Stimulation of C. Elegans mechanoreceptor neurons using microfluidics

Figure 1 : Picture of a trapped C. Elegans worm in the microfluidic chip of Adam L. Nekimken et al. Arrowheads point to the location of the touch receptor neurons (TRNs). Scale bar = 100 μm.

This article covers the work of Adam L. Nekimken et al. published in Lab Chip in January 2017.

The research team employed pressure-driven flow controlled microfluidics to introduce a novel microfluidic device that enables high-resolution imaging of cellular deformations in response to precise mechanical stimuli to the surface of the C. Elegans worm cuticle.

Mechanical signals during touch and pain provide animals with crucial information about their environment.

The C. Elegans transmission process from mechanical stimuli of the cuticle to physiological signals has been difficult to investigate. This process uses the touch receptor neurons (TRNs) that tile the body of the worm in anterior and posterior tactile receptive fields (TRF).

Besides, although previous studies shown explanations on TRN physiology, experiments didn’t perform efficient high-resolution imaging and weren’t able to recover the worm for subsequent long-term studies.

OB1 Mk3+ pressure-driven flow controller

PDMS microfluidic chip

Reservoirs

Tubings & Fittings

Figure 2 : Channel design overview.
A: Overall device design with inlet, outlet, and air reservoirs. Scale bar = 1 mm.
B: Enlarged view of the pillars for worm orientation, the tapered trap, and the air channels.
C: Enlarged view of two of the six independent pneumatic actuators with their diaphragms and the tip of the tapered trap, which is 24 μm in width at its smallest point.
D: Representative micrograph of a trapped worm; arrowheads point to the location of the TRN cell bodies. The mechanosensitive neurites extend anteriorly from the cell bodies toward the animal’s head. Scale bar = 100 μm.

The microfluidic device of Adam L. Nekimken et al. consists of two integrated modules: an immobilization channel and six pneumatic stimulation channels connected to pressure reservoirs. When a worm’s head is near the trapping channel, it is moved into the immobilization channel by applying pressure (Elveflow’s pressure pump). The tapered end of the immobilization channel is 24 μm wide and restricts the animal from further forward movement, holding it in place for imaging. At this exit of this channel, a gravity flow creates suction to keep the animal snug.

In this position, the animal is located adjacent to three actuators on each side of the trap (six total). These actuators are thin PDMS membranes that deflect into the animal’s body when pressurized from behind. They are positioned such that they are within the putative TRF along the process of each neuron.

To perform the calcium-sensitive fluorescence imaging, Adam L. Nekimken et al. produced transgenic worms expressing the genetically encoded calcium indicator GCaMP6s exclusively in TRNs (TRN::GCaMP6s). In order to have a negative control, they used mec-4(u253) mutants, which lack the pore-forming subunit of the mechanosensitive ion channel. To identify the stimulus type for TRN activation, they stimulated ALM, AVM and PVM (anterior lateral, anterior ventral, posterior ventral) using one of the six actuators which was located closest to their cell bodies within their presumptive TRFs.

Results for microfluidics Stimulation of C. Elegans mechanoreceptor neurons

Finally, Adam L. Nekimken et al. introduced a device that facilitates streamlined and high-resolution imaging of the mechanosensory response in combination with mechanical stimulation in C. Elegans. Using Elveflow’s precise pressure pump, they identified TRN activation with a calcium-sensitive fluorescence indicator and confirmed that this process is frequency dependent in vivo (“buzz” stimulus).

Additional actuators beyond the six can be implemented to improve the device’s ability to stimulate the animal at various locations. Also, future studies will have to investigate how TRN activation differs in mutants that are not easily scored in classical behavioral assays.

These results empowered investigations of the physiological and molecular basis of touch sensation.