Temporal activity patterns in thermosensory neurons of freely moving Caenorhabditis elegans encode spatial thermal gradients

Abstract

Our understanding of the operation of neurons and neuronal circuits has come primarily from probing their activity in dissected, anesthetized, or restrained animals. However, the behaviorally relevant operation of neurons and neuronal circuits occurs within intact animals as they freely perform behavioral tasks. The small size and transparency of the nematode Caenorhabditis elegans make it an ideal system for noninvasive, optical measurements of neuronal activity. Here, we use a high signal-to-noise version of cameleon, a fluorescent calcium-binding protein, to quantify the activity of the AFD thermosensory neuron of individual worms freely navigating spatial thermal gradients. We find that AFD activity is directly coupled to the worm's exploratory movements in spatial thermal gradients. We show that the worm is able, in principle, to evaluate and guide its own thermotactic behaviors with respect to ambient spatial thermal gradients by monitoring the activity of this single thermosensory neuron.

Figure 1.

Temporal changes in temperature evoke ratiometric emission signals from yellow cameleon expressed in the AFD neuron of immobilized worms. A, In an immobilized worm expressing YC2.12 in the AFD neuron, as the temperature increases above a threshold of ∼17°C, the ratiometric emission signal from YC2.12 changes by ∼30% in a representative experiment, indicating changes in the level of intracellular calcium in the AFD neuron. B, The ratiometric emission signal from YC3.60 in the AFD neuron reports the same qualitative changes in the level of intracellular calcium, but with changes in the ratiometric emission signal of 200% in a representative experiment.

Figure 2.

AFD neuronal activity in semirestrained worms moving in spatial thermal gradients. A, The intracellular calcium dynamics of the AFD neuron were quantified in semirestrained worms. The worm tails were glued, allowing free undulating motion of the anterior part of the worm's body in imposed spatial thermal gradients. Data from a single experiment are shown, with color and spatial position indicating the time-varying ratiometric emission signal and position of the AFD soma, respectively, over several undulation cycles. To indicate scale, the gray silhouette shows the relative size and orientation of the anterior portion of the semirestrained worm at one time point during this experiment. B, In the top and bottom panels, the data from A are plotted as temperature over time and ratiometric emission signal over time, respectively. C, The ratiometric emission signal from A is plotted parametrically against head position. D, The slope of the ratiometric emission signal with respect to position was calculated for worms either expressing YC3.60 or GFP in the AFD neurons, with spatial thermal gradients pointed in opposite directions or with no gradient at all. Error bars indicate 1 SEM. n = 14–27 worms were used for each measurement.

Figure 3.

AFD neuronal activity in unrestrained worms navigating spatial thermal gradients. A, The undulating path of an unrestrained worm crawling ∼8 mm on a spatial temperature gradient in the vicinity of T*_AFD. The color and position of each data point represent the ratiometric emission signal and position of the AFD thermosensory neuron of the moving worm, respectively. The black circle indicates initial position. To indicate scale, the gray silhouette shows the approximate relative size of the worm body. B, The top and bottom panels show the time-varying temperature at the worm's head and the ratiometric emission signal from AFD for the data shown in A. C, A compilation of data showing the ratiometric emission signals for worms cultivated at 20°C navigating spatial thermal gradients at different absolute temperatures from ∼15 to ∼25°C. The ratiometric emission signal exhibits position dependence only at temperatures above ∼17°C (i.e., T > T*_AFD). A black circle indicates the initial position of each data trace.

Figure 4.

Operating range and short-term adaptation in the AFD thermosensory neuron. A, The range of all ratiometric FRET measurements exhibited by worms freely navigating spatial thermal gradients at different absolute temperatures, showing the 1st, 25th, and 99th percentiles (mean ± SEM). The FRET measurements that correspond to the lowest intracellular calcium levels, the 1st and 25th percentiles, are invariant with absolute temperature (p > 0.1 using ANOVA). The 25th percentile exhibited by each worm was assigned as an arbitrary baseline for its ratiometric emission signals (R₀) (see Materials and Methods). B, Top, Representative parametric plots of the ratiometric emission signal from the AFD neuron expressing YC3.60 and GFP against temperature for worms freely navigating spatial thermal gradients at different absolute temperatures. Bottom, The measurements of slope in the plot of ratiometric emission signal versus temperature of all data, calculated at different absolute temperatures (solid line, n = 77 worms expressing YC3.60; dotted line, n = 31 worms expressing GFP). C, Using all worm tracks at T > T*_AFD (n = 58), we calculated the linear weighting of 75 s of thermosensory history that best fit the measured ratiometric emission signal. The top trace shows the ratiometric emission signal from an immobilized worm subjected to an actual step change in temperature. The bottom trace shows the predicted ratiometric emission signal to a temperature step, based on the best-fit calculation. The gray line shows the predicted response, and the black line shows the predicted response smoothed with a 2 s filter.

Figure 5.

Representation of behaviorally relevant thermosensory information in the AFD neurons. A, Approximately 40 s of data showing the ratiometric emission signal of a worm moving slantwise on a spatial thermal gradient, with intracellular calcium transients phase-locked to rhythm of the undulatory gait. Left, The color and position of each data point represent the ratiometric emission signal and position of the AFD thermosensory neuron of the moving worm. Right, The temperature at the worm's head and the ratiometric emission signal are plotted over time. B, Ratiometric emission signals are plotted as a function of instantaneous heading with respect to the direction of the spatial thermal gradient for all worms cultivated at 20°C navigating at T > T*_AFD (n = 58; right) and at T < T*_AFD (n = 19; left). The distance from the center of each scatter plot indicates the magnitude of the measured ratiometric emission signal; the angular position represents instantaneous worm heading with respect to the direction of the spatial thermal gradient. C, The distribution of measured ratiometric emission signals for each heading angle is shown by plotting every 10th percentile of measured values, from the 10th to 90th percentiles, both above and below T*_AFD. The bottom panel shows behavioral measurements indicating the worm's reorientation probability as a function of worm heading on a spatial thermal gradient, both at temperatures above and below the previous cultivation temperature [what we call the thermotactic set point or T_S (Biron et al., 2006)]. Error bars indicate 1 SEM. D, Using all measurements of worms navigating temperatures above T*_AFD, we plot the relative likelihood that the worm has a particular heading conditioned on the measurement of a specific value of the ratiometric emission signal. Headings are indicated by gray scale (see legend to the left).