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Figure 1. Outline of the arrangement of microscope to efficiently record/capture C.elegans locomotory behaviour.
a. Burette stand and clamps. b. Dino Microscope attached to the stand placed on the bottom of burette stand. c. A manual XY stage framed on a custom-made stand. d. LED (7W) bulb fixed on the top of the burette stand with a clamp. e. White and red colour acrylic sheets positioned under the illumination source with clamps to diffuse light. f. Image of a single worm. g. Image of multiple worms from the video captured in monochrome set up. h. Reproduced result of wild type N2 shows slowing behaviour in the presence of food compared with absence of food, modulated by dopaminergic neurons.
Caenorhabditis elegans is an excellent model to study animal chemotaxis behaviour. These nematodes have highly predictable behaviour pattern towards olfactory cues both attractants as well as repellent (Ward., 1973; Colbert et al., 1995; Troemel et al., 1997). Moreover, for salt chemotaxis worms show unsurpassed behaviour with a pattern of movement based on concentration gradient in the assay plate (Saeki et al., 2001; Iino et al., 2009). Such behavioural patterns are highly intriguing because they give better understanding on how various neuronal signalling elicit such pattern of behaviour and how factors such as past experience of the animal, mutations affecting neuronal activity and connectome cross talks modify them. (Brenner., 1974; de Bono et al., 1998). Hence, behavioural assays have critical role in elucidating the alterations in neuronal activities in C. elegans.
There is a series of attempts have been done to develop a set up for recording animal behaviour (Buckingham et al., 2005; Yemini et al., 2011). Automated single worm tracker allows long term behavioural recording (Husson et al., 2005; Wang et al., 2013). However often one need to record body bends and omega turns to assay behavioural alterations under experimental conditions. Here we report a simple setup to manually record and count these behavioural changes in worms. Efficient recording often eliminates researcher’s bias and makes it easy to re-evaluate the results if needed (Piere-Shimoura et al., 1999; Hardaker LA et al., 2001; Baek et al., 2002). In this study we measured basal slowing response and enhanced slowing responses, the two different locomotory changes in response to food. Neuronal circuitry underlying these locomotory changes were identified as dopamine and serotonin respectively by direct analysis of body bends of mutants (Sawin et al., 2000). In addition, these particular assays are useful for validating the involvement of neurotransmitters in other behavioural changes.

Detailed protocol.
The skeleton of this system is a burette stand and clamps to hold various parts (Fig.1a). For video capturing we used Dino-Lite digital microscope (Model no AM4113-FVT) with USB connector and its software module Dinocapure2.0 (Fig.1b) to record the data. Dino-Lite digital microscope was fixed on a table top stand (Dino-Lite RK-10) enables smooth focus adjustment and quick vertical movements (Fig.1b). An X-Y stage was placed over on a custom-made metal frame (Fig.1c). Petri dish containing the worms were placed on a transparent acrylic sheet placed on this XY stage (if the stage has a universal holder, petri dish can be directly held on the stage).
As illumination source, 7 W led white light was used. To enhance the image contrast as well as uniform illumination we used white and a red acrylic sheets ((Fig 1 e). The gap between LED light source and the white acrylic sheet was adjusted between 4-7 cm; adjusted based on the required intensity of light to visualize the animals clearly. The distance between white and red acrylic sheets was 7-15 cm (Fig.1e). Parallel alignment of both these sheets found to be crucial for the set up.
The USB microscope was connected to a computer though USB cable. All recording of the video was done using the Dino-Lite software module. Using this system, we tested the animal behaviour in the presence and absence of food and could establish significant variation in body bends (Fig.1h).
Dino-Lite microscope (Model AM4113-FVT ), Dinocapture2.0 software (available online), Dino microscope stand (RK-10), Burette stand and clamps, Acrylic sheets (transparent, red and white), Metal stand (Steel made; Length 30cm X Breadth 15cm X Height 30cm), Manual stage-Olympus; Model No: IX2-KSP-9C13573, Philips 7W LED bulb, Computer with 2GB RAM, N2 Bristol (strain obtained from CGC, Minnesota, USA), Petri dishes, NG Media.

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This research was funded by Sree Chitra Tirunal Institute for Medical Sciences and Technology.

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