During their lives, animals encounter a broad range of stimuli from their surroundings including heat, light and touch. The ability to appropriately respond to such stimuli is crucial for survival as it allows the animals to avoid predators and other dangers, locate food and shelter, and find mates.

Fruit fly larvae are a useful model for studying how animals respond to unpleasant (known as painful) heat stimuli. When something hot touches a larva, the larva rolls away to avoid the stimulus. The heat stimulates electrical activity in a type of neuron known as C4da neurons on the surface of the larva. Ultraviolet light and several other stimuli are also able to activate electrical activity in C4da neurons, resulting in the larvae changing the direction they move to avoid the stimuli.

Only older fly larvae respond to painful heat stimuli and previous studies found that a hormone receptor protein is required for this response. However, it remains unclear how this response develops as the larvae age.

Jaszczak et al. studied the behavior of fly larvae and electrical activities of C4da neurons in response to painful heat and ultraviolet light. The experiments found that painful heat triggered more rolling behavior from older larvae than those of younger larvae. In contrast, ultraviolet light triggered lower levels of electrical activity in the C4da neurons of older larvae than those of younger larvae. The team raised the levels of a hormone known as ecdysone and found that this increased the rolling behavior in younger larvae. They then increased the amount of receptor protein for this hormone in the neurons and found that it decreased the levels of another protein called Subdued in the C4da neurons. This in turn increased the neurons’ response to painful heat and decreased their response to ultraviolet light.

Jaszczak et al. propose that as the larva develops, ecdysone reduces the levels of Subdued, which promotes C4da neurons to switch their sensitivity from detecting ultraviolet light to painful heat. In the future, better understanding of what causes pain sensations in developing animals will help us search for factors that cause long-term pain conditions in humans.

Detection of internal and external stimuli depends on developmental programing of the proper physiological and morphological properties of sensory neurons. It is an intriguing open question as to how the characteristics of sensory transduction arise during development, and which neuronal properties are crucial for the initiation of sensory detection.

Nociception, the ability to detect and escape potentially harmful stimuli, is a highly conserved function of sensory systems present in all animals (Arenas et al., 2017). A nociception system is composed of nociceptors, which are neurons with molecular components that directly sense harmful stimuli; a processing center where the stimuli are interpreted, typically the neural circuits of the central nervous system (CNS); and the reflexive neuromuscular circuit which generates a reaction to escape injury (Baliki and Apkarian, 2015). Development of sensory systems requires the coordination of processes on the cellular, morphogenic, and circuit level.

Thermal nociception generates an avoidance behavior, such as a fast limb retraction or change of movement trajectory. Previous studies of thermal nociception behaviors and neural activity demonstrate a change in nociceptive responses during development of both mice and Drosophila. For example, a mouse paw exhibits low sensitivity to painful heat early in postnatal development, but the rate of reflex gets faster as the animal develops (Jankowski et al., 2014; Ford et al., 2019). A developmental transition is also observed in nociceptor activity, as revealed by the change in the number of heat-sensitive C-fibers with age in mice (Hiura et al., 1992). Likewise, a behavioral transition in heat thermal nociception has been observed in Drosophila melanogaster larvae (Sulkowski et al., 2011). Drosophila third instar larvae exhibit a stereotyped ‘corkscrew’ behavior, when a thermal probe heated above 38°C touches the cuticle (Tracey et al., 2003; Babcock et al., 2009). However, this behavior is not observed in second instar or earlier larval stages, and appears to be acquired only during the last stage of larval development.

The larval nociceptive behavior has been a useful model for dissection of the molecular mechanisms and circuitry of nociception (Himmel et al., 2017). Among the heat-sensitive neurons in the larval cuticle (Liu et al., 2003) are the Class IV dendritic arborization (C4da) neurons, which function as the primary nociceptors for noxious heat-induced escape behavior (Tracey et al., 2003; Hwang et al., 2007). C4da neurons are multimodal sensors for harmful stimuli, with the capacity to respond to multiple forms of sensory information. In addition to responding to temperature, C4da neurons respond to high-intensity blue light (Xiang et al., 2010), noxious UV light (Xiang et al., 2010; Gu et al., 2019), and harsh touch (Zhong et al., 2010).

While noxious heat and touch both elicit the rolling escape behavior, stimulation of C4da neurons with short wavelength (blue or UV) light causes a locomotion trajectory avoidance behavior. These different stimuli cause different electrical signals in C4da neurons. Noxious heat causes high-frequency spike trains interspersed with quiescent periods, in contrast to blue light which elicits lower frequency firing with few quiescent periods in third instar larvae (Terada et al., 2016). Therefore, distinct behaviors appear to be encoded by the C4da neurons according to their differential responses to differing stimuli.

