Author response: Connectomic analysis of the Drosophila lateral neuron clock cells reveals the synaptic basis of functional pacemaker classes
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation This paper analyzes the synaptic connections of two subsets of the Drosophila clock neurons, the small ventral lateral neurons, and the dorsal lateral neurons. The study reveals high heterogeneity of the dorsal lateral neurons and the potential involvement of non-clock neurons that might 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. https://doi.org/10.7554/eLife.79139.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Most organisms on Earth possess an internal timekeeping system which ensures that bodily processes such as sleep, wakefulness or digestion take place at the right time. These precise daily rhythms are kept in check by a master clock in the brain. There, thousands of neurons – some of which carrying an internal ‘molecular clock’ – connect to each other through structures known as synapses. Exactly how the resulting network is organised to support circadian timekeeping remains unclear. To explore this question, Shafer, Gutierrez et al. focused on fruit flies, as recent efforts have systematically mapped every neuron and synaptic connection in the brain of this model organism. Analysing available data from the hemibrain connectome project at Janelia revealed that that the neurons with the most important timekeeping roles were in fact forming the fewest synapses within the network. In addition, neurons without internal molecular clocks mediated strong synaptic connections between those that did, suggesting that ‘clockless’ cells still play an integral role in circadian timekeeping. With this research, Shafer, Gutierrez et al. provide unexpected insights into the organisation of the master body clock. Better understanding the networks that underpin circadian rhythms will help to grasp how and why these are disrupted in obesity, depression and Alzheimer’s disease. Introduction Most organisms undergo predictable daily changes in gene expression, physiology, and behavior that are driven by endogenous circadian clocks (Aschoff, 1981; Takahashi et al., 2001). In animals, the molecular feedback loops underlying circadian clocks are present in all organ systems but are required within a relatively small population of brain neurons to maintain daily rhythms in behavior and physiology (Herzog, 2007). In mammals, the hypothalamic suprachiasmatic nuclei (SCN) are the brain’s central circadian clock since the ablation of the SCN or knockouts of the molecular clock within the SCN results in loss of circadian rhythms (reviewed by Herzog et al., 2017). Compared to peripheral tissues, timekeeping in the brain’s central clock is more resilient to the loss of clock gene function and to environmental perturbations. This resilience comes from physiological connections between SCN neurons (Buhr et al., 2010; Liu et al., 2007). Determining how clock neurons are connected is, therefore, essential to understand circadian timekeeping in animals. A fundamental challenge to addressing this question is the complexity of the circadian neuronal networks. The paired mammalian SCN consists of tens of thousands of neurons with diverse neurochemical outputs that form a complex network (reviewed by Hastings et al., 2019). The clock neuron network of Drosophila melanogaster offers a powerful model to understand the network properties of neural circadian timekeeping. The molecular clocks of insects and mammals are highly conserved and their neuronal networks appear to share common motifs (Nitabach and Taghert, 2008). The fly’s clock neuron network consists of relatively few neurons that can be organized in a small number of discrete anatomical classes (Shafer et al., 2006; Figure 1A). Furthermore, genetic tools to specifically manipulate subclasses of the fly circadian network have made it possible to understand the functional roles of these subclasses in the production of endogenous circadian rhythms in sleep and activity (Ahmad et al., 2021; Shafer and Keene, 2021). Though transgenic markers of synaptic connectivity and live-imaging-based assessments of functional connectivity have been used to address neural connectivity within the clock neuron network (e.g. Reinhard et al., 2022a; Yao et al., 2012), a systematic assessment of synaptic connectivity within this network is lacking. Figure 1 Download asset Open asset The circadian clock neuron network and