Energetic Demands Regulate Sleep-Wake Rhythm Circuit Development

Joint Public Review:

Summary:

This manuscript investigates how energetic demands affect the sleep-wake cycle in Drosophila larvae. L2 stage larvae do not show sleep rhythm and long-term memory (LTM), however, L3 larvae do. The authors manipulate food content to provide insufficient nutrition, which leads to more feeding, no LTM, and no sleep even in older larvae. Similarly, activation of NPF neurons suppresses sleep rhythm. Furthermore, they try to induce a sleep-like state using pharmacology or genetic manipulations in L2 larvae, which can mimic some of the L3 behaviours. A key experimental finding is that activation of DN1a neurons activate the downstream DH44 neurons, as assayed by GCaMP calcium imaging. This occurs only in third instar and not in second instar, in keeping with the development of sleep-wake and feeding separation. The authors also show that glucose metabolic genes are required in Dh44 neurons to develop sleep rhythm and that DH44 neurons respond differently in malnutrition or younger larvae.

Strengths:

Previous studies from the same lab have shown the sleep is required for LTM formation in the larvae, and that this requires DN1a and DH44 neurons. The current work builds upon this observation and addresses in more detail when and how this might develop. The authors can show that low quality food exposure and enhanced feeding during larval stage of Drosophila affects the formation of sleep rhythm and long-term memory. This suggests that the development of sleep and LTM are only possible under well fed and balanced nutrition in fly larvae. Non-sleep larvae were fed in low sugar conditions and indeed, the authors also find glucose metabolic genes to be required for a proper sleep rhythm. The paper presents precise genetic manipulations of individual classes of neurons in fly larvae followed by careful behavioural analysis. The authors also combine thermogenetic or peptide bath application experiments with direct calcium imaging of specific neurons.

Weaknesses:

The authors tried to induce sleep in younger L2 larvae, however the behavioral results suggest that they were not able to induce proper sleep behaviour as in normal L3 larvae. Thus, they cannot show that sleep during L2 stage would be sufficient to form LTM.
The authors suggest that larval Dh44 neurons may integrate "information about the nutritional environment through the direct sensing of glucose levels to modulate sleep-wake rhythm development". They identify glucose metabolism genes (e.g., Glut1) in the downstream DH44 neurons as being required for the organization of the sleep-wake-feeding rhythm, and that CCHa signaling in DN1a signaling to the DH44 cells via the receptor. However, how this is connected is not well explained. Do the authors think that the nutrient sensing is only occurring in the DH44 neurons and not in DN1a or other neurons? Would not knocking down glucose metabolism in any neuron lead to a functional defect? What is the evidence that Dh44 neurons are specific sensors of nutritional state? For example, do the authors think that e.g. the overexpression of Glut1 in Dh44 neurons, a manipulation that can increase transport of glucose into cells, would rescue the effects of low-sugar food?
Some of the genetic controls seem to be inconsistent suggesting some genetic background effects. In Figure 2B, npf-gal4 flies without the UAS show no significant circadian change in sleep duration, whereas UAS-TrpA flies do. The genetic control data in Figure 2D are also inconsistent. Npf-Gal4 seems to have some effect by itself without the UAS. The same is not seen with R76G11-Gal4. Suppl Fig 2: Naïve OCT and AM preference in L3 expressing various combinations of the transgenes show significant differences. npf-Gal4 alone seems to influence preference.
The sleep duration and bout number/length data are highly variable.