Circadian photoreceptor CRYPTOCHROME promotes wakefulness under short winter-like days via a GABAergic circuitry

Reviewer #1 (Public Review):

Summary:
In this paper, Chen et al. identified a role for the circadian photoreceptor CRYPTOCHROME (cry) in promoting wakefulness under short photoperiods. This research is potentially important as hypersomnolence is often seen in patients suffering from SAD during winter times. The mechanisms underlying these sleep effects are poorly known.

Strengths:
The authors clearly demonstrated that mutations in cry lead to elevated sleep under 4:20 Light-Dark (LD) cycles. Furthermore, using RNAi, they identified GABAergic neurons as a primary site of cry action to promote wakefulness under short photoperiods. They then provide genetic and pharmacological evidence demonstrating that cry acts on GABAergic transmission to modulate sleep under such conditions.

Weaknesses:
The authors then went on to identify the neuronal location of this cry action on sleep. This is where this reviewer is much more circumspect about the data provided. The authors hypothesize that the l-LNvs which are known to be arousal-promoting may be involved in the phenotypes they are observing. To investigate this, they undertook several imaging and genetic experiments.

Major concerns:
1. Figure 2 A-B: The authors show that knocking down cry expression in GABAergic neurons mimics the sleep increase seen in cryb mutants under short photoperiod. However, they do not provide any other sleep parameters such as sleep bout numbers, sleep bout duration, and more importantly waking activity measurements. This is an essential parameter that is needed to rule out paralysis and/or motor defects as the cause of increased "sleep". Any experiments looking at sleep need to include these parameters.

2. For all Figures displaying immunostaining and imaging data the resolution of the images is quite poor. This makes it difficult to assess whether the authors' conclusions are supported by the data or not.

3. In Figure 4-S1A it appears that the syt-GFP signal driven by Gad1-GAL4 is colabeling the l-LNvs. This would imply that the l-LNvs are GABAergic. The authors suggest that this experiment suggests that l-LNvs receive input from GABAergic neurons. I am not sure the data presented support this.

4. In Figure 4-S1B. The GRASP experiment is not very convincing. The resolution of the image is quite poor. In addition, the authors used Pdf-LexA to express the post t-GRASP construct in l-LNvs, but Pdf-LexA also labels the s-LNvs, so it is possible that the GRASP signal the authors observe is coming from the s-LNvs and not the l-LNvs. The authors could use a l-LNvs specific tool to do this experiment and remove any doubts. Altogether this reviewer is not convinced that the data presented supports the conclusion "All in all, these results demonstrate that GABAergic neurons project to the l-LNvs and form synaptic connections." (Line 176). In addition, the authors could have downregulated the expression of Rdl specifically in l-LNvs to support their conclusions. The data they are providing supports a role for RDL but does not prove that RDL is involved in l-LNvs.

5. In Figures 4 A and C: it appears that GABA is expressed in the l-LNvs. Is this correct? Can the authors clarify this? Maybe the authors could do an experiment where they co-label using Gad1-GAL4 and Pdf-LexA to clearly demonstrate that l-LNvs are not GABAergic. Also, the choice of colors could be better. It is very difficult to see what GABA is and what is PDF.

6. Figure 4G: Pdf-GAL4 expresses in both s-LNvs and l-LNvs. So, in this experiment, the authors are silencing both groups, not only the l-LNvs. Why not use a l-LNvs specific tool?

7. Figure 4H-I: The C929-GAL4 driver expresses in many peptidergic neurons. This makes the interpretation of these data difficult. The effects could be due to peptidergic cells being different than the l-LNvs. Why not use a more specific l-LNvs specific tool? I am also confused as to why some experiments used Pdf-GAL4 and some others used C929-GAL4 in a view to specifically manipulate l-LNvs? This is confusing since both drivers are not specific to the l-LNvs.

8. Figure 5-S1B: Why does the pdf-GAL80 construct not block the sleep increase seen when reducing expression of cry in Gad1-GAL4 neurons? This suggests that there are GABAergic neurons that are not PDF expressing involved in the cry-mediated effect on sleep under short photoperiods.

In conclusion, it is not clear that the authors demonstrated that they are looking at a cry-mediated effect on GABA in s-LNvs resulting in a modulation of the activity of the l-LNvs. Better images and more-suited genetic experiments could be used to address this.

Reviewer #2 (Public Review):

Summary:
The sleep patterns of animals are adaptable, with shorter sleep durations in the winter and longer sleep durations in the summer. Chen and colleagues conducted a study using Drosophila (fruit flies) and discovered that a circadian photoreceptor called cryptochrome (cry) plays a role in reducing sleep duration during day/night cycles resembling winter conditions. They also found that cry functions in specific GABAergic circadian pacemaker cells known as s-LNvs inhibit these neurons, thereby promoting wakefulness in the animals in the winter. They also identified l-LNvs, known as arousal-promoting cells, as the downstream neurons.

Strengths:
Detailed mapping of the neural circuits cry acts to mediate the shortened sleep in winter-like day/night cycles.

Weaknesses:
The supporting evidence for s-LNvs being GABAergic neurons is not particularly strong. Additionally, there is a lack of direct evidence regarding changes in neural activity for s-LNvs and l-LNvs under varying day/night cycles, as well as in cry mutant flies.

Reviewer #3 (Public Review):

Summary:
In humans, short photoperiods are associated with hypersomnolence. The mechanisms underlying these effects are, however, unknown. Chen et al. use the fly Drosophila to determine the mechanisms regulating sleep under short photoperiods. They find that mutations in the circadian photoreceptor cryptochrome (cry) increase sleep specifically under short photoperiods (e.g. 4h light : 20 h dark). They go on to show that cry is required in GABAergic neurons. Further, they suggest that the relevant subset of GABAergic neurons are the well-studied small ventral lateral neurons that they suggest inhibit the arousal-promoting large ventral neurons via GABA signalling.

Strengths:
Genetic analysis to show that cryptochrome (but not other core clock genes) mediates the increase in sleep in short photoperiods, and circuit analysis to localise cry function to GABAergic neurons.

Weaknesses:
The authors' conclusion that the sLNvs are GABAergic is not well supported by the data. Better immunostaining experiments and perhaps more specific genetic driver lines would help with this point (details below).

1. The sLNvs are well known as a key component of the circadian network. The finding that they are GABAergic would if true, be of great interest to the community. However, the data presented in support of this conclusion are not convincing. Much of the confocal images are of insufficient resolution to evaluate the paper's claims. The Anti-GABA immunostaining in Fig 4 and 5 seem to have a high background, and the GRASP experiments in Fig 4 supplement 1 low signal.

Transcriptomic datasets are available for the components of the circadian network (e.g. PMID 33438579, and PMID 19966839). It would be of interest to determine if transcripts for GAD or other GABA synthesis/transport components were detected in sLNvs. Further, there are also more specific driver lines for GAD, and the lLNvs, sLNVs that could be used.

2. The authors' model posits that in short photoperiods, cry functions to suppress GABA secretion from sLNvs thereby disinhibiting the lNVs. In Fig 4I they find that activating the lLNvs (and other peptidergic cells) by c929>NaChBac in a cryb background reduces sleep compared to activating lLNVs in a wild-type background. It's not clear how this follows from the model. A similar trend is observable in Fig 4H with TRP-mediated activation of lNVs, although it is not clear from the figure if the difference b/w cryb vs wild-type background is significant.