Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

Summary

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (“electrotaxis”) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

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

1. Photolithography for Master Mold Fabrication Bathe a 3 in. silicon wafer in acetone for 30 sec and then methanol for 30 sec. Rinse with dH20 water for 5 min. Dry the wafer’s surface with a N2 blow gun. Heat the wafer on a hot plate at 140 °C for 2 min. Plasma oxidize the surface of the silicon wafer (1 min, 50 W). Spin-coat the wafer’s surface with 3 ml SU-8 100 photoresist (40 sec; 1,750 rpm). Pre-bake the coated wafer on a hot plate at 65 °C for …

Representative Results

A representative video of a wild-type young adult nematode’s electrotaxis and its position and velocity outputs from the worm tracking software are shown in Supplementary Video 1 and Figure 3. The movement analysis software itself does not recognize the direction of field polarity and the time of polarity reversal; rather, this information must be obtained from the source video. This could be done using an audio or visual cue in the video or writing down experimental conditions and manip…

Discussion

Taking advantage of the behavioural phenomenon first described by Gabel and colleagues and building on the dielectrophoretic manipulation work of Chuang and colleagues ^11,12, our microfluidic-based electrotaxis assay provides an easy, robust and sensitive method to probe neuronal activity in worms using movement as an output. The analysis of movement parameters allows quantitative comparison between different genotypes. The precision of microchannel fabrication and electric field application together provide b…

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