Connectomic analysis of the Drosophila lateral neuron clock cells reveals the synaptic basis of functional pacemaker classes
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Evaluation Summary:
This paper analyses the synaptic connections of two subsets of clock neurons in the Drosophila brain, the small ventral lateral neurons and the dorsal lateral neurons that control the sleep-wake behavior. The study reveals interesting features of the clock network, including the high heterogeneity of the LNd subset and the existence of non-clock cells that are predicted to act as "inter-clock neurons". The manuscript will be of interest to chronobiologists and neuroscientists working on neuronal networks, and it provides new insights into circadian clock network organization that may be of general value. The data analysis is rigorous, and the conclusions are justified by the data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
The circadian clock orchestrates daily changes in physiology and behavior to ensure internal temporal order and optimal timing across the day. In animals, a central brain clock coordinates circadian rhythms throughout the body and is characterized by a remarkable robustness that depends on synaptic connections between constituent neurons. The clock neuron network of Drosophila , which shares network motifs with clock networks in the mammalian brain yet is built of many fewer neurons, offers a powerful model for understanding the network properties of circadian timekeeping. Here, we report an assessment of synaptic connectivity within a clock network, focusing on the critical lateral neuron (LN) clock neuron classes within the Janelia hemibrain dataset. Our results reveal that previously identified anatomical and functional subclasses of LNs represent distinct connectomic types. Moreover, we identify a small number of non-clock cell subtypes representing highly synaptically coupled nodes within the clock neuron network. This suggests that neurons lacking molecular timekeeping likely play integral roles within the circadian timekeeping network. To our knowledge, this represents the first comprehensive connectomic analysis of a circadian neuronal network.
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Evaluation Summary:
This paper analyses the synaptic connections of two subsets of clock neurons in the Drosophila brain, the small ventral lateral neurons and the dorsal lateral neurons that control the sleep-wake behavior. The study reveals interesting features of the clock network, including the high heterogeneity of the LNd subset and the existence of non-clock cells that are predicted to act as "inter-clock neurons". The manuscript will be of interest to chronobiologists and neuroscientists working on neuronal networks, and it provides new insights into circadian clock network organization that may be of general value. The data analysis is rigorous, and the conclusions are justified by the data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private …
Evaluation Summary:
This paper analyses the synaptic connections of two subsets of clock neurons in the Drosophila brain, the small ventral lateral neurons and the dorsal lateral neurons that control the sleep-wake behavior. The study reveals interesting features of the clock network, including the high heterogeneity of the LNd subset and the existence of non-clock cells that are predicted to act as "inter-clock neurons". The manuscript will be of interest to chronobiologists and neuroscientists working on neuronal networks, and it provides new insights into circadian clock network organization that may be of general value. The data analysis is rigorous, and the conclusions are justified by the data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
By using the Janelia hemibrain dataset, the authors assess the synaptic connectivity of Drosophila's lateral clock neurons in unprecedented detail. As suspected by previous studies, they show remarkable heterogeneity in the connections made by specific subsets of clock neurons. For example, they provide the anatomical basis for previously predicted subsets of clock neurons within evening (E) neurons that are coupled to morning (M) neurons to varying degrees. Of these E neurons, the E2 subgroup is characterized by a particularly strong connection with other clock neurons, suggesting that it acts as a synaptic hub in the clock. In contrast to E neurons, M neurons, which are thought to be the most important clock neurons in the fruit fly circadian clock, have surprisingly few synaptic connections inside and …
Reviewer #1 (Public Review):
By using the Janelia hemibrain dataset, the authors assess the synaptic connectivity of Drosophila's lateral clock neurons in unprecedented detail. As suspected by previous studies, they show remarkable heterogeneity in the connections made by specific subsets of clock neurons. For example, they provide the anatomical basis for previously predicted subsets of clock neurons within evening (E) neurons that are coupled to morning (M) neurons to varying degrees. Of these E neurons, the E2 subgroup is characterized by a particularly strong connection with other clock neurons, suggesting that it acts as a synaptic hub in the clock. In contrast to E neurons, M neurons, which are thought to be the most important clock neurons in the fruit fly circadian clock, have surprisingly few synaptic connections inside and outside the clock network. Instead, they are mainly mutually connected with each other. Furthermore, the few downstream neurons of the M neurons all signal back onto identified clock neurons. The authors hypothesize that these neurons, which are not involved in the clock, may be important for stabilizing the signals of the clock network and thus are an integral part of the network. This hypothesis reminds us of central pattern generators and can be tested in the future. In summary, this well-conducted study provides important new insights into the organization of clock networks that could be of general value.
Strengths:
The study is carefully performed, the methods are described in detail, the results are excellently documented, and all conclusions are justified by the data. In addition, it provides new hypotheses about clock function that can be tested in the future.Weakness:
As true for many anatomical studies, the paper is not easy to read. One reason for the difficult comprehensibility is the use of many abbreviations, another reason is complicated and partly redundant descriptions. -
Reviewer #2 (Public Review):
The paper by Shafer et al. analyzes the connectomic data of the Drosophila clock neurons from the electronic microscopy dataset that has been generated by Janelia Farm. Among the different subsets of neurons, the study focuses on the s-LNvs and the LNds for which all cells (5 and 6 respectively) are included in the Janelia dataset. The analysis reveals very interesting features of the clock network such as the strong connectivity within the clock neuron network of one LNd as well as the 5th PDF-negative sLNv, the higher general connectivity (both input and output) of the LNd + 5th sLNv compared to PDF cells. It also reveals the existence of yet unidentified non-clock neurons that are post-synaptic to some clock neurons and pre-synaptic to others, suggesting the contribution of non-clock cells to the …
Reviewer #2 (Public Review):
The paper by Shafer et al. analyzes the connectomic data of the Drosophila clock neurons from the electronic microscopy dataset that has been generated by Janelia Farm. Among the different subsets of neurons, the study focuses on the s-LNvs and the LNds for which all cells (5 and 6 respectively) are included in the Janelia dataset. The analysis reveals very interesting features of the clock network such as the strong connectivity within the clock neuron network of one LNd as well as the 5th PDF-negative sLNv, the higher general connectivity (both input and output) of the LNd + 5th sLNv compared to PDF cells. It also reveals the existence of yet unidentified non-clock neurons that are post-synaptic to some clock neurons and pre-synaptic to others, suggesting the contribution of non-clock cells to the functioning of the clock network. These findings provide a very useful anatomical basis for future functional studies at the cellular and behavioral levels and predict network features that will be exciting to investigate.
The information is very clearly presented with the relevant tables, graphs, and drawings and the paper is relatively easy to read for such a connectomic analysis.
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Reviewer #3 (Public Review):
In this manuscript, the authors used a publicly available dataset to extract information about connectivity in the circadian network. I found this paper hard to evaluate. It does not rely on newly obtained data, only on the analysis of existing information. I am not an expert in EM data analysis and do not know if the analysis employed here is particularly sophisticated. Even relatively simple analysis could in principle be OK if it leads to significant biological insight. In this case, there are some ideas that are proposed but are not tested, so it is difficult to know what the value of the paper will be in the future. I could see it being relevant for people in the circadian field, but the authors should convince us by experimentally testing at least one of the predictions generated by their analysis.
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