The majority of studies of nociception in Drosophila larvae have focused on the last (i.e., the third) larval instar, where nearly all larvae will perform the behavioral response. However, Sulkowski et al. found that during the first half of larval development (i.e., the first and second instars), larvae exhibited either no or a low rate of thermal nociceptive behavior, leading to the hypothesis that a developmental transition occurs between the second and third larval instars (Sulkowski et al., 2011). The mechanisms which regulate this transition remain unknown. Optogenetic activation of C4da neurons can induce the corkscrew behavior or a locomotion trajectory avoidance behavior in third instar larvae. However, optogenetic activation could only induce a locomotion trajectory avoidance behavior in the second instar. This difference between second and third instars in the optogenetic activation of behavior, taken together with the difference in coding activity in C4da neurons in response to thermal stimulation or light (Terada et al., 2016), raises the possibility that the developmental transition may involve a change in sensory coding. Supporting this possibility, misexpression of low temperature activated TrpA1 (isoform A) channels in the C4da neurons enables second instar larvae to exhibit corkscrew behavior in response to an innocuous temperature stimulus (Luo et al., 2017). It is conceivable that the sensory switch may be caused by a cell autonomous change in the C4da neurons. Whether C4da neurons change in their neural activity during the developmental transition from second to third instar is unknown, and the regulators of such transition are unknown as well.

Transcription factors facilitate the formation of distinct neuronal functions (Parrish et al., 2014) and regulate the dynamic expression profiles of many developmental transitions between and during larval instars (Sullivan and Thummel, 2003). One such transcription factor is the nuclear hormone receptor ecdysone receptor (EcR). EcR forms a heterodimer with ultraspiracle (USP) and binds the ligand ecdysone, a steroid hormone, to coordinate a wide variety of developmental programs (Yamanaka et al., 2013). EcR and USP are required for thermal nociception in the third instar (McParland et al., 2015), but the mechanism and role of ecdysone signaling in the second instar have not been determined.

EcR expression in C4da neurons has been reported at the beginning and end of larval development (Kuo et al., 2005; Ou et al., 2008; Kirilly et al., 2009; McParland et al., 2015). In the absence of ligand, EcR and USP distribute between the cytoplasm and nucleus, while ligand binding increases nuclear localization (Nieva et al., 2007; Nieva et al., 2008). Without ligand binding, EcR and USP recruit nucleosome remodeling proteins to compact the chromatin and locally suppress transcription. When bound to ecdysone, EcR-USP recruit coactivators, such as histone acetylases, to expand the chromatin structure and promote transcription. In this way, steroid hormone signaling can create positive and negative feedback to promote cascades of transcriptional programs at developmentally precise periods (Sullivan and Thummel, 2003; Hill et al., 2013). Whether the ligand-induced activity and transcriptional regulation are involved in EcR-mediated thermal nociception remains to be determined.

In this study, we performed a series of experiments to determine the activity of the C4da neurons during the developmental nociceptive transition as well as the mechanisms which regulate this sensory switch. These experiments demonstrate that steroid hormone signaling in nociceptive neurons regulates the channel composition in C4da neurons to modulate the sensory properties and behavioral response at distinct stages of larval development.

The impact of peripheral nervous system (PNS) development on behavior is underscored by the recent finding that defects in the PNS are sufficient to produce symptoms in mouse models of autism spectrum disorders (Orefice et al., 2016). The role of hormonal regulation of sensory systems during periods of postnatal development has also been highlighted by studies of growth hormone regulation of thermal sensitivity (Liu et al., 2017; Ford et al., 2019). Considering that sensory neurons progress through transcriptionally distinct identities during development (Sharma et al., 2020), characterizing how the specific changes in receptors or channels that produce these developmentally specific outcomes is of prominent importance for understanding the development of neurological disease.

In this study, we address how a nociceptive behavior is temporally acquired during development. We demonstrate how steroid hormone regulation of nociceptor activity in a class of sensory neurons in the PNS adjusts the developing larval response to nociceptive stimuli. Heat-induced nociceptive behavior in D. melanogaster larvae arises during the final (third) instar of development (Sulkowski et al., 2011). Here, we show that this behavioral transition reflects a nociceptive sensory switch through regulation of Subdued by the steroid hormone ecdysone and the EcR isoform EcR-A.

