(A) A proposed thermotaxis neural circuit. The arrows represent synaptic inputs.

(B) In vivo AFD neuron forms synapses with its synaptic partners (left), and isolated AFD neuron has no neural connections (right). Triangles indicate sensory neurons, and hexagons indicate interneurons. The numbers represent the number of synapses.

(C) Expression of ttx-1p::GFP in a live animal (top) or a cultured cell (bottom). Scale bars, 20 μm (top) and 10 μm (bottom), respectively. D, dorsal; V, ventral; A, anterior; P, posterior.

(D) Response temperature is defined as the temperature at which GCaMP3/TagRFP fluorescence ratio change first exceeded 1.0 in single-trial calcium imaging and 0.5 in multiple-trial calcium imaging, respectively.

(E and F) Calcium imaging of in vivo AFD cultivated (Cult.) at 17°C (n = 28), 20°C (n = 58), 23°C (n = 46), or shifted from 17°C to 23°C (n = 17) (E) and isolated AFD neurons cultured at 17°C (n = 30), 20°C (n = 38), 23°C (n = 22), or shifted from 17°C to 23°C (n = 16) (F). In boxplots, center lines indicate the medians, box limits indicate the 25th and 75th percentiles as determined by R software, whiskers indicate 1.5 times the interquartile range from the 25th and 75th percentiles, and outliers are represented by dots. Mean ± SEM of ratio change (top) and boxplots of response temperatures (bottom). The red arrows indicate the mean values of the response temperatures. ∗p < 0.05; ∗∗p < 0.01.