Neuropeptidergic circuit modulation of developmental sleep in Drosophila

Reviewer #1 (Public review):

Summary:

The study investigates how neuropeptidergic signaling affects sleep regulation in Drosophila larvae. The authors first conduct a screen of CRISPR knock-out lines of genes encoding enzymes or receptors for neuropeptides and monoamines. As a result of this screen, the authors follow up on one hit, the hugin receptor, PK2-R1. They use genetic approaches, including mutants and targeted manipulations of PK2-R1 activity in insulin-producing cells (IPCs) to increase total sleep amounts in 2nd instar larvae. Similarly, dilp3 and dilp5 null mutants and genetic silencing of IPCs show increases in sleep. The authors also show that hugin mutants and thermogenetic/optogenetic activation of hugin-expressing neurons caused reductions in sleep. Furthermore, they show through imaging-based approaches that hugin-expressing neurons activate IPCs. A key finding is that wash-on of hugin peptides, Hug-γ and PK-2, in ex vivo brain preparations activates larval IPCs, as assayed by CRTC::GFP imaging. The authors then examine how the PK2-R1, hugin, and IPC manipulations affect adult sleep. Finally, the authors examine how Ca2+ responses through CRTC::GFP imaging in adult IPCs are influenced by the wash-on of hugin peptides. The conclusions of this paper are somewhat well supported by data, but some aspects of the experimental approach and sleep analysis need to be clarified and extended.

Strengths:

(1) This paper builds on previously published studies that examine Drosophila larval sleep regulation. Through the power of Drosophila genetics, this study yields additional insights into what role neuropeptides play in the regulation of Drosophila larval sleep.

(2) This study utilizes several diverse approaches to examine larval and adult sleep regulation, neural activity, and circuit connections. The impressive array of distinct analyses provides new understanding into how Drosophila sleep-wake circuitry in regulated across the lifespan.

(3) The imaging approaches used to examine IPC activation upon hugin manipulation (either thermogenetic activation or wash-on of peptides) demonstrate a powerful approach for examining how changes in neuropeptidergic signaling affect downstream neurons. These experiments involve precise manipulations as the authors use both in vivo and ex vivo conditions to observe an effect on IPC activity.

Weaknesses:

Although the paper does have some strengths in principle, these strengths are not fully supported by the experimental approaches used by the authors. In particular:

(1) The authors show total sleep amount over an 18-hour period for all the measures of 2nd instar larval sleep throughout the paper. However, published studies have shown that sleep changes over the course of 2nd instar development, so more precise time windows are necessary for the analyses in this study.

(2) Previously published reports of sleep metrics in both Drosophila larvae and adults include the average number of sleep episodes (bout number) and the average length of sleep episodes (bout length). Neither of these metrics is included in the paper for either the larval sleep or adult sleep data. Not including these metrics makes it difficult for readers to compare the findings in this study to previously published papers in the established Drosophila sleep literature.

(3) Because Drosophila adult & larval sleep is based on locomotion, the authors need to show the activity values for the experiments supporting their key conclusions. They do show travel distances in Figure 2 - Figure Supplement 1, however, it is not clear how these distances were calculated or how the distances relate to the overall activity of individual larvae during sleep experiments. It is also concerning that inactivation of the PK2-R1-expressing neurons causes a reduction in locomotion speed. This could partially explain the increase in sleep that they observe.

(4) The authors rely on homozygous mutant larvae and adult flies to support many of their conclusions. They also rely on Gal4 lines with fairly broad expression in the Drosophila brain to support their conclusions. Adding more precise tissue-specific manipulations, including thermogenetic activation and inhibition of smaller populations of neurons in the study would be needed to increase confidence in the presented results. Similarly, demonstrating that larval development and feeding are not affected by the broad manipulations would strengthen the conclusions.

(5) Many of the experiments presented in this study would benefit from genetic and temperature controls. These controls would increase confidence in the presented results.

(6) The authors claim that their findings in larvae uncover the circuit basis for larval sleep regulation. However, there is very little comparison to published studies demonstrating that neuropeptides like Dh44 regulate larval sleep. Because hugin-expressing neurons have been shown to be downstream of Dh44 neurons, the authors need to include this as part of their discussion. The authors also do not explain why other neuropeptides in the initial screen are not pursued in the study. Given the effect that these manipulations have on larval sleep in their initial screen, it seems likely that other neuropeptidergic circuits regulate larval sleep.