identified clock neurons in the hemibrain. (A) The circadian clock network is shown in both hemispheres. Currently identified cells are shown in color (s-LNvs red, l-LNvs light red, LNds in orange, DN1a in purple, a subset of DN1p in blue) and known clock cells not yet identified within the hemibrain connectome data are shown in gray (DN2, DN3, and a subset of DN1p). The pars intercerebralis (PI) and optic lobe (OL) are indicated in gray. (B) Identified clock neurons in the hemibrain. Neuropils are shown in gray. Color codes as indicated in (A). (C) Heatmap indicates synaptic connections strength among all identified circadian clock neurons, including weak (weight <3) connections. (D) The timekeeping lateral neurons that compose the morning (M) and evening (E) Oscillators: s-LNvs (M cells), and 5th LNv and LNds (E cells). The comprehensive mapping of synaptic connectivity using serial electron microscopy and reconstruction of neuronal volumes allows us to assess the network structures of brain circuits (Kasthuri et al., 2015). Scheffer et al., 2020 provided a dense reconstruction of chemical synapses within a large portion of the fly’s central brain, called the hemibrain. Annotation of the Janelia hemibrain connectome contains most of the key anatomical subsets of the fly’s clock neuron network and powerful computational tools are available to navigate this connectome (Lazar et al., 2021; Scheffer et al., 2020) making it possible to analyze the structure of the fly’s brain clock network. The Drosophila Circadian Neuron Network is a group of ~150 neurons that display synchronous oscillation in PER and TIM abundance and can be subdivided based on gene expression, anatomy, and location in the brain (Helfrich-Förster, 2005). These are (i) the lateral neurons (LNs), which can be subdivided into ventral lateral (LNv) and dorsal lateral (LNd) neurons, (ii) the lateral posterior neurons (LPNs), and (iii) the dorsal neurons (DNs), which can be subdivided into dorsal neurons 1, 2, and 3 (DN1, DN2, and DN3) and further subdivided into anterior (DN1a) and posterior (DN1p) DN1s (Helfrich-Förster et al., 2007b; Helfrich-Forster, Yoshii, Helfrich-Förster et al., 2007a; Shafer et al., 2006). The LNvs release the key circadian neuropeptide Pigment Dispersing Factor (PDF) and can be further subdivided into small and large LNvs (s-LNvs and l-LNvs). Release of PDF from the s-LNvs, which takes place in the dorsal protocerebrum where the LNds and DNs reside, is required for endogenous circadian timekeeping (Park et al., 2000; Renn et al., 1999). The current annotation of the hemibrain dataset identifies all the expected lateral neuron classes: four l-LNvs, four s-LNvs, the 5th-s-LNv, and six LNds (Scheffer et al., 2020; Figure 1B and C). However, some clock neurons have not yet been unequivocally identified within the hemibrain dataset: approximately half of the DN1ps (Reinhard et al., 2022a; Scheffer et al., 2020) and the DN2s and DN3s. The LN classes of clock neurons represent the minimum sub-network required to produce endogenous bimodal rhythm of sleep and activity. A functional molecular clock only in the s-LNvs, 5th s-LNv, and LNds is sufficient to produce such rhythms (Grima et al., 2004; Rieger et al., 2006). Here, we report an assessment of chemical synaptic connectivity for the critical LN clock neuron classes within the hemibrain volume. We have determined the patterns of synaptic connectivity within this core LN circadian sub-network and the connectivity with other clock neurons annotated within the hemibrain dataset. This analysis provides a significant and comprehensive insight into the synaptic circuitry underlying the organization and function of the Drosophila central brain clock. Our results reveal that previously identified anatomical and functional subclasses of LNs are distinct connectomic classes that receive unique sources of synaptic inputs and outputs. Furthermore, our analysis reveals a remarkable heterogeneity in how clock neuron subclasses form connections to other clock neurons and identifies a small number of clock cell subtypes that are highly synaptically coupled nodes. Finally, we find ‘non-clock neuron’ targets which themselves form synapses back onto clock neurons. We propose that those non-clock cells are important components of the timekeeping network. Results The clock neuron network and the hemibrain dataset Representatives of all classes of clock neurons, with the exception of the DN2 and DN3 classes, have been identified in