Stimulation of C. Elegans mechanoreceptor neurons using microfluidics

c elegans microfluidic elveflow chip

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 introduced 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.

In this context, microfluidic devices offer innovative strategy for immobilizing worms, either in a tapered channel, by compressive immobilization, or within an agar bed. Using a microfluidic chip, Adam L. Nekimken detected TRN activation with a calcium-sensitive fluorescence indicator and produced mechanical stimuli patterns thanks to the Elveflow’s OB1 pressure controller. Thus, they confirmed that C. Elegans TRN activation is dependent on a “buzz” stimulus in vivo and not on a “step” or “ramp” stimulus (Cf. Microfluidic results section).

APPLICATIONS

  • Tool performing simultaneous imaging of worm tissue and cell deformation as well as cellular activity
  • Valuable platform for investigating the mechanobiology of living animals and their mechanosensitive neurons
  • Genetic screen studies after mechanoreceptor activation

MATERIALS & METHOD

MATERIALS

OB1 Mk3+ Flow Controller

OB1-Pressure-controller-MK3-perspective-Elveflow

PDMS microfluidic chip

chip microfluidics elveflow

METHOD

C. ELEGANS MECHANORECEPTOR NEURONS microfluidics elveflow

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.

MICROFLUIDIC RESULTS

Calcium dynamics of TRNs microfluidics elveflow

Figure 3 : Calcium dynamics of TRNs as a response to a step, ramp and buzz stimulus within and outside their presumptive tactile receptive fields (TRF).

A–C: Response dynamics of ALM (A), AVM (B) and PVM (C) touch receptor neurons stimulated within and outside their TRF.
(ii) Stimulus protocol including 2 second diaphragm excitation representing a 275 kPa step, a 275 kPa ramp and a sine (75 kPa; 10 Hz) superimposed with a 275 kPa step (buzz).
(iii) Multiple false color-coded normalized fluorescence intensity traces (F/F0) during the mechanical stimulation (shown in (ii)) of TRN::GCaMP6s (control) and mec-4(u253);TRN::GCaMP6s (mec-4) mutant animals and of control animals stimulated outside their TRF.
(iv) Average fluorescence intensity (F/F0) of the traces shown in (ii) for control (mean ± SEM as green shaded area, N = 17 for ALM, N = 14 for AVM, N = 12 for PVM) and mec-4 mutant animals (mean ± SEM in blue with N = 10 animals for ALM, AVM, and PVM) when stimulated within their TRF and the average fluorescence intensity of five traces of each TRN when stimulated outside their TRF (magenta).

The application of the step or the ramp stimulus (Fig. 5A–C) gave no detectable response from the TRNs at these pressure amplitudes when stimulated within their TRF. In agreement with previous reports, a strong activation of each TRN after application of the buzz stimulus was observed, which was visible as an increase in GCaMP6s fluorescence in the cell body and the neurite.

They reported a throughput of >20 worms recorded in 3 h (data not shown). All worms survived the trapping and release procedure and laid eggs, although stimulated worms laid fewer eggs than untreated animals (38 ± 14 vs. 73 ± 12; mean ± SD; N = 10 animals).

CONCLUSION

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.

RELATED PUBLICATION

[1] Adam L. Nekimken et al. “Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap”. Lab Chip. (2017).