A thermometer circuit for hot temperature adjusts Drosophila behavior to persistent heat

Abstract

Small poikilotherms such as the fruit fly Drosophila depend on absolute temperature measurements to identify external conditions that are above (hot) or below (cold) their preferred range and to react accordingly. Hot and cold temperatures have a different impact on fly activity and sleep, but the circuits and mechanisms that adjust behavior to specific thermal conditions are not well understood. Here, we use patch-clamp electrophysiology to show that internal thermosensory neurons located within the fly head capsule (the AC neurons¹) function as a thermometer active in the hot range. ACs exhibit sustained firing rates that scale with absolute temperature-but only for temperatures above the fly's preferred ∼25°C (i.e., "hot" temperature). We identify ACs in the fly brain connectome and demonstrate that they target a single class of circadian neurons, the LPNs.² LPNs receive excitatory drive from ACs and respond robustly to hot stimuli, but their responses do not exclusively rely on ACs. Instead, LPNs receive independent drive from thermosensory neurons of the fly antenna via a new class of second-order projection neurons (TPN-IV). Finally, we show that silencing LPNs blocks the restructuring of daytime "siesta" sleep, which normally occurs in response to persistent heat. Our previous work described a distinct thermometer circuit for cold temperature.³ Together, the results demonstrate that the fly nervous system separately encodes and relays absolute hot and cold temperature information, show how patterns of sleep and activity can be adapted to specific temperature conditions, and illustrate how persistent drive from sensory pathways can impact behavior on extended temporal scales.

Keywords: AC neurons; Drosophila; LPNs; circadian rhythms; clock neurons; daytime sleep; electrophysiology; sleep and activity; temperature; thermosensation.

Figure 1:. AC neurons display persistent firing which scales with absolute temperature in the hot range

(A) 2-photon frame of GFP fluorescence overlaid with a Dodt gradient contrast image demonstrating the targeting of ACs for recording. AC expresses GFP, while the electrode is filled with Alexafluor 594 (an = antennal nerve). (B) Diagram of ACs and other thermosensory circuit components (TRNs = thermosensory receptor neurons; PAL = posterior antennal lobe; TPNs = thermosensory projection neurons; SMP = superior median protocerebrum). (C) Representative whole-cell current clamp recording from an AC neuron in response to a hot step (~3°C; 1 min) demonstrating persistent responses. (D) Firing rate histograms from ACs in response to hot steps of different sizes and settling on distinct absolute temperatures above or below 25°C (gray line), showing that persistent activity only appears in the hot range (above 25°C; 9 cells/6 animals, ±SEM; temp. trace av. of 9, ±SEM). Note that the firing rate at a similar hot temperature is comparable, independent of stimulus history (arrowheads). (E) AC responses appear to have a distinct threshold of ~27.2°C. Circles represent 9 cells/6 animals (linear fit ± 95% CI; for 20–25°C r2=0.7; for ~28–33°C r2=0.83). (F-G) AC firing rates show no evidence of adaptation in hot temperature. (F) Representative firing rate histogram from an AC subjected to extended hot steps (AV±SD of 4 sweeps) and (G) responses from 4 different cells/animals (circles are average firing rates at stable temperature, connected circles are recordings from the same cell).

Figure 2:. Direct drive from ACs imparts sustained heat responses to LPN circadian neurons

