterile, vacuum filtration units used to sterilize extracellular solution used for electrophysiology electrode ringer |
voltage-clamp current-clamp headstage | Molecular Devices | CV-7B | supplied with MultiClamp 700B amplifier used as left and right headstages |
α-bungarotoxin | ThermoFisher | B1601 | for immobilizing the larvae prior to recording |