While previous studies have focused on EcR localization in the third instar and pupal stages of C4da neurons, we show EcR is present in both the nucleus and cytoplasm in the second instar, followed with an increase of EcR nuclear localization 8 hr into the third instar (Figure 2). Previous work has shown that loss of EcR-A reduces thermal nociception in the third instar (McParland et al., 2015). While we did not find detectable transcriptional changes in nociceptor genes in third instar C4da neurons during EcR-A RNAi expression, we found broad suppression of nociceptor genes during EcR ligand-binding mutant (W650A) expression (Figure 5—figure supplement 2). Through behavioral and transcriptional analysis, we show that EcR-A nociception regulation is ligand dependent, by demonstrating that increased ecdysone titer and overexpression of ligand-competent EcR both accelerate nociception in the second instar (Figures 3 and 4). Ligand binding of EcR leads to widespread epigenetic changes through release of corepressors and recruitment of coactivators (Uyehara et al., 2017; Uyehara and McKay, 2019). The precocious activation of nociception in the second instar requires the unique A/B domain of the isoform EcR-A, as other isoforms lacking this domain do not increase nociception (Figure 4A). The A/B domain of EcR-A is less activating and more repressive then the EcR-B1 A/B domain (Dela Cruz et al., 2000; Mouillet et al., 2001; Hu et al., 2003). It thus appears that ligand-dependent derepression could be the action which promotes nociception. Moreover, expression of the EcR coactivator mutant (F645A) phenocopies expression of wild-type EcR in its ability to promote nociception (Figure 4B), suggesting that in contrast to coactivator recruitment, preservation of the ability to remove corepressors is required for nociception. This regulation of repression may account for the ability of the EcR ligand-binding mutant (W650A) to suppress multiple nociceptive genes including subdued in the second and third instars. The release of corepressors could allow other transcription factors to gain access to response elements which suppress subdued, or directly promote transcription of a repressor of subdued. How derepression leads to the suppression of subdued is an intriguing open question.

For third instar larval nociception, our data suggest there are both ligand-dependent and -independent EcR-A pathways. We find third instar expression of EcR-A ligand-binding mutant to have a temperature-specific phenotype. Expression of the EcR-A ligand-binding mutant inhibits nociception at 42°C, but does not reduce nociception at 46°C (Figure 3B, D). This temperature-specific effect is reminiscent of the phenotype of TrpA1 mutant larvae, which lose nociceptive behavior at probe temperatures of 44°C and lower, but are still responsive to 46°C stimuli (Gu et al., 2019). We found that expression of EcR-A ligand-binding mutant reduced subdued expression in the third instar even more than during the developmental transition (Figure 5A, C), suggesting that this loss of subdued expression may contribute to the phenotype at 42°C but not at 46°C. Consistent with this possibility, we observed that Subdued-RNAi was able to increase nociception in second instar larvae while Subdued knockout mutation did not increase nociception. Together these results suggest that the amount of subdued expression is important for promoting or inhibiting nociception. Furthermore, there are Subdued and EcR-A ligand-independent pathways which promote nociception in the third instar. We observe alteration of TrpA1, painless, ppk1 and ppk26 expression during development but not as a result of EcR overexpression (Figure 5). These separate pathways may involve mechanisms of regulating temperature specificity in C4da neurons.

Subdued has homology to the Calcium-activated Chloride Channels (CaCC) of the mammalian TMEM16 family, with channel activity similar to both TMEM16A and TMEM16F (Wong et al., 2013; Le et al., 2019). The effect of CaCC on neural activity is dependent on the intracellular Cl⁻ concentration ([Cl⁻]_i) (Berg et al., 2012). When [Cl⁻]_i is high, elevation of internal Ca²⁺ level activates CaCCs and causes Cl⁻ efflux, leading to excitation. In contrast, when [Cl⁻]_i is low, Ca²⁺ activation of CaCCs causes an influx of Cl⁻ which has an inhibitory effect on neuronal excitability. Differential expression of symporters and cotransporters in the CNS and PNS, as well as in immature and mature neurons, is important for creating the inhibitory or excitatory effects of CaCCs. In third instar larvae, Cl⁻ efflux has been observed during thermal nociceptive stimulation of C4da neurons (Onodera et al., 2017). However, Onodera et al. found that loss of Subdued did not significantly change the magnitude of the Cl⁻ efflux during heat stimulus, suggesting other channels are responsible for the Cl⁻ efflux in third instar C4da neurons. The direction of Cl⁻ movement in the second instar remains to be determined, and it is conceivable that Subdued could produce an inhibitory influx of Cl⁻ in the second instar. Characterizing Cl⁻ homeostasis during C4da neuron development and its impact on nociception will be of interest in future studies.