the hemibrain volume used here (Figure 1A and B). See Table 1 for the naming scheme used here for the clock neurons identified in the dataset with their corresponding unique body IDs. The patterns of connectivity among some of these neurons have recently been briefly described (Ahmad et al., 2021; Reinhard et al., 2022a; Reinhard et al., 2022b). Scheffer and colleagues (Scheffer et al., 2020) define synaptic strength within the hemibrain volume by the number of synaptic connections formed between neurons, defining connections consisting of only one or two chemical synapses as weak and subject to error, three to nine synapses as medium strength, and of ten or more synapses as strong. We used these definitions of synaptic strength in this study. As a whole, the identified clock neurons appear to be sparsely interconnected by chemical synapses. Most clock neuron pairs form either no synapses or weak synaptic connections (Figure 1C). However, there are a few clear exceptions: a single LNd (LNd6) and the 5th s-LNv form strong connections with one another and with members of the DN1 group (Figure 1C; see also Reinhard et al., 2022a) and stand out among the clock neurons as hubs of inter-clock synaptic connectivity. Table 1 Identification of Clock Neurons within the Hemibrain Volume. BodyIdTypeSequential labelSubphase2068801704s-LNvs-LNv1M1664980698s-LNvs-LNv2M2007068523s-LNvs-LNv3M1975347348s-LNvs-LNv4M5813056917LNdLNd4E15813021192LNdLNd5E15813069648LNdLNd6E25110514775th LNv5th LNvE2296544364LNdLNd1E3448260940LNdLNd2E35813064789LNdLNd3E3356818551LPNLPN1480029788LPNLPN2450034902LPNLPN3546977514LPNLPN4264083994DN1aDN1a15813022274DN1aDN1a25813010153DN1pADN1pA1324846570DN1pADN1pA2325529237DN1pADN1pA3387944118DN1pADN1pA4387166379DN1pADN1pA5386834269DN1pBDN1pB15813071319DN1pBDN1pB21884625521l-LNvlLNv12065745704l-LNvlLNv25813001741l-LNvlLNv35813026773l-LNvlLNv4 To asses synaptic connectivity in the Drosophila clock network, we focused on three critical classes of lateral clock neurons (LNs); the four Pigment Dispersing Factor (PDF) -expressing small ventral lateral neurons (s-LNvs), the six dorsal lateral neurons (LNds) and the PDF negative 5th s-LNv (Figure 1D). We have chosen to focus on these clock neuron classes for two reasons. First, these classes are completely accounted for in the hemibrain annotation used here. Second, the 11 neurons that comprise the LN class are sufficient to drive the fly’s endogenous bimodal rhythm in locomotor activity (Grima et al., 2004; Rieger et al., 2006) and therefore represent a critical sub-network within the fly’s circadian system. The small LNvs are a highly unified connectomic class distinct from the 5th s-LNv The s-LNvs have long been considered critical circadian pacemakers within the Drosophila clock neuron network (Helfrich-Förster, 1998). These cells express the neuropeptide PDF (Helfrich-Förster, 1995; Renn et al., 1999), maintain strong molecular timekeeping under constant conditions (Shafer et al., 2002; Yang and Sehgal, 2001) and are required for robust endogenous circadian rhythms (Grima et al., 2004; Shafer and Taghert, 2009). The s-LNvs also contribute to the morning peak of the fly’s crepuscular daily activity rhythm (Grima et al., 2004; Stoleru et al., 2004). In addition to PDF, these cells also release the neuropeptide short Neuropeptide F (sNPF) (Johard et al., 2009) and the neurotransmitter glycine (Frenkel et al., 2017). Hyper excitation of the s-LNvs leads to a morning-like gene expression profile for many circadian genes, while hyperpolarization leads to an evening-like transcriptional profile (Mizrak et al., 2012). Though by no means the only clock neurons capable of producing an endogenous sense of time (Delventhal et al., 2019; Schlichting et al., 2019; Yao and Shafer, 2014), a large body of evidence supports the notion that s-LNvs are among the most critical neurons for the maintenance of endogenous circadian rhythms (reviewed by Ahmad et al., 2021). The 5th s-LNv was named because it was initially thought to be anatomically similar to the PDF positive s-LNvs in the larval brain (Kaneko and Hall, 2000; Kaneko et al., 1997; Rieger et al., 2006). However, subsequent work has suggested that the 5th s-LNv is likely functionally and anatomically more akin to the LNds (Grima et al., 2004; Schubert et al., 2018; Yao and Shafer, 2014). The four PDF-expressing s-LNvs are characterized by a relatively simple morphology (Figure 2A–C; Helfrich-Förster et al., 2007b). Their cell bodies are located