Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms

摘要

We present a method for the flexible chemical and multimodal stimulation and recording of simultaneous neural activity from many Caenorhabditis elegans worms. This method uses microfluidics, open-source hardware and software, and supervised automated data analysis to enable the measurement of neuronal phenomena such as adaptation, temporal inhibition, and stimulus crosstalk.

摘要

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons to entire brain circuits. However, neural responses may vary due to prior experience, internal states, or stochastic factors, thus generating the need for methods that can assess neural function across many individuals at once. Whereas most recording techniques examine a single animal at a time, we describe the use of wide-field microscopy to scale up neuronal recordings to dozens of Caenorhabditis elegans or other sub-millimeter-scale organisms at once. Open-source hardware and software allow great flexibility in programming fully automated experiments that control the intensity and timing of various stimulus types, including chemical, optical, mechanical, thermal, and electromagnetic stimuli. In particular, microfluidic flow devices provide precise, repeatable, and quantitative control of chemosensory stimuli with sub-second time resolution. The NeuroTracker semi-automated data analysis pipeline then extracts individual and population-wide neural responses to uncover functional changes in neural excitability and dynamics. This paper presents examples of measuring neuronal adaptation, temporal inhibition, and stimulus crosstalk. These techniques increase the precision and repeatability of stimulation, allow the exploration of population variability, and are generalizable to other dynamic fluorescent signals in small biosystems from cells and organoids to whole organisms and plants.

引言

Calcium imaging techniques have allowed the noninvasive recording of in vivo neural dynamics in real time using fluorescence microscopy and genetically encoded calcium indicators expressed in target cells^1,2,3. These sensors typically use a green fluorescent protein (GFP), such as the GFP-calmodulin-M13 peptide (GCaMP) family, to increase the fluorescence intensity upon neuronal activation and elevated intracellular calcium levels. Calcium imaging has been especially powerful in the nematode C. elegans for examining how neurons and neural circuits function i....

方案

1. Neural imaging equipment

NOTE: See Lawler and Albrecht¹⁵ for detailed instructions on building the imaging and stimulation system, which controls the microscope illumination timing, image acquisition, and stimulus delivery (Figure 1). An inexpensive Arduino Nano stimulus controller actuates the fluidic valves through digital signals to a valve controller and controls the optogenetic illumination through analog voltage signals to an LED controller. Other stimuli, such as vibration motors and thermal heaters, can be controlled using digital or analog signals. The stimulus....

结果

We present several examples of stimulus patterns that assess different neural phenomena, including temporal inhibition, adaptation, and disinhibition. Temporal inhibition is the momentary suppression of a neural response to a second stimulus presentation occurring shortly after the initial presentation¹⁴. To test this phenomenon, in a paired-pulse experiment, eight patterns consisting of two 1 s odorant pulses separated by an interval ranging from 0 s to 20 s were presented (

讨论

In this protocol, we describe an open-access microscopy system for the assessment of neural activity phenomena using the temporally precise delivery of different stimulus patterns. The microfluidic platform delivers repeatable stimuli while keeping tens of animals in the microscope field of view. Few commercial microscopy software packages allow for the easy programming of various stimulus timing patterns, and those that do often require the manual entry of each pattern or proprietary file formats. In contrast, experimen.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
<强>细菌菌株
E. 大肠杆菌 （OP50）	隐球菌炎 遗传学中心 （CGC）	Cat# OP50
实验模型：生物体/菌株
C. 在所需神经元中表达 GCaMP（以及可选的 Chrimson）的秀丽隐杆线虫<	<> Caenorhabditis /em>遗传学中心 （CGC） 或已发表工作	NZ1091
化学品、治疗和蠕虫制备用品
2,3-丁二酮	Sigma-Aldrich	Cat# B85307	二乙酰，示例化学刺激
氯化钙，CaCl2	Sigma-Aldrich	Cat# C3881
荧光素，钠盐	Sigma-Aldrich	Cat# F6377
玻璃防水剂	Rain-X	Cat #800002250	玻璃疏水处理（一次性使用）
氯化镁，MgCl2	Sigma-Aldrich	Cat# M2393
线虫生长培养基 （NGM） 琼脂	Genesee	Cat #： 20-273NGM
培养皿 （60 mm）	Tritech	Cat #T3305
聚（二甲基硅氧烷）（PDMS）：Sylgard 184	Dow Chemical	Cat# 1673921
磷酸二氢钾	Sigma-Aldrich	Cat# P5655
磷酸氢钾	Sigma-Aldrich	Cat# P8281
氯化钠，NaCl	Sigma-Aldrich	Cat# S7653
（十三氟-1,1,2,2-四氢辛基）三氯氢硅 （TFOCS）	Gelest	CAS# 78560-45-9	玻璃疏水处理（耐用）
软件和算法
Arduino IDE	Arduino	https://www.arduino.cc/en/software
ImageJ	NIH	https://imagej.nih.gov/ij/
MATLAB	MathWorks	https://www.mathworks.com/products/matlab.html
Micro-manager Micro-manager		https://micro-manager.org/
显微镜控制软件	Albrecht Lab	https://github.com/albrechtLab/MicroscopeControl
Neurotracker 数据分析软件	Albrecht Lab	https://github.com/albrechtLab/Neurotracker
自动显微镜和刺激系统
Axio Observer.A1 倒置显微镜设置用于落射荧光（GFP 滤光片立方体，5&次物镜或类似物镜）	蔡司	猫 #491237-0012-000
Excelitas X-cite XYLIS LED 照明器	Excelitas	Cat #XYLIS
Orca Flash 4.0 数码 sCMOS 相机	滨松	猫#C11440-22CU
Arduino nano	Arduino	Cat #A000005
3 通微型隔膜隔离阀 （LQX12）	派克	Cat #LQX12-3W24FF48-000	阀门 1：控制
2 通常闭 （NC） 夹管阀	Bio-Chem Valve Inc	Cat #075P2-S432	阀门 2：流出
3 通夹管阀	NResearch	Cat #161P091	阀 3：刺激选择
光遗传学刺激 LED 和控制器 （615 nm）	Mightex	Cat #PLS-0625-030-S 和 #SLA-1200-2
ValveLink 8.2 数字/手动阀门控制器	AutoMate Scientific	Cat #01-18
各种电线和连接器		参见 Cell STARS 协议（Lawler，2021
微流体装置制备
Dremel 变速旋转切割机 4000	Dremel	Cat #F0134000AB	设定速度为5k RPM切割玻璃
Dremel 钻床旋转工具工作站	Dremel	Cat #220-01
钻石钻头	Dremel	Cat #7134
玻璃滑轨，1 mm 厚	VWR	Cat #75799-268
玻璃划线器（Diamond 划线器）	Ted Pella	Cat #54468
鲁尔三通旋塞	Cole-Parmer	Cat #EW-30600-07
鲁尔 23 G 钝针	VWR	Cat #89134-100
微流体装置	与本文相关的 CAD 文件更正作者或制造	N/A
微流体装置夹具	华纳仪器（或机械车间）	P-2
微流体管，0.02″ID	Cole-Parmer	猫 #EW-06419-01
管 19 克，0.5″	新英格兰小管	猫 #NE-1027-12