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Sleep; Drosophila; Ontogeny development

Introduction

Sleeping time peaks in adolescence and decreases with age across all species. 1, 2, 3, 4, 5 Early life sleep is also distinguished from mature sleep by changes in sleep architecture. For instance, in humans, newborn infants experience the highest levels of sleep duration and REM sleep percentage, which both decline with age. 4 Early life sleeps is essential for healthy neurodevelopment, according to a number of lines of research. 2, 6, 7, 8, 9, 10, 11 Thus, juvenile sleep may have traits that meet particular requirements for nervous system development. The mechanisms underpinning sleep ontogeny, or the evolution of sleep characteristics, remain mainly unknown. [1].

Methods

Drosophila

The likelihood of waking up during sleep affects how long you sleep. These changes are regulated by the interaction of the neurological substrates that regulate sleep. 12, 13, and 14 Additionally, transitions between various sleep stages are visible in both mammals and invertebrates like the Drosophila melanogaster and are identified by electrophysiologic and behavioural studies. 15, 16, 17, 18, 19, 20, 21, 22 Hidden Markov modelling (HMM) of sleep/wake substates and conditional probabilities of activity/inactivity state transitions have both been shown to be effective, non-invasive techniques for examining the neurobiology underlying sleep architecture in Drosophila. 23 It has not yet been investigated how to apply such methods to a thorough examination of sleep/wake transitions and sleep states in young flies.

How do changes in sleep architecture across the lifespan affect the growth of sleep-regulatory circuits? Sleep ontogenetic alterations occur in flies as a result of the core complex of the brain’s important sleep circuit maturing. Particularly, when compared to mature flies, juvenile flies show higher activity in the dorsal fan-shaped body (dFB) sleeppromoting neurons. 2 The maturation of dopaminergic (DA) inputs that suppress dFB activity is one factor influencing this modification in sleep output. 24, 25, 26 In comparison to mature flies, these DA inputs are both less frequent and less active in juvenile flies, which results in higher dFB activity. 2, 27 However, it is unknown if dFB neurons that promote sleep also go through intrinsic maturation [2, 3].

We address the issue of how sleep architecture varies between juvenile and mature Drosophila using a conditional probability approach applied to locomotor data and HMM of sleep/wake substates23. We discover that in young flies, excessive sleep is mostly caused by a lower likelihood of flies transitioning out of sleep. Additionally, compared to mature flies, juvenile flies spend proportionally more time in a deep sleep state. In mature flies, stimulation of the R23E10-GAL4-defined sleep-promoting neurons lengthens sleep duration while producing a sleep architecture different from that of the juvenile sleep state. On the other hand, in young flies, inhibition of the same dFB neurons does not lead to the development of adult fly sleep architecture. Finally, we discover that the dFB displays distinctive chemical signatures throughout the sleep maturation phase, supporting the notion.

Finally, we discover that the dFB displays distinctive molecular signatures throughout the sleep maturation period, supporting the notion that the dFB plays a changing role throughout development. Ringer is linked to the morphological and functional maturation of dFB sleep neurons, according to an RNAi-based analysis of differentially expressed genes (DEGs). Our findings suggest that intrinsic maturation of sleep output neurons influences the ontogenetic alterations related to sleep.

We used a high-resolution multibeam Drosophila activity monitoring (DAM) system to record sleep in unmated iso31 female flies in order to examine how sleep/wake transition probabilities varied between juvenile (1 day post-eclosion) and mature (5-7 days posteclosion) adult flies. In line with earlier studies2,3,28, we found that juvenile flies sleep longer on average overall, have longer average sleep bouts, and have fewer sleep bouts both during the day (ZT0-12) and night (ZT12-24) (Figures 1A–1C and S1A). These measurements are consistent with juvenile flies sleeping more consolidatedly than mature flies. The chance of changing from an inactive to an active state is known as P(wake), whereas the probability of changing from an active to an inactive state is known as P(doze) [4,5,6].

23 Juvenile flies’ P (wake) dramatically decreased both throughout the day and at night (Figures 1D and S1B), indicating that their longer sleep periods are caused by a less likely chance of waking up. In juvenile flies, P(doze) was generally lowered both during the day and at night (Figure 1E); however, this measure showed much greater temporal variability, with particular times when juvenile flies showed increased P(doze) (for example, ZT3–6 and ZT15–18; Figure S1C). Our observation that P(wake) is continuously decreased in juvenile flies and drives greater sleep duration is consistent with previous research, which has shown that P(doze) is less closely connected with sleep length than P(wake)23. Additionally, while measuring P(doze), we found that juvenile flies exhibited greater variance than mature flies [7,8,9].

Discussion

Fly brains were dissected in 1xPBS, then fixed for 20 minutes at room temperature in 4% PFA in PBS with 0.3% Triton-X 100 (PBST). Brains were removed at ZT8 for CaLexA experimentation. Brains were incubated overnight at 4°C with rabbit anti-GFP primary antibody (Invitrogen, Cat# A11122) at a dose of 1:500 after 3x10 minute washes in PBST. Brains were rinsed three times for ten minutes in PBST before being incubated for two hours at room temperature with donkey antirabbit Alexa Fluor 488 (Thermo Fisher). Brains were cleaned in 50% glycerol and mounted in Vectashield after three PBST washes lasting 10 minutes each [10].

Acknowledgement

We are grateful for the support of the PWAN management, the Adamawa state ministry of education, the village heads and teachers who allowed us to conduct the study, as well as our respondents who cooperated.

Potential Conflict of Interest

No conflict or competing interests in the publication of this paper.

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