An in vivo assay to study locomotion in Caenorhabditis elegans

Abstract

Adaptation in the sensory-mechanical loop during locomotion is a powerful mechanism that allows organisms to survive in different conditions and environments. Motile animals need to alter motion patterns in different environments. For example, crocodiles and other animals can walk on solid ground but switch to swimming in water beds. The nematode Caenorhabditis elegans also shows adaptability by employing thrashing behaviour in low viscosity media and crawling in high viscosity media. The mechanism that enables this adaptability is an active area of research. It has been attributed previously to neuro-modulation by dopamine and serotonin. This study introduces an experimental assay to physiologically investigate the neuronal mechanisms of modulation of locomotion by dopamine. The technique is utilized to test gait switching while imaging the mechanosensory dopaminergic neurons PDE. Results revealed their role to be not limited to touch sensation, but to sensing surrounding environment resistance as well. The significance of such characterization is improving our understanding of dopamine gait switching which gets impaired in Parkinson's disease.-A locomotion pattern switching system was devised to allow studying this process in vivo in the nematode C. elegans.-This system allowed the study of dopaminergic neurons PDE response as the worms switched from crawling to swimming.

Keywords: Behavioral assay; Locomotion; Neuromodulation.

Graphical abstract

Fig. 1

A description of the steps of the viscosity separation experimental protocol where the worm is initially confined in a small droplet of 65% dextran (a) and then dextran 30% is added with a micropipette (zero time point) (b). The worm then starts to move out of the small droplet (c) until10 the whole body gets to the lower viscosity and swimming is maintained (d).

Fig. 2

PDE neurons and frequency of undulation response to crossing viscosity separation for three sample worms. Solid lines represent neuronal fluorescence signals of PDE neurons of the worms that cross the separation while dashed lines represent the control signal with mid-body auto-fluorescence signal for the same three worms. Matching colors denote the same worm sample. Dotted lines represent three control worms that remain at high viscosity. Since control worm samples are different so they are not color coded. (b) Undulation frequency responses of the same three worms in (a) as the worms switch from crawling to swimming while crossing the viscosity separation. The frequency was measured every 10 s and then linear interpolation was done to get the smooth curve. The vertical dashed line denotes the zero time point.

Fig. 3

Mean fluorescence signal of PDE neurons and corresponding behavioural response to the worm crossing viscosity separation: (a) Average PDE response to crossing the separation where the zero point is the point of addition of the droplet of liquid (n = 10). The control represents worms that remain at high viscosity (n = 10). Same-worm control is the measurement of auto-fluorescence signal from the mid-body in order to ensure that the measured decrease in activation is not due to loss of focus. (b) Average frequency response to crossing the separation of the worms in (a). Error bars correspond to ±2 SE.

Fig. 4

Comparison of mean of data from 10 s before the zero time point and from 60 to 70 s after zero time point where the worm in each case is in a completely homogeneous viscosity. (a) Mean activation of PDE neurons in high and low viscosities shows a significant difference. (b) The mean frequency of undulation also shows two different frequencies that are characteristic of both crawling in the high viscosity case and swimming in the low viscosity case. (c) The mean difference in fluorescence signal of the PDE neurons in the worm crossing separation in comparison to the mid-body auto-fluorescence signal and to the PDE neurons signal from the control worms that remain in the high viscosity medium. It shows that the difference in the observed decrease in activation is a result of the physiological change associated with sensing the environment and not photobleaching or loss of focus.