Reviewer #2 (Public review):

Summary:

This study examines larval sleep patterns and compares them to sleep regulation in adult flies. The authors demonstrate hallmark sleep characteristics in larvae, including sleep rebound and increased arousal thresholds. Through genetic and behavioral analyses, they identify PK2-R1 as a key receptor involved in sleep modulation, likely via the HuginPC-IPC signaling pathway. Loss of PK2-R1 results in increased sleep, which aligns with previous findings in hugin knockout mutants. While the study presents significant contributions to the field, further investigation is needed to address discrepancies with earlier research and strengthen mechanistic claims.

Strengths:

(1) The study explores a relatively understudied aspect of sleep regulation, focusing on larval development.

(2) The use of an automated behavioral measurement system ensures precise quantification of sleep patterns.

(3) The findings provide strong genetic and behavioral evidence supporting the role of the HuginPC-IPC pathway in sleep regulation.

(4) The study has broader implications for understanding the evolution and functional divergence of sleep circuits.

Weaknesses:

(1) The manuscript does not sufficiently discuss previous studies, particularly concerning hugin mutants and their metabolic effects.

(2) The specificity of IPC secretion mechanisms is unclear, particularly regarding potential indirect effects on Dilp2.

(3) Alternative circuits, such as the HuginPC-DH44 pathway, require further consideration.

(4) Functional connectivity between HuginPC neurons and IPCs is not directly validated.

(5) Developmental differences in sleep regulatory mechanisms are not thoroughly examined.

Reviewer #3 (Public review):

Summary:

Sleep affects cognition and metabolism, evolving throughout development. In mammals, infants have fast sleep-wake cycles that stabilize in adults via circadian regulation. In this study, the author performed a genetic screen for neurotransmitters/peptides regulating sleep and identified the neuropeptide Hugin and its receptor PK2-R1 as essential components for sleep in Drosophila larvae. They showed that IPCs express Pk2-R1 and silencing IPCs resulted in a significant increase in the sleep amount, which was consistent with the effect they observed in PK2-R1 knock-out mutants. They also showed that Hugin peptides, secreted by a subset of Hugin neurons (Hug-PC), activate IPCs through the PK2-R1 receptor. This activation prompts IPCs to release insulin-like peptides (Dilps), which are implicated in the modulation of sleep. They showed that Hugin peptides induce a PK2-R1 dependent calcium (Ca²⁺) increase in IPCs, which they linked to the release of Dilp3, showing a connection between Hugin signaling to IPCs, Dilp3 release, and sleep regulation. Additionally, the activation of Hug-PC neurons reduced sleep amounts, while silencing them had the opposite effect. In contrast to the larval stage, the Hugin/PK2-R1 axis was not critical for sleep regulation in Drosophila adults, suggesting that this neuropeptidergic circuitry has divergent roles in sleep regulation across different stages of development.

Strengths:

This study used an updated system for sleep quantification in Drosophila larvae, and this method allowed precise measurement of larval sleep patterns which is essential for the understanding of sleep regulation.

The authors performed unbiased genetics screening and successfully identified novel regulators for larval sleep, Hugin and its receptor PK2-R1, making a substantial contribution to the understanding of neuropeptidergic control of sleep regulation.

They clearly demonstrated the mechanism by which Hugin-expressing neurons influence sleep through the activation of IPCs via PK2-R1 with Ca2+ responses and can modulate sleep.

Based on the demonstrated activation of PK2-R1 by the human Hugin orthologue Neuromedin U, research on human sleep disorders may benefit from the discoveries from Drosophila since sleep-regulating mechanisms are conserved across species.

Weaknesses:

The study primarily focused on sleep regulation in Drosophila larvae, showing that the Hugin/PK2-R1 axis is critical for larval sleep but not necessary for adult sleep. The effects of the Hugin axis in the adult are, however, incompletely explained and somewhat inconsistent. PK2-R1 knockout adults also display increased sleep, as does HugPC silencing, at least for daytime sleep. The difference lies in Dilp3/5 mutant animals showing decreased sleep and IPCs seemingly responding with reduced Dilp3 release to PK-2 treatment (Figure 6). It seems difficult to reconcile the author's conclusions regarding this point without additional data. It could be argued that PK2-R1 still regulates adult sleep, but not via Hugin and IPCs/Dilps.

Another issue might be that the authors show relative sleep levels for adults using Trikinetics monitoring. From the methods, it is not clear if the authors backcrossed their line to an isogenic wild-type background to normalize for line-specific effects on sleep. Thus, it is likely that each line has differences in total sleep time due to background effects, e.g., their Kir2.1 control line showed reduced sleep relative to the compared genotypes. This might limit the conclusions on the role of Hugin/PK2-R1 on adult sleep.