Микрофлюидных основе электротаксис по требованию Количественный анализ<em> Caenorhabditis Элеганс</em> 'Передвижение
Summary
Полуавтоматические микро-электро-жидкостный способ индукции по требованию локомоция<em> Caenorhabditis Элеганс</em> Описан. Этот метод основан на нейрофизиологических явление червей в ответ на мягкий электрические поля ("электротаксис") внутри микроканалов. Микрофлюидных электротаксис служит быстрый, чувствительный, недорогих и масштабируемых технику для выявления факторов, влияющих на здоровье нейронов.
Abstract
The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson’s disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans‘ locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode (“electrotaxis”) 4. Reversing the field’s polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.
Protocol
Representative Results
Discussion
Воспользовавшись тем, что поведенческий феномен впервые описан Габель и коллег и здания на работу диэлектрофореза манипуляции Чжуан 11,12 и коллеги, наши микрожидкостной основе анализа электротаксис обеспечивает простой, надежный и чувствительный метод для исследования активно?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить естественным наукам и инженерным исследованиям Совета Канады, Канада Исследование программы кафедр, Канадский институт исследований в области здравоохранения, и Онтарио министерство исследований и инноваций через их раннего премии Исследователи Программы финансовой поддержки.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Acetone | CALEDON Labs | 1200-1-30 | |
| Methanol | CALEDON Labs | 6700-1-30 | |
| Isopropanol | CALEDON Labs | 8600-1-40 | |
| SU-8 | Microchem Corp. | Y131273 | SU-8 100 |
| SU-8 Developer | Microchem Corp. | Y020100 | |
| 92×16 mm Petri dish | Sarstedt | 82.1473.001 | |
| Sylgard 184 Silicone Elastomer Kit | Dow Corning | Contains elastomer base and curing agent | |
| Function generator | Tektronix Inc. | Model AFG3022B | |
| Amplifier | Trek Inc. | Model 2210-CE | |
| Syringe pump | Harvard Apparatus | 70-4506 | Model 11 ELITE |
| Hot plate | Fisher Scientific | 11675916Q | Model HP131725Q |
Riferimenti
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