Fig. 1: TPN-IIIs display ON responses to temperature change.

a EM reconstruction of a group of 5 right-hemisphere TPN-IIIs (orange; arrowhead: cell bodies) and of the afferents from aristal hot and cold TRNs (red and blue, respectively) partially overlapping in the hot and cold glomeruli of the posterior antennal lobe (PAL). b Expression pattern of VT040053-Gal4/UAS-CD8:GFP, R22C06-Gal4/UAS-CD8:GFP and VT040053.DBD ∩ R22C06.AD/UAS-CD8:GFP; the split driver displays selective expression in TPN-IIIs (2-photon stacks of top: brain, bottom: VNC; inverted so that GFP expression is in black; arrowhead: cell bodies; ∩ = intersection; images represent 5 independent repeats; scale bars = 50 µm). c–e Silencing TPN-IIIs produces defects in both hot and cold avoidance in a 2-choice test for temperature preference. c Assay schematic. Groups of 15 flies are given a choice between 25 °C (gray shading) and a test temperature (TT; blue shading on top and red shading on the bottom), the time in each temperature is used to quantify an avoidance index (AI). d AIs for flies in which TPN-IIIs are silenced by expression of Kir2.1 (under the control of VT040053-Gal4.DBD ∩ R22C06-AD); * = p < 0.05, p = [0.001,2.09E−07,5.31E−05,0.717,3.42E−12,2.83E−06,0.069] for 25 °C vs 10,15,20,25,35,40 °C, respectively. e Control genotypes (d and e: black/red line = mean, inner box = 95% CI, outer box = SD; individual points = # of groups; N = [8,10,16] groups at each temperature for Gal4/Kir, Ga4/+, Kir/+, respectively, groups, 20 animals/group; 2-way ANOVA). f–k 2-photon guided patch clamp electrophysiology of TPN-IIIs. f–h Optogenetic activation of hot or cold TRNs of the antenna drives firing in TPN-IIIs. f Experiment schematic. g Example TPN-III whole cell recording from flies expressing CsChrimson in hot or cold TRNs and in which TPN-IIIs are independently labeled by GFP (control lacks CsChrimson expression), pink boxes represent red light stimulation (N = 14,7,6 cells for firing rates; line and shading = mean ± SEM). h Quantifications of peak firing rates during light stimulation (gray dots indicate individual cells, orange circles indicate mean ± SD; N = 14,7,6 cells, * = significantly different from Control, p = 9.04E−06, 1-way ANOVA). i–k TPN-III display comparable transient “ON” responses to temperature change, irrespective of direction (heating or cooling) and of absolute temperature. i Schematic of the recording (j), raw traces, (k) average firing rate histograms, and (l) quantification of firing rate. Peak responses to heating (red shading) and cooling (blue shading) are comparable (circled numbers in k and top row plots l). Firing rates at stable temperatures of 22 °C, 25 °C, and 28 °C are also comparable (boxes in k and bottom row plots l; in k and l n = 15 cells/hot and 18 cells/cold from 9 animals, mean ± SEM; colored circles in (l) are mean ± SD). Source data are provided as a source data file.