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Temperature cues are integrated in a flexible circadian neuropeptidergic feedback circuit to remodel sleep-wake patterns in flies [1]

['Xin Yuan', 'Department Of Neurology Of Children S Hospital', 'School Of Brain Science', 'Brain Medicine', 'Zhejiang University School Of Medicine', 'Hangzhou', 'Moe Frontier Science Center For Brain Research', 'Brain-Machine Integration', 'State Key Laboratory Of Brain-Machine Intelligence', 'Zhejiang University']

Date: 2024-12

Organisms detect temperature signals through peripheral neurons, which relay them to central circadian networks to drive adaptive behaviors. Despite recent advances in Drosophila research, how circadian circuits integrate temperature cues with circadian signals to regulate sleep/wake patterns remains unclear. In this study, we used the FlyWire brain electron microscopy connectome to map neuronal connections, identifying lateral posterior neurons LPNs as key nodes for integrating temperature information into the circadian network. LPNs receive input from both circadian and temperature-sensing neurons, promoting sleep behavior. Through connectome analysis, genetic manipulation, and behavioral assays, we demonstrated that LPNs, downstream of thermo-sensitive anterior cells (ACs), suppress activity-promoting lateral dorsal neurons LNds via the AstC pathway, inducing sleep Disrupting LPN-LNd communication through either AstCR1 RNAi in LNds or in an AstCR1 mutant significantly impairs the heat-induced reduction in the evening activity peak. Conversely, optogenetic calcium imaging and behavioral assays revealed that cold-activated LNds subsequently stimulate LPNs through NPF-NPFR signaling, establishing a negative feedback loop. This feedback mechanism limits LNd activation to appropriate levels, thereby fine-tuning the evening peak increase at lower temperatures. In conclusion, our study constructed a comprehensive connectome centered on LPNs and identified a novel peptidergic circadian feedback circuit that coordinates temperature and circadian signals, offering new insights into the regulation of sleep patterns in Drosophila.

Funding: This work is supported by funding from the National Key Research and Development Program of China (2019YFA0802400), the National Natural Science Foundation of China (31970941, 32171008, 32471210), the Zhejiang Provincial Outstanding Youth Science Foundation (LR20C090001), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2023-PT310-01), the Fundamental Research Funds for the Central Universities (2023ZFJH01-01, 2024ZFJH01-01) and the Fundamental Research Funds for the Central Universities to F.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Yuan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In our study, we utilized Drosophila FlyWire [ 23 ] connectomics analysis to demonstrate that LPNs primarily receive input from both circadian and temperature-sensing neurons. Additionally, we identified a circadian feedback circuit that dynamically modulates the sleep-wake profile in response to varying temperature conditions. At elevated temperatures, LPNs are highly active, receiving heat signals from TrpA1 + ACs and promoting sleep by inhibiting wake-promoting LNds via the AstC signaling pathway. Conversely, LNds receive cold signals from DN1a and become active at lower temperatures [ 17 ], then activate LPNs through the NPF pathway, establishing a feedback inhibition mechanism. This integration of temperature information into a circadian feedback circuit enables adaptive behavioral responses to different thermal environments. Our findings provide significant insights into the dynamic interplay between temperature cues and circadian signaling within the central circadian circuit.

Advances in brain structure reconstruction using electron microscopy (EM) have made it possible to clearly map the neural networks of model organisms like Drosophila [ 22 – 24 ]. Detailed dissection of neural connectomes based on EM data has significantly advanced our understanding of neural circuits involved in sensory processing, such as olfactory and visual systems [ 25 , 26 ], as well as innate behaviors [ 27 , 28 ]. Recently, the synaptic connectome of Drosophila circadian neuron revealed an extensive synaptic connectivity within the network, uncovering novel light input pathways and key neurosecretory output cells [ 29 ].

The recent studies have highlighted a strong connection between LPNs and heat-sensitive ACs [ 16 ]. Additionally, LPNs have been reported to promote daytime sleep [ 20 ], while other research suggests a slight inhibitory effect on sleep behavior [ 21 ]. These conflicting results prompted us to investigate the potential communication within the circadian neuron network, focusing on the role of LPNs in temperature-sensitive sleep regulation.

Peripheral temperature-sensitive cells, including cold cells, hot cells, and anterior cells (AC), respond to thermal changes and relay signals to higher brain regions [ 18 , 19 ]. The secondary thermosensory projection neurons process the information about prolonged cold temperatures and relay signals to higher brain centers, including the circadian neuron DN1a, which reshapes normal daytime sleep under cold conditions [ 14 ]. Conversely, high temperatures activate ACs via Transient Receptor Potential A1 (TrpA1), sending signals to DN1ps [ 15 ] and LPNs [ 16 ]. DN1p promotes nighty wakefulness by inhibiting DH44 + neurons through a CNMa-dependent mechanism [ 15 ]. Those cues suggest that the perception of temperature information by circadian neurons is critical for the plastic regulation of arousal and sleep for survival. However, the dynamic communication among circadian neurons and their adjustments in response to temperature changes remains incompletely understood.