(A-C) LPNs are a major synaptic target of ACs. (A) Partial EM brain volume reconstruction showing reconstructed ACs, LPNs and relevant brain regions (the mushroom body, MB, is shown as a reference; see below for abbreviations). (B) Pie charts representing synaptic connectivity between AC and LPN (each slice corresponds to a cell type, cell type names are abbreviated and clustered by region where possible, see Table S2 for full names and details). (C) Connectivity diagram illustrating drive from the four ACs to the three left hemisphere LPNs (right hemisphere LPNs are not included in the EM brain volume). (D, E) Optogenetic activation of LPNs can drive action potential firing in ACs. (D) Experimental schematic. ACs selectively express CsChrimson (driven by SH-Gal4), while LPNs are targeted for recording by independent GFP expression (under 65D05-LexA). (E) Left: example recording and firing rate histogram (6 cells/3 animals, ±SEM; temp. trace is shown as control). Right: max. response from 8 cells/4 animals for experimental and 3 cells/ 3 animals for control (control: no Cs Chrimson; empty circles: max firing rate during light stimulation; Filled circles AV±SD; 1-way, 2-sample t-test, asterisk: p<0.05). (F-L) Heat responses of LPNs can be explained by AC input and additional input from the antenna. (F) Circuit diagram for LPN hot drive. (G) Representative whole-cell current clamp recording from a control LPN (top trace), and from an LPN in an animal were ACs had been independently silenced (by expression of Kir2.1; bottom trace). The initial burst of activity (arrowheads) is not affected by AC silencing but persistent firing in hot conditions is no longer elevated compared to the baseline at 25°C (grey boxes). (H) Quantification of the effects of AC silencing. Top: firing rate histograms and corresponding temperature stimuli (control: 6 cells/4 animals ±SEM;-AC: 6 cells/2 animals ±SEM). Bottom: firing rates at stable temperatures (circles: average firing rates at stable temperature, connected circles: recordings from the same cell, color line: AV±SEM; 1-way, 2-sample t-test, asterisk: p<0.01). (I) Partial EM brain volume reconstruction showing hot receptors of the arista, TPN-IV and LPNs. (J) Connectivity diagram. (K) Removal of the antennae in AC silenced animals abolishes all heat responses in LPNs. Top: firing rate histograms and corresponding temperature stimuli (5 cells/2 animals ±SEM). Bottom: firing rates at stable temperatures (circles: average firing rates at stable temperature, connected dots: recordings from the same cell, color line: AV±SEM; 1-way, 2-sample t-test, asterisk: p<0.01). (L) LPN’s display heating responses from a “cold” baseline (<25°C; see arrowhead). Top: firing rate histograms and corresponding temperature stimuli (5 cells/N animals ±SEM). Bottom: firing rates at stable temperatures (circles: average firing rates at stable temperature, connected circles: recordings from the same cell, color line: AV±SEM;). (Abbreviations, MB: mushroom body, PAL: posterior antennal lobe, SLP: superior lateral protecerebrum, SMP: superior median protocerebrum; ACR/L: AC of the right/left hemisphere; LPN: lateral posterior neuron;TRNs: thermosensory receptor neurons; TPNs: projection neurons; arHC: hot cell of the arista).

Figure 3:. ACs and LPNs are required for daytime sleep restructuring by hot temperature