in the ventral brain, near the accessory medulla (AMe), into which they extend short neurites which likely receive input from photoreceptors (Helfrich-Förster et al., 2002; Malpel et al., 2002). These four sLNvs project dorsally to the posterior dorsal protocerebrum, where they turn toward the midline and form fine ramified termini that extend toward the midline (Helfrich-Förster, 1997; Helfrich-Förster et al., 2007b). These dorsal ramifications are thought to be the major site of s-LNv synaptic output but are also known to contain synaptic inputs (Yasuyama and Meinertzhagen, 2010). Work by Schubert et al., 2018 showed that the 5th s-LNv (Figure 2F), which expresses the neuropeptide ion transport peptide (ITP) and choline acetyltransferase (Johard et al., 2009), an enzyme involved in the biosynthesis of acetylcholine, has more extensive ramifications within the dorsal brain and accessory medulla than the PDF expressing s-LNvs and provided evidence that they are anatomically more akin to LNds than to s-LNvs. Based on these findings, Schubert et al. suggested that the 5th sLNv be reclassified as a LNv (Schubert et al., 2018), a convention we follow for the remainder of this study. Based on this work, we predict that the 5th LNv within the hemibrain will be connectomically distinct from the PDF expressing s-LNvs. Figure 2 with 1 supplement see all Download asset Open asset Connectivity patterns of the s-LNvs. (A–C). Synaptic connections of the four pdf + s LNvs, s-LNv_R_1 through s-LNv_R_4. Neuronal morphology is shown in gray. In A and B, inputs to the s-LNvs are shown in blue, outputs are shown in magenta. (A) All connections, including non-clock cells, (B) Connections to/from clock cells only, excluding connections to s-LNvs, (C) Connections among the four pdf + s LNvs. Input and output sides coincide and are indicated in orange. (D–E) Sankey diagram indicating the strong synaptic partners of all s-LNvs, including the 5th LNv. The total weight of synapses formed by each cell with its inputs (D) or outputs (E) is shown. (D) Presynaptic partners (inputs) of s-LNvs. No shared connections were found between the pdf + s LNvs and the 5th LNv. (E) Post synaptic partners (outputs) of s-LNvs. Only one cell (Body ID 571372889) receives synaptic input from the 5th LNv plus a pdf + s LNv. (F–G) Synaptic connections of the 5th LNv. (F) All connections, including non-clock cells, (G) connections to clock cells only. (H) Connectivity map of the four pdf + s LNvs indicating their peptide and receptor expression. The four PDF-expressing s-LNvs and the 5th LNv have been identified within the hemibrain dataset (Figure 1B–C and Figure 2A and F; Scheffer et al., 2020). Visualization of T-bars and postsynaptic densities within these neurons reveals that their ventral neurites, which innervate the accessory medulla, are biased toward receiving synaptic input (Figure 2A and F). Individual PDF-positive s-LNvs display relatively simple dorsal medial termini with relatively few branch-points that tend to run in parallel to neighboring branches (Figure 2—figure supplement 1). As previously described (Yasuyama and Meinertzhagen, 2010), these dorsal medial s-LNv termini contain both presynaptic and postsynaptic structures with the former significantly outnumbering the latter (Figure 2A and Figure 2—figure supplement 1). The dorsal termini of the 5th LNvs also contains both pre- and postsynaptic structures but appear to be less biased toward output compared to the PDF expressing s-LNvs (Figure 2F). Taken together, the four pdf +s LNvs form a total of 2238 synapses within the hemibrain volume: 505 inputs (postsynaptic densities) and 1733 outputs (projections onto postsynaptic densities). The single 5th s-LNv contains 1413 postsynaptic densities and forms synapses onto 1992 postsynaptic densities. Thus, the 5th LNv forms about four times the number of synapses of a single PDF-positive s-LNv (Figure 4C; Supplementary file 1, Supplementary file 2). The four pdf +s LNvs display uniformity in their synaptic inputs for strong synaptic connections. Only three neurons in the hemibrain provide ten or more synapses onto at least one of these cells (Figure 2D), and only two of those three neurons form strong connections onto all four sLNvs (Figure 4D; Supplementary file 1). They also appear to be remarkably uniform in their patterns of strong synaptic output. Only 11 neurons receive 10 or more synapses from at least one pdf +s LNv (Figure 2E). Compared to the other LN classes, these