The AFD sensory neurons encode multiple functions underlying thermotactic behavior in Caenorhabditis elegans

Abstract

The thermotactic behaviors of Caenorhabditis elegans indicate that its thermosensory system exhibits exquisite temperature sensitivity, long-term plasticity, and the ability to transform thermosensory input into different patterns of motor output. Here, we study the physiological role of the AFD thermosensory neurons by quantifying intracellular calcium dynamics in response to defined temperature stimuli. We demonstrate that short-term adaptation allows AFD to sense temperature changes as small as 0.05 degrees C over temperature ranges as wide as 10 degrees C. We show that a bidirectional thermosensory response (increasing temperature raises and decreasing temperature lowers the level of intracellular calcium in AFD) allows the AFD neurons to phase-lock their calcium dynamics to oscillatory thermosensory inputs. By analyzing the thermosensory response of AFD dendrites severed from their cell bodies by femtosecond laser ablation, we show that long-term plasticity is encoded as shifts in the operating range of a putative thermoreceptor(s) in the AFD sensory endings. Finally, we demonstrate that AFD activity is directly coupled to stimulation of its postsynaptic partner AIY. These observations indicate that many functions underlying thermotactic behavior are properties of one sensory neuronal type. Encoding multiple functions in individual sensory neurons may enable C. elegans to perform complex behaviors with simple neuronal circuits.

Figure 1.

Operating range of AFD Ca²⁺ dynamics. A, Worms cultivated at 20°C were subjected to sinusoidal temperature variations (gray line) while simultaneously monitoring fluorescence emission at the AFD soma from cameleon in the yellow fluorescent protein (YFP) emission channel (green line) and cyan fluorescent protein (CFP) emission channel (blue line). The FRET response (black line) is calculated as the ratio of yellow and cyan emission. At temperatures >2–3°C below T_S, the fluorescence of cameleon is unaffected by temperature changes, indicating an absence of intracellular [Ca²⁺] changes. At temperatures near or above T_S, the yellow and cyan emissions varied inversely, indicating that cameleon is responding to variations in intracellular [Ca²⁺] stimulated by the temperature waveform. B, Representative FRET signal of worms cultivated at 20°C subjected to temperature waveforms consisting of a positive linear ramp (above) and of a small amplitude sine wave added to a positive linear ramp (below). In each case, when the temperature (gray line) reached ∼17°C, calcium dynamics could be quantified by measuring changes in the ratio (black line) of the yellow and cyan emission channels (green and blue lines, respectively). In each case, the lower bound of AFD calcium dynamics was taken as the temperature that first triggers a change in the ratio of the yellow and cyan emission channels (illustrated by dashed gray lines). C, We used the temperature waveforms of B to measure the lower bound of the operating range of AFD in different worm strains with different T_S. Representative parametric plots of the FRET signal versus temperature stimulus near each lower bound are shown for worms with wild-type background cultivated at 15, 20, and 25°C (black lines, each corresponding to an experiment using the stimulus comprising a sine wave added to a positive linear ramp). Below the parametric plots are the lower bounds of the operating range corresponding to each cultivation temperature for wild-type (green), ttx-3(mg158) (red), and odr-7(ky4) (blue) strains. In each case, the circle represents the mean value of the measurements of lower bound, the line represents the full range of the measurements, and the number of worms tested is shown in italics. The filled circles correspond to measurements of the lower bound using stimuli comprised of sine waves added to positive linear ramps. The open circle corresponds to a measurement of the lower bound for wild-type worms cultivated at 20°C using the stimulus comprising only a positive linear ramp.

Figure 2.

Dynamic range and sensitivity of the AFD response. A, Wild-type worms were subjected to temperature stimuli within the AFD operating range consisting of a 30 s period sine wave multiplied by a linear ramp. The size of the FRET signal increases with the amplitude of the sine wave. B, The peak-to-peak fractional change in the FRET signal is plotted against the peak-to-peak temperature change of the stimulus used in A. Three representative signals from individual worms are shown by dashed lines. Mean ± 1 SEM error bars calculated from 21 worms are shown by circles connected by the thick black line. The FRET signal saturation corresponds to temperature changes of ∼0.4°C in 15 s. C, FRET signals were apparent well below saturation. Here, we show a 10% FRET signal in an AFD soma stimulated by a temperature change of ∼0.05°C in 15 s.

Figure 3.