Subdued has previously been found to regulate thermal nociception in third instar larvae (Jang et al., 2015). Jang et al. found the strongest decrease of nociception in the third instar when expression of subdued was reduced in all dendritic arborization neurons. Additionally, when subdued was overexpressed in C4da neurons the number of third instar larvae that responded to nociceptive heat did not significantly change. Interesting, when subdued was overexpressed with a subdued-Gal4, the number of fast responding third instar larvae increased (Jang et al., 2015). This suggests there may be a substantial thermal nociception role for Subdued in dendritic arborization neurons other than C4da neurons during third instar. Our data suggest that as C4da neurons develop from second to third instars, the ability of Subdued to influence detection of stimuli may change. We find that thermal nociception is enhanced by reduction of subdued expression in second instar C4da neurons, via either EcR-A overexpression or Subdued-RNAi knockdown. Additionally, we find that expression of the EcR-A-ligand mutant reduces subdued expression even further in third instar C4da neurons, and that this is associated with a loss of nociception. The greater amount of subdued expression in the second instar may mean that Subdued could have a greater impact on Cl⁻ regulation during the second instar. This mechanism of Subdued activity would be dependent on the intracellular Cl⁻ concentration created by symporters and cotransporters. The importance of Cl⁻ regulation has recently been highlighted in the Class III da neurons, as both Subdued and depolarizing Cl⁻ currents have recently been found to promote cold-evoked nociception (Himmel et al., 2021). Therefore, identifying regulators of Cl⁻ concentration during the development of C4da neurons and their impact on nociception will be of interest in future studies.

Our work adds to the evidence that the developmental transition of thermal nociception represents a shift in sensory state rather than a change in sensory system construction: (1) optogenetics can stimulate aversive behavior in both second and third instar C4da neurons (Sulkowski et al., 2011), (2) TrpA1 misexpression can confer nociceptive behavior in both second and third instar larvae exposed to innocuous temperatures (Luo et al., 2017), (3) UV-A stimulation induces greater Ca²⁺ response in second instar than in third instar C4da neurons (Figure 1), and (4) reduction of subdued expression in second instar renders C4da neurons responsive to thermal nociceptive stimuli (Figure 6). Additional properties of C4da neurons during this developmental transition remain to be determined. These properties include: whether thermally induced calcium activity in C4da neurons (Terada et al., 2016; Gu et al., 2019) also changes over this period of development, whether EcR regulates the ‘burst and pause’ encoding (Terada et al., 2016), and whether EcR regulation alters synaptic connections of C4da neurons (Valdes-Aleman et al., 2021).

Recent findings have highlighted the occurrence of developmentally timed modulation of sensory state in both larvae and adult Drosophila. A sensory switch in thermotaxis behavior of Drosophila larvae is regulated by transcriptional regulation of thermoreceptors (Tyrrell et al., 2021). In adults, male courtship behavior is modulated through olfactory neuron pheromone sensitivity and hormone-mediate chromatin reprograming (Zhao et al., 2020). Our study highlights that temporally programed sensory switches may be a widely used developmental mechanism to match behavioral outcomes with life history events.

We speculate that the nociceptive shift in sensory state is an important mechanism for matching sensory system function with life history transitions. D. melanogaster larvae do not develop robust thermal nociception until the second half of the larval phase. This means that for the first half of larval life, larvae do not have thermal nociception behavior. In contrast, nociceptor activity to UV-A light decreases from the second to third instars, suggesting that nociceptive sensitivity to short wavelength light decreases during the second half of the larval phase. This transition correlates with larval behavioral changes that emerge during the third instar, such as cessation of feeding beneath the surface and wandering above ground (Wegman et al., 2010). Larvae are transitioning from predominantly feeding behavior in the second instar, often shielded from light and high temperatures, to movement away from food in search of pupation sites in the third instar. In a natural habitat, this transition can mean leaving the food source, which necessitates greater exposure to heat, increasing the risk of predation and desiccation (Ballman et al., 2017). This sensory switch may function as a developmental strategy, shaping behavioral outcomes as animals encounter changing environments, thus increasing survival.

All data generated or analyzed during this study are included in the manuscript and supporting files.

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Author details

1. Jacob S Jaszczak

   1. Department of Physiology, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
   2. Howard Hughes Medical Institute, Chevy Chase, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Validation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3752-1152

2. Laura DeVault

   1. Department of Physiology, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
   2. Department of Developmental Biology, Washington University Medical School, Saint Louis, United States

   Contribution

   Data curation, Investigation, Methodology, Validation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6801-5098

3. Lily Yeh Jan

   1. Department of Physiology, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
   2. Howard Hughes Medical Institute, Chevy Chase, United States

   Contribution

   Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3938-8498

4. Yuh Nung Jan

   1. Department of Physiology, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
   2. Howard Hughes Medical Institute, Chevy Chase, United States

   Contribution

   Funding acquisition, Methodology, Supervision, Writing - review and editing

   For correspondence

   YuhNung.Jan@ucsf.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1367-6299

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