Thermotaxis in Caenorhabditis elegans analyzed by measuring responses to defined Thermal stimuli

Abstract

In a spatial thermal gradient, Caenorhabditis elegans migrates toward and then isothermally tracks near its cultivation temperature. A current model for thermotactic behavior involves a thermophilic drive (involving the neurons AFD and AIY) and cryophilic drive (involving the neuron AIZ) that balance at the cultivation temperature. Here, we analyze the movements of individual worms responding to defined thermal gradients. We found evidence for a mechanism for migration down thermal gradients that is active at temperatures above the cultivation temperature, and a mechanism for isothermal tracking that is active near the cultivation temperature. However, we found no evidence for a mechanism for migration up thermal gradients at temperatures below the cultivation temperature that might have supported the model of opposing drives. The mechanisms for migration down gradients and isothermal tracking control the worm's movements in different manners. Migration down gradients works by shortening (lengthening) the duration of forward movement in response to positive (negative) temperature changes. Isothermal tracking works by orienting persistent forward movement to offset temperature changes. We believe preference for the cultivation temperature is not at the balance between two drives. Instead, the worm activates the mechanism for isothermal tracking near the cultivation temperature and inactivates the mechanism for migration down gradients near or below the cultivation temperature. Inactivation of the mechanism for migration down gradients near or below the cultivation temperature requires the neurons AFD and AIY.

Fig. 1.

Run termination in crawling and swimming worms. Crawling worms terminate runs by turns (a) or by pirouettes (b), which involve reversals and coiling. c, Swimming worms terminate runs by abrupt bends larger than those of the rhythmic bends of forward swimming.

Fig. 2.

A sample histogram of run durations of N2 worms crawling in an isotropic environment (21°C). The data shown correspond to ∼150 runs of worms raised at 22°C; the mean of this distribution corresponds to an entry in Table 1. The distribution is fit by a single exponential line: run termination might be a first-order kinetic process obeying Poisson statistics. The histogram was binned with 10 sec width.

Fig. 3.

Times of occurrence of run termination events of N2 worms cultivated at 20°C swimming in droplets. Experiments at temperatures below T_c are at theleft, and experiments at temperatures aboveT_c are at the right. Experiments at fixed temperatures are at the top (19°C for T < T_c; 24°C for T >T_c). Experiments subjecting the drops to positive temporal ramps of +4°C/min are in themiddle (17–19°C for T <T_c; 22–24°C forT > T_c). Experiments subjecting the drops to negative temporal ramps of −4°C/min are at the bottom (19–17°C forT < T_c; 24–22°C for T >T_c). Within a panel, individual experimental trials are aligned along the vertical axis. Run termination events during a trial are indicated by closed squares; their horizontal positions indicate their times of occurrence from the beginning of each trial. (In the case of the ramp experiments, trials begin at the onset of the ramp.) All trials lasted 30 sec. For T <T_c, the rate of run termination was 0.26 ± 0.04 and 0.33 ± 0.04 Hz for positive and negative ramps, respectively; for T >T_c, the rate of run termination was 0.56 ± 0.03 and 0.08 ± 0.02 Hz for positive and negative ramps, respectively. The rate of run termination at fixed temperatures was 0.33 ± 0.08 and 0.22 ± 0.03 Hz for 19°C and 24°C, respectively. Values are means ± SEM.

Fig. 4.

a, Initial and final run orientations interrupted by single abrupt reorientations (n = 413). Here, N2 worms cultivated at 22°C navigated a linear spatial gradient between 24 and 26°C of steepness 0.4°C/cm. The direction down the gradient was 0 radians. Final run orientations were not more clustered at 0 radians than initial run orientations, as would be the case if the angle of abrupt reorientation was not random and angle selection was weighted toward the favorable direction of movement. Along both axes there is a greater frequency of events between −π/2 and +π/2 radians, but this is an artifact: only abrupt reorientations in which both initial and final runs were longer than approximately three body lengths were used. This criterion enabled accurate measurement of run orientation, but on such steep gradients also reduced events corresponding to run orientations less than −π/2 or greater than +π/2. However, the relevant feature of this plot that survives this artifact is that the dispersion around 0 radians, the desired direction, is apparently the same for the final as for the initial run orientations. A numerical analysis of this data set shows that the rms deviations from 0 radians for the initial and final run orientations are 1.4 radians and 1.5 radians, respectively: worms are not better oriented toward 0 radians after abrupt reorientations. Similar results were obtained for worms cultivated at 22°C and navigating gradients between 17 and 19°C (data not shown).b, Traces of runs of duration of ∼20 sec. Here, N2 worms cultivated at 22°C navigated a linear spatial gradient between 24 and 26°C of steepness 0.4°C/cm, a steepness at which worms robustly migrated down gradients but did not always terminate runs within a few seconds when moving up gradients. Forpresentation purposes, we have centered all runs as emerging from a single point. The arrow indicates the direction down the gradient. More tracks are to theright (n = 27) than to theleft (n = 15), but the essential data are the shape and not the number of the tracks. The number of tracks to the right is larger because in any trial there were more runs of 20 sec duration down the gradient than up the gradient attributable to the worm's strategy of shortening or lengthening runs up or down the gradient. Based on the shape of the individual tracks, we conclude that worms did not steer individual runs toward the direction of the gradient. Some runs were curved, but the curvature was randomly CW or CCW and was not correlated with the gradient. Similar results were obtained for worms cultivated at 22°C and navigating gradients between 17 and 19°C (data not shown).

Fig. 5.

a, A single isothermal track of an N2 worm cultivated at 22°C on a linear thermal gradient of steepness 0.4°C/cm. b, Isothermal tracks were narrower as the steepness of thermal gradients increased. One metric of the width of isothermal tracks is 〈y²〉^1/2, the mean square deviation from the midline of the track. Here, excursion amplitude is defined as the mean 〈y²〉^1/2 of ∼50 isothermal tracks of N2 worms cultivated at 22°C on three different gradients (0.4, 0.75, and 2°C/cm). Note that the metric of 〈y²〉^1/2 (i.e., 1 SD) is not the overall side-to-side width of a track. A better estimate is 4〈y²〉^1/2, which corresponds to the width in which the worm spends 95% of its time during a track. The corresponding temperature boundaries of isothermal tracks (the product of gradient steepness and 4〈y²〉^1/2) is 0.1 ± 0.01°C. c, In steeper gradients, the frequency of the side-to-side movement increased. Here, we show the normalized power spectrums of the isothermal tracks described in Figure4b.

Fig. 6.

a, N2 worms cultivated at 22°C tracked isotherms in a range within 2°C of that cultivation temperature. The histogram shows the positions of the isotherms of ∼40 worms drawn from a single plate tracked on a linear thermal gradient of steepness 0.4°C/cm and binned at a width of 0.4°C.b, Individual worms often stopped tracking one isotherm and resumed tracking a different one. This trace is an unusually clear example: the worm initially tracked at 21°C, fell off the isotherm, and continued to track at 22°C.

Fig. 7.

Trajectories of crawling ttx-1worms. Circling is either CW or CCW and is independent of ambient temperature or spatial gradients.