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

Justin Tong; Pouya Rezai; Sangeena Salam; P. Ravi Selvaganapathy; Bhagwati P. Gupta

doi:10.3791/50226

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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 10 min, then ramp the temperature up to 95 °C over 2 min. Maintain this setting for an additional 1 hr.
Align a photomask containing the desired channel design. Expose the resist to 550-600 mJ/cm² of UV light (350-400 nm). Photomasks can be designed in AutoCAD and printed on a transparency with high resolution printing.
Post-bake the wafer on a hot plate at 65 °C for 1 min and 95 °C for 10 min, ramping the temperature as before.
Immerse the wafer in SU-8 developer solution for 10-15 min. Check for completion of development by rinsing with isopropanol. If a white precipitate appears, continue developing. The master mold is shown in Figure 2A.

2. Soft Lithography for Microchannel Fabrication

Mix 35 ml polydimethylsiloxane (PDMS) elastomer base with 3.5 ml PDMS curing agent.
Place the fabricated master mold (pattern facing up) and a blank silicon wafer into Petri dishes lined with aluminum foil.
Pour 20 ml PDMS prepolymer into the master mold dish and 15 ml into the second dish. Eliminate air pockets underneath the wafers by gently pressing on them with a disposable wooden applicator.
Cover both dishes and set aside for a day to cure. Alternatively, for faster curing, remove air bubbles from the PDMS using a vacuum degasifier and then leave the dishes on a hot plate at 80 °C for 2 hr.
Remove the foil and peel the PDMS from the wafers.
Use the Harris Uni-Core (2.5 mm) to punch fluid access ports at both ends of the channel. Cut the channel and blank PDMS into similarly sized strips.
Load the channel, the blank PDMS strip and a glass slide (75×25 mm²) into a plasma oxidizer, likely located in a cleanroom. Expose to oxygen plasma for 40 sec at 40 W power.
Stick the channel piece and glass slide to opposite sides of the blank strip. Set aside for 2 hr to complete the bonding.
Place the assembly onto a hot plate at 120 °C. Attach plastic tubing (inner diameter 1/32", outer diameter 3/32"), each at least 6 in. long, to the punched reservoirs using PDMS prepolymer. Affix a fluidic plastic connector to one or both tubes to allow syringe attachment, or use commercially available fittings.
Allow the PDMS securing the tubing to cure. Insert 3" lengths of 22 gauge insulated copper wire into each reservoir, between the inlet tube and the channel, and secure with PDMS prepolymer. The finished product is shown in Figure 2B.

3. Electrotaxis Experiment

Place the microchannel on the stage (preferably XY-movable) of a microscope with a mounted camera connected to a monitor (Figure 1).
Connect the power supply or amplifier's output wires to the microchannel's electrodes. A simple DC power supply is sufficient if only a DC signal is desired, but an amplifier connected to a function generator allows application of pulsed DC and AC signals as well.
Attach the microchannel's output tube to a disposable syringe. Submerge the mouth of the inlet tube in M9 physiological buffer and gently aspire liquid into the channel by applying a negative pressure inside the syringe (either manually or using a syringe pump). When the inlet and outlet tubes are both filled with M9, disconnect the syringe from the tube. Level both tubes to the same height to prevent hydrostatically driven flow.
Apply a DC voltage to the channel and ensure that resistance (R= V/I) is around 0.6 MΩ (for a 50 mm long, 0.3 mm wide and ~0.1 mm deep microchannel).
If satisfied with the channel's integrity, follow the above steps to load worms from a diluted suspension into the channel.
Disconnect the syringe and hydrostatically manipulate the flow by adjusting the tubes' relative height. Use this method to place a worm in the center of the channel and then lay both tubes flat at the same elevation.
Set the power supply to the appropriate voltage: 4-12 V/cm for L3 stage animals, 4-10 V/cm for L4s, and 2-4 V/cm for young adults. Activate the electric signal and allow 1 min of pre-exposure for the worm to acclimatize to the field. The worm should begin moving towards the cathode. When the minute has passed, use the camera to begin recording.
For AC and pulsed DC experiments, the maximum responsive electric field can be adopted from above and frequency and duty cycle of the signal can be modulated as desired ^{7, 8}.
When experiment is finished, remove all liquid (and worms) from the channel, rinse it with dH₂0, and leave the device on a hot plate at 125 °C to dry.
Extract locomotory data from recorded videos manually using NIH ImageJ (http://rsbweb.nih.gov/ij/) or custom MATLAB-based worm tracking software.