Investigations Of Neuronal Activity Of C.elegans With Microfluidic Chips

Caeborhabditis elegans (C. elegans) is a popular model organism in developmental biology, genetics and genomics research. With a simple neuronal system consist of 302 neurons, it became an ideal model animal to research the connection of behavior and neuronal activity. However, it was difficult to immobilize the tiny and free-moving body for anatomy observation, microsurgery, neuronal fluorescence imaging, or stimulation. In conventional methods, C. elegans were usually glued on an agar pad, anaesthetized by drugs, or partially snapped by a micropipette. Although the methods have been widely adopted, it is relatively labor-intensive and time-consuming, and the side effect is unclear. Immobilization of living C. elegans with a high-throuput and without damage for in vivo imaging of neurons in high-resolution becomes a technical bottleneck.Recently, microfluidic technology has drawn increasing attentions of researchers in studying neuronal and behavioral activities of C. elegans, due to advantages of ease of fabrication, good transparency, flexible fluid control and size compatibility. Two-layer PDMS membrane valve combined with sucktion side channels can immobilize the whole animals'bodies for laser nanosurgery and multi-photo imaging. However, it is just suitable for the immobilization of whole body, and cannot expose its head. Partial stimulation is important in studying responses of a single neuron when C. elegans faces to outside environment, because it simplifies the complexity of neuronal network. Researchers ever tried to trap C. elegans in a low and taper-shaped channel to match the shape and size of its head, and kept neurons in the head staying in the focus during imaging, but cannot unload the animals conveniently.Here, we demonstrated a microfluidic system integrating a comb-shaped microfluidic valve controlled by gas input for improved immobilization of young adult C. elegans in the underlayer. The advantages of five force points were not only in the better effect of immobilization and higher success rate. More importantly, it could expose head outside of the valve, thus was suitable for introducing chemical stimulation to the head. In addition, because this valve can match the rectangular channels under it, it can be widely used in many experiment based on immobilization living C. elegans, such as high-throughput screening of strains, drugs, laser nanosurgery, microinjection, etc. For the evaluation of immobilization effect, movement of the C. elegans in the z-plane could be minimized, comparing to the glue method, and it can keep high activity after long term immobilization, such as the body wave frequency, egg-laying, life-span. Thus, our PDMS comb-shaped valve is an efficient and high-throughput method without damage to C. elegans.Based on the well immobilization method, we built an automated loading platform for the subsequent fluorescence imaging experiments. To study the responses of C. elegans in the liquid environment, chemical stimuli were then delivered to the head of the animal by interface shifting of laminar flows. With a single-drug delivery system, the developed method was evaluated for neuronal analysis, resulting in observation of the neuronal responses of the ASH neurons and their adaptations to high concentration glycerol. Using a multi-drug delivery system, we further demonstrated rapid differentiation between mutant and wild type animals. This chip can be widely used in high-throughput screnning of mutantion or drugs, connection of the neuronal network, etc. To study the responses of C. elegans in the vapor environment, we output controllable gas plugs in laminar manner to its head, resulting in observation of the neuronal responses of the URX/BAG to the oxygen in different concentration and the ASH to the gas octanol stuimulation in a low concentration. This method opened a new avenue of gas stimulation.We also built simple behavior models by microfluidic chips, and studied C. elegans sensing of odors or light, electrotaxis, thermotaxi, and mechanical stimulation, for understanding the neuronal network's function in the behavior level.