Drosophila melanogaster wild type and control flies respond to 30°C heat by increasing morning and afternoon sleep, while genetic silencing of ACs or LPNs impairs normal responses to heat. (A,D,F,H) Flies of the noted genotype were entrained to a LD 12:12 cycle at 25°C before the temperature was increased to 30°C for the subsequent day or time-delimited as illustrated (during the first or second half of the day). Top drawing in A is an outline of the temperature and light/dark protocol used for the experiments. For each panel (D,F), the schematic to the left is a circuit diagram representing the specific manipulation. Activity and sleep were quantified in 30-minute bins using one day per condition. Graphs represent the averages ± SEM of 2 consecutive days of sleep (top) or activity (bar graphs, bottom) at 25°C (black/gray) and 30°C (overlaid in red); filled circles in sleep plots and black dots above activity bars indicate timepoints that are significantly different between 25° and 30°C conditions (p < 0.05, paired two-sided t-test). Dark shading indicates lights off (night); red shading indicates 30°C; ZT = zeitgeber time. Specific sleep quantifications are presented in (B,C,G). (A) In WT flies, daytime hot conditions suppress activity and increase both morning (ZT0-3) and afternoon sleep (ZT6-12; n=32). Effects are replicated during time-delimited heat steps (n = 31 flies for morning and n=29 for afternoon; black arrowheads denote the afternoon increase). (B) Quantification of the effects of heat on total sleep. Box edges: 25th and 75th percentiles; thick lines: median; whiskers: data range; gray dots: individual data points/flies; *p < 0.05 in paired two-sided t-test comparing 25°C versus 30°C within genotype. (C) Quantification of per-fly sleep increases demonstrate a stronger effect of heat on afternoon sleep (arrowhead). Outer box edges: one standard deviation range; inner colored box edges: 95% confidence interval of the mean; thick lines: mean; gray dots: individual data points/flies; *p < 0.05 different from zero, one-sample t-test. (D) Silencing AC synaptic output via expression of tetanus toxin light chain perturbs afternoon sleep restructuring by hot temperature (empty arrowhead; n=30; see G and H for quantifications and controls). (E-F) Silencing LPN synaptic output under the control of a selective driver impairs afternoon sleep restructuring by heat (n = 63; empty arrowhead); E is a two-photon z stack from the brain of a fly expressing GFP under the control of LPN^split, overlaid with a 3D reconstruction of EM volumes for ACs and LPNs for comparison. (G) Quantification of per-fly sleep increases for silenced and control flies. Outer box edges: one standard deviation range; inner colored box edges: 95% confidence interval of the mean; thick lines: mean; gray dots: individual data points/flies; **p < 0.05 for differences between silenced and control genotypes, 2-way ANOVAs with a Bonferroni correction. (H) Control genotypes for D and F (n = 25 for AC/+, n = 55 for LPN/+, and n = 62 for TNT/+).

Figure 4:. Distinct thermometer circuits for hot and cold temperature in the Drosophila brain

Cold-inhibited DN1as and hot-activated LPNs are characterized by largely non-overlapping connectivity. (A) Pie charts representing the major synaptic input and output pathways of DN1a and LPN as reconstructed from the EM connectome hemibrain dataset (each slice corresponds to a cell type, cell type names are abbreviated and clustered by region where possible, see Table S2 for tables and methods for details). Inputs: DN1a’s major input is from the cold-activated inhibitory projection neuron TPN-II (blue slice, 300 synapses, 17% of total input). DN1a also receive significant input from TPN-IV and TPN-V (hot TPNs, gold and orange slices -respectively) and in addition from a number of less characterized PNs (including potential hygrosensory PNs). LPNs’ top input is from ACs (136 synapses, 7% of total; red slice), but they also receive additional hot input from TPN-IV (121 synapses, 6%; gold slice). Outputs: DN1a and LPN outputs are largely non-overlapping. DN1a has a number of targets in the LHPV/LHAV region, in the SLP and aMe, while the large majority of the synaptic targets of LPNs are in the SMP. The only common target appears to be SLP266. Both DN1a and LPNs demonstrate some reciprocal connectivity and some connectivity with DN3a (brown slices). (B) Circuit diagram illustrating how cold and hot temperature are relayed by distinct “thermometer” circuits that are active in a non-overlapping thermal range (<25°C for cold-TRNs>TPN-IIs, and >27°C for ACs, respectively) and drive adjustments in daytime sleep by directly modulating the activity of DN1a and LPNs. Abbreviations: TRN thermosensory receptor neurons, TPN thermosensory projection neuron, AC anterior cell, PN projection neuron, PAL posterior antennal lobe, MB mushroom body, CA Calyx, SLP/SMP superior lateral/medial protocerebrum, LHA/P V lateral horn anterior/posterior ventral, AVLP anterior ventrolateral protocerebrum, aMe accessory Medulla, WED wedge, CL claw; “unc” stands for uncharacterized. Clock neurons: DN1a, LPN, DN3a, LNd, s-LNv, DN1pB.