Maintaining appropriate sleep and activity levels across different seasonal environments is crucial for animal health and survival [ 1 – 5 ]. However, the precise neural mechanism underlying the adaptive regulation of sleep/wake behavior in response to external cues, like hibernation behavior in mammals, remains incompletely understood [ 6 – 9 ]. In fruit flies, environmental temperature changes induce distinctly adaptive modifications in sleep/wake profiles [ 10 ]. The circadian neuron system receives temperature signal and integrates this information with circadian information to orchestrate suitable behavioral responses [ 11 ]. In Drosophila, the fundamental circadian neurons are categorized into groups of lateral neurons (LNs) and dorsal neurons (DNs), based on their anatomy [ 12 ]. Among these, DN1, LPN, LNd, and DN3, are reported to be temperature-sensitive [ 13 – 17 ].

Results

LNd signal back to LPN to form negative feedback circuit If high temperature activates sleep-promoting LPNs via ppkAC neurons, an increase in overall sleep duration throughout the high-temperature day would be expected. However, our observations revealed a specific increase in evening sleep, indicating a potential heightened excitability of LPNs in response to elevated temperatures during the evening hours. Given the documented higher calcium levels in the LNds during the evening [48], we proposed that LNd may activate LPN during this time, with LPN potentially exerting negative feedback on LNds to maintain their activity within an appropriate range. We firstly analyzed the main postsynaptic neurons of LNds in Flywire database [23], which included LNd, DN3A, DN1p, and sLNv but not LPN (S8A and S8B Fig). To investigate the functional implications of the LNd-LPN connection, we employed optogenetic techniques to activate LNds while concurrently monitoring the neuronal activity of LPNs using GCaMP. Our results demonstrated a significant increase in calcium levels in LPNs following LNd activation with DvPdf-LexA while activation of LNvs with Pdf-LexA had minimal impact on LPN calcium levels (Fig 5A–5D and S6 Video). Those results clearly demonstrate a negative feedback circuit between LNds and LPNs. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. LNd stimulate LPN to form a circadian feedback circuit. (A–D) Optogenetic activation of LNds using CsChrimson (DvPdf-LexA>LexAop-CsChrimson) increases LPN calcium levels in vivo, with no effect by LNvs activation (Pdf-LexA>LexAop-CsChrimson). Heat maps (B), summarized line graphs (C), and normalized maximum calcium fluorescence (D) are shown. Z-stacks of 10 frames with 1.1 s intervals. (E) A working model of LPN-LNd feedback circuit. The red line represents the activation effect and the blue line represents the inhibition effect. If the LPN neurons and AstCR1 receptor in the negative feedback circuit are interfered, it is speculated that the LNd neurons will be affected. (F, G) Optogenetic activation of LNds using CsChrimson (DvPdf-LexA>LexAop-CsChrimson) reduces sleep (red line) and this decrease effect was enhanced by LPN inhibition (DvPdf-LexA>LexAop-CsChrimson, LPN-spGal4>Uas-hid; green line). Quantification of relative second-day sleep compared to baseline (G). (H, I) Optogenetic LNd activation caused sleep decrease (DvPdf-Gal4>Uas-CsChrimson, red line) and this decrease effect was enhanced by AstCR1 interference (DvPdf-Gal4>Uas-CsChrimson, Uas-AstCR1 RNAi; green line). The quantification of relative second day sleep compared with the baseline day (I). Data (D) were quantified with unpaired t test. Data (G, I) were analyzed using Welch’s one-way ANOVA. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The raw data in this figure including B, C, D, F, G, H, and I can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3002918.g005 Given that LNds activates LPNs for feedback inhibition, the removal of LPNs’ inhibition should render LNds more excitable. This hypothesis was verified by activating LNds while simultaneously obstructing the incoming LPN signal, either through LPN ablation (Fig 5F and 5G) or reducing AstCR1 expression (Fig 5H and 5I) in LNds. Despite variations in the wake-promoting effects between DvPdf-Gal4>Uas-CsChrimson and DvPdf-LexA>LexAop-CsChrimson, our epistasis results revealed an enhanced ability of LNds to promote wakefulness when LPN input was disrupted. Those data underscore the role of the LNd-LPN negative feedback in maintaining appropriate levels of LNd activation, as the activation of LNds by CsChrimson should override the inhibitory effect of LPNs if there is no excitatory input from LNds to LPNs. In summary, our findings substantiate the presence of a LNd-LPN negative feedback circuitry. LNd exhibit the capability to activate LPN, while LPN, via the AstC-AstCR pathway, exert a negative modulatory influence on LNd activity. This feedback mechanism plays a crucial role in regulating evening activity under seasonal change in nature.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002918

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