Ionotropic Receptors Specify the Morphogenesis of Phasic Sensors Controlling Rapid Thermal Preference in Drosophila

Abstract

Thermosensation is critical for avoiding thermal extremes and regulating body temperature. While thermosensors activated by noxious temperatures respond to hot or cold, many innocuous thermosensors exhibit robust baseline activity and lack discrete temperature thresholds, suggesting they are not simply warm and cool detectors. Here, we investigate how the aristal Cold Cells encode innocuous temperatures in Drosophila. We find they are not cold sensors but cooling-activated and warming-inhibited phasic thermosensors that operate similarly at warm and cool temperatures; we propose renaming them "Cooling Cells." Unexpectedly, Cooling Cell thermosensing does not require the previously reported Brivido Transient Receptor Potential (TRP) channels. Instead, three Ionotropic Receptors (IRs), IR21a, IR25a, and IR93a, specify both the unique structure of Cooling Cell cilia endings and their thermosensitivity. Behaviorally, Cooling Cells promote both warm and cool avoidance. These findings reveal a morphogenetic role for IRs and demonstrate the central role of phasic thermosensing in innocuous thermosensation. VIDEO ABSTRACT.

Keywords: Ir21a; Ir25a; Ir93a; iGluR; ionotropic receptor; morphogenesis; sensory neuron; temperature; thermoreceptor; thermosensation.

Fig. 1. Cold Cells are Brivido-independent phasic thermoreceptors.

A,Representative recording from a wild type arista. In upper panels, instantaneous spike frequency was smoothed using a 1s triangular window to generate weighted average spike rate. Lower panels show data from upper panels displayed on expanded time scale, revealing individual spikes (open circles). Spike voltage threshold of 3.5 times the standard deviation of spike-free regions of recording indicated by dotted lines. B, C, Peristimulus time histograms (PSTHs) of responses from wild type aristae (n=7 animals per condition; one trial per animal). Average +/− SEM. In panel C, results of four different temperature steps are superimposed. D, Upper panels show representative recordings from wild type, brv1^L563stop, and brv2^W205stop aristae. In lower panels, data from upper panels is displayed on expanded time scale, as in A. E, PSTHs from brv1^L563stop n=6) and brv2^W205stop (n=7) recordings. F, Cooling response quantification: cooling response = (average spike rate during first 2 sec of 30˚C to 25˚C cooling) – (average spike rate, 10 sec pre-cooling). wild type, n=8 animals, brv1^L563stop, n=6, brv2^W205stop, n=7. In violin plots, white circles represent median, black boxes denote 25th to 75th percentiles, and whiskers extend 1.5x interquartile range. See also Supplemental Figures 1 and 2.

Fig. 2. IR21a, IR25a and IR93a proteins localize to Cold Cell sensory endings.

A-C, IR21a (A), IR25a (B), and IR93a (C) immunostaining. Top panels, anti-IR immunostaining. Bottom, merge with Hot Cell-specific HC-Gal4;UAS-GFP. Blue asterisks, Cold Cells. Red asterisks, Hot Cells. Blue arrows, Cold Cell outer segments. See also Supplemental Figure 3.

Fig. 3. Ir21a, Ir25a and Ir93a are required for Cold Cell thermosensing.

A, Representative recordings from indicated genotypes, with indicated portions displayed below using an expanded time scale, as in Fig. 1. B-I,PSTHs of temperature responses. (J) Cooling response quantification as in Fig. 1. Genotypes detailed in methods. **alpha=0.01, distinct from wild type, Tukey HSD (n=6 or 7 animals per genotype). See also Supplemental Figures 4 and 5.

Fig. 4. IRs are required for thermoreceptor morphogenesis.

A. Arista immunostaining. Merge: Green, beta-tubulin; magenta, IR21a. Green arrows denote tubulin staining and magenta arrows denote IR21a staining of thermoreceptor sensory endings. B-D, EM sections through arista sensilla. Hot Cell, red shading. Cold Cell, blue shading. Upper panels, sections nearer sensillum’s distal tip. Strains: Ir21a^−/−: Ir21a^∆1/ Ir21a^∆1. Ir25a^−/−: Ir25a²/Ir25a². Ir93a^−/−: Ir93a^MI05555/Ir93a^MI05555. B, Left panel, transmission electron micrographs. Middle, drawing depicts microtubules (circles) and “BOSS” structures (thin lines) between Cold Cell membranes. Right, drawing depicts arista thermoreceptor pairs and approximate micrograph locations. C,D, Representative electron micrographs of similar locations (upper panels, more distal) in Ir25a^−/− and Ir21a^−/−. Scale bar: panel A, 10 µm, panels B-D, 200 nm.

Fig. 5. Ectopic IR21a expression alters thermoreceptor morphology.

A, Left panel, drawing of arista indicating approximate locations of adjacent electron micrographs. Right panels, representative transmission electron micrograph of animals expressing IR21a in both Hot and Cold Cells: Ir21a^∆1/Ir21a^∆1;GMR11F02-Gal4/UAS-Ir21a. Cold Cell, blue. Hot Cell, red. B, EM sections through wild-type and GMR11F02>Ir21a animals, near distal tip of sensory endings. Middle panels represent inset region of left panels. Right panels represent the numbered insets from middle panels. Yellow arrowheads denote high magnification views of BOSS structures between membranes. Scale bars: A (right panels) and B (left panels), 200 nm; B (middle panels), 100 nm; B (right panels), 20 nm. See also Supplemental Figure 6.

Figure 6:. Ionotropic Receptors mediate both cool and warm avoidance.

A, Top panel, cool avoidance assay schematic. Bottom panel, cool avoidance of indicated genotypes. B, Top panel, warm avoidance assay schematic. Bottom panel, warm avoidance of indicated genotypes. ** P<0.01 distinct from wild type, Steel with control. n=24 individuals assayed for each genotype and condition, except n=48 for wild type, arista ablation cool avoidance and Gr28b^MB03888 warm avoidance.