Neuronal and molecular bases of circadian, wake, and sleep behaviors in Drosophila melanogaster
Creators: name: Le, Jasmine Quynh
Publisher: name: Brandeis University
Year: 2023
Subjects: Behavior
Drosophila melanogaster
RNA sequencing
Sleep
Circadian Rhythms
Dopamine
Molecular Biology

description: Each day, Earth rotates about its own axis, thereby creating a cycle of light and dark phases across its face. The organisms that live on our world have adapted to these cycling phases and have evolved intricate molecular rhythms of their own that keep their physiological processes operating on time. These rhythms are entrained and strengthened by environmental cues, such as light, but are also able to continue cycling without those cues. In animals, the clearest manifestation of circadian rhythms is the timing of wake and sleep behaviors. This timing is driven by the activity of circadian neurons in the brain. In mammals, approximately 20,000 neurons in the superchiasmatic nucleus are responsible for this. In Drosophila melanogaster, there are only about 150 neurons responsible for timing wake and sleep, making them a tractable model with which to study this phenomenon. Circadian timing in flies is carried out by different subtypes of the ~150 aforementioned neurons firing synchronously during different times of day. Additionally, different neuronal subtypes express distinct genes important for neural communication, such as neurotransmitters, neuropeptides, and G protein-coupled receptors (GPCRs). Here we focus on the role of two circadian lateral neurons (LNs), which have previously been identified as members of the evening cells (E cells). E cells were originally named so because of their necessity for the stereotyped evening locomotor activity peak that Drosophila exhibit. The two LNs in question have been shown to be molecularly distinct for being the only two circadian neurons that express ion transport peptide (ITP). Here we show that these two ITP+ LNs are strongly interconnected with each other and sit at the top of a connectivity hierarchy with the other E cells downstream. Additionally, the ITP+ LNs by themselves are able to strongly promote activity. Based on single-cell RNA sequencing profiling of these neurons around the clock, we were able to identify that they differentially express transcripts for the dopamine receptor Dop1R1 at a higher level in the early morning. Using a CRISPR/Cas9-based method to functionally knock out these receptors, we find a loss of early morning wakefulness, indicating a new role of the ITP+ LNs.
description: On the other side of wake is sleep. In Drosophila, the sleep-promoting dorsal fan-shaped body (dFB) neurons have been shown to be the output arm of the sleep homeostat. In order to understand how these neurons regulate sleep, we assessed their molecular profiles with sleep loss and homeostatic rebound sleep and found that there are changes in multiple neuropeptides and GPCRs under both circumstances. Some of these neuropeptides and GPCRs have previously been identified in the regulation of sleep or circadian rhythms. The function of one receptor, though, has not been characterized in the Drosophila brain: the proctolin receptor (Proc-R). Using RNA interference to knock down the expression of Proc-R, we find that normal sleep promotion by dFB neurons is dependent on this receptor. Additionally, we characterize proctolin as a sleep-promoting neuropeptide because activating proctolin-expressing neurons increases sleep. Lastly, we find that dFB neurons consist of at least two types of neurons, cholinergic neurons and glutamatergic neurons, that also differentially express different neuropeptides. These experiments reveal a molecular mechanism for sleep promotion through dFB neurons and shines a light on their molecular heterogeneity.
description: Circadian, wake, and sleep behaviors are governed by dedicated circuits with neurons that have substantial molecular heterogeneity. Overall, we find that the expression levels of signaling molecules, such as neuropeptides and GPCRs, in as few as two neurons can strongly affect these behaviors.