Responses to sine and step stimuli. A, FRET measurements were made of the AFD soma responding to nonsaturating temperature sine waves within their operating range with 30 and 240 s periods (note the different scale bars for time). The gray lines show one cycle of the temperature waveforms, dashed lines show the representative FRET signal from one temperature cycle of a single worm, and the black lines show the mean FRET signals collected from 35 temperature cycles on 7 worms (for the 30 s waveform) and 13 temperatures cycles on 13 worms (for the 240 s waveform). Error bars (±1 SEM) are shown at intervals along the mean response. B, Worms were subjected to upsteps and downsteps in temperature, 0.2°C in size. The gray lines show one cycle of the temperature waveforms, broken lines show the representative FRET signal from one temperature cycle of a single worm, and the black lines shown the mean FRET signals collected from 10 stimulus presentations on 10 worms. Error bars (±1 SEM) are shown at intervals along the mean response. C, Worms were subjected to positive and negative pulses, 0.1°C in amplitude and 30 s in duration. Representative individual traces are shown. D, Worms were subjected to successive upsteps (top) and to successive temperature downsteps (bottom), ∼0.15°C in amplitude, separated by 45 s intervals. Significant recovery of the FRET signal within 45 s after each upstep lead to a reliable response to the successive upstep. The FRET signal drops after the first downstep but does not recover significantly in 45 s, and so no FRET signal is measured with subsequent downsteps.

Figure 4.

Spatial localization of FRET signal. A, Representative images display the severing of an AFD dendrite using femtosecond laser pulses, showing the dendrite before surgery and ∼1 min after surgery. Scale bar, 10 μm. B, FRET measurements in response to a sinusoidal temperature stimulus (gray line) were taken at the soma of an AFD neuron (s) and the sensory endings of the AFD neuron (e). All worms in B and C were cultivated at 15°C. C, FRET measurements after severing one or both dendrites. In 13 cases in which we measured a FRET signal from the contralateral AFD soma (s2), its FRET signal was unaffected by severing the dendrite of its partner. In 20 of 26 cases in which an AFD dendrite was severed, the FRET signal was abolished in the parent soma (s). In the other 6 of the 26 cases in which an AFD dendrite was severed, a FRET signal persisted in the parent soma. In three of those six worms, we were also able to sever the dendrite of the contralateral AFD neuron, which in all three cases abolish the FRET signal in the soma (broken line in s). In 14 cases in which we measured a FRET signal from the sensory endings (e), the FRET signal was unaffected by severing the dendrite.

Figure 5.

Calcium dynamics in the sensory endings. A, FRET signals from the sensory endings of an unsevered AFD dendrite were measured in response to a sinusoidal temperature stimulus, with 0.2°C peak-to-peak amplitude and 30 s period. The thick black line shows the average FRET signal from 30 stimulus cycles on nine worms cultivated at 15°C; the dashed line shows a representative response from one cycle of one worm. The 1 SEM error bars show the average FRET signal. B, A 20 s sine wave added to a positive linear ramp was used to detect the existence of a threshold temperature in the AFD sensory endings after the dendrite was severed from its cell body. The sensory endings continued to exhibit an activity threshold correlated with T_S. The top trace shows the response of the sensory endings of an unsevered dendrite, whereas the bottom trace shows the response of the sensory endings after severing the dendrite. This worm was cultivated at 20°C. In a few cases, such as the one shown, the activity threshold of the sensory endings would shift upward or downward by as much as 1°C after severing the dendrite. C, The activity threshold temperature was measured for worms cultivated at 15, 20, and 25°C, as in Figure 1C. For each cultivation temperature, sample parametric plots of FRET versus temperature are shown; the circle represents the mean value of the measurements of activity threshold, the line represents the full range of the measurements, and the number of worms tested is shown in italics.

Figure 6.

Postsynaptic Ca²⁺ dynamics in the AIY axon. A, Worms with wild-type background expressing cameleon in AIY were subjected to temperature stimuli consisting of a small sine wave added to a linear ramp, as in Figure 1C. We recorded FRET signals from a varicosity of the AIY axon near the ventral edge of the nerve ring. The FRET signal from AIY phase-locks to the sinusoidal oscillation, but only above a threshold temperature comparable to the threshold temperature of AFD activity (see Fig. 1C). B, As in Figure 1C, we show a representative FRET signal from an AIY exhibiting a threshold temperature for a worm cultivated 20°C. Representative parametric plots of the FRET signal versus temperature of individual worms cultivated at 15, 20, and 25°C are shown. Below each representative parametric plot, we show the activity threshold temperature corresponding to each cultivation temperature. For each cultivation temperature, the circle represents the mean value of the measurements of activity threshold, the line represents the full range of the measurements, and the number of worms tested is shown in italics. C, To test whether the signal in AIY could be attributed to AFD synaptic output, we subjected worms to the stimulus in Figure 5A, killed the ipsilateral AFD soma with femtosecond laser pulses, and repeated the stimulus. In 10 of 10 surgeries, killing AFD eliminated the FRET signal in the second run. In mock surgery experiments, in which the experimental protocol was the same, except that AFD was not irradiated with femtosecond laser pulses, only in 2 of 13 cases did we not measure a phase-locked FRET signal in the second run.