s-LNvs are highly similar to each other in both their strong and medium-strength connections (Figure 4A, B) and form few but nearly uniform patterns of strong shared synaptic connections (Figure 4). Notably, within the hemibrain volume, the pdf +s LNvs are not strongly connected to other clock neurons, though they do form medium strength connections with one another (Figures 1C and 2H). The 5th LNv displays patterns of strong synaptic connectivity that are almost completely distinct from those of the four pdf +s LNvs, sharing a single strong output connection with only one of these cells (Figure 2E). Though quite uniform in their patterns of strong synaptic input and outputs, the pdf +s LNvs do display some within-class differences in their patterns of weak and medium strength connections. the inputs only one s-LNv, and mediated by only three is the a the model that this provides input onto s-LNvs and to light of circadian rhythms (Helfrich-Förster et al., 2002; Malpel et al., 2002; Rieger et al., though recent work has indicated that the to s-LNv connection may be et al., The connectivity between the and the s-LNvs found within the hemibrain is with recent work suggesting that the to light et al., 2019). our analysis supports the notion that the pdf +s LNvs represent a uniform connectomic cell with to patterns of strong synaptic connections, with recent work a uniformity in gene expression across the s-LNvs et al., 2021). A recent connectomic analysis of the larval brain similar conclusions the uniformity of anatomy, and connections among the four larval pdf et al., which through to the s-LNvs (Helfrich-Förster, The LNds comprise connectomic subclasses As their the of the dorsal lateral neurons (LNds) are dorsally to the LNvs, in the lateral cell body (Helfrich-Förster et al., 2007b; Kaneko and Hall, 2000; Schubert et al., All six LNds dorsal medial across the protocerebrum and three an toward the ventral brain (Schubert et al., 2018; Figure 3 and Figure supplement 1). Though consisting of only two more neurons than the PDF-positive s-LNv the LNds form times more synaptic connections postsynaptic densities and to Figures and are characterized by a number of strong synaptic are distinct neurons that receive strong synaptic inputs from at least one LNd to 11 neurons for pdf +s Supplementary file and distinct neurons provide strong synaptic input onto at least one LNd to three for Supplementary file 2). Figure 3 with 2 see all Download asset Open asset Connectivity patterns of the (A–C). Synaptic connections of the six Neuronal morphology is shown in gray. In A and B, inputs to the s-LNvs are shown in blue, outputs are shown in magenta. (A) All connections, including non-clock cells, (B) Connections to clock cells only, excluding connections to Input and output sides coincide and are indicated in orange. (C) Connections within (D) Connectivity map of the six LNds indicating their peptide and receptor expression. Sankey diagram indicating the strong synaptic partners of all The total weight of synapses formed by each cell with its inputs (E) or outputs (F) is shown. (E) Presynaptic partners of No shared connections were found between and Most of the shared connections are between and cells provide strong inputs to (F) partners of the Figure with 2 see all Download asset Open asset cells are while cells can be in three distinct for in synaptic partners of and Only synaptic partners that medium or strong connections. more in either inputs (A) or outputs (C) input and output for and (D–E) shared connections of the four pdf +s LNvs. Only cells that share one connection with at least two cells are shown. The strength of the connection is Only medium and strong connections are (D) The two cells that strong connections to at least two cells strong connections to all (E) The six cells that receive strong connections from at least two cells receive strong connections from all (F–G) shared connections of the six LNds plus the 5th LNv to as cells). Only cells that share a strong connection with at least two cells are shown. The strength of the connection is Only medium and strong connections are (F) that strong connections to at least two cells are in the (G) that receive strong connections from at least two cells are in the In addition to forming more synapses with more neurons than the PDF-positive s-LNvs, the LNds are less uniform in their patterns of strong synaptic connections (Figures is not a single of strong synaptic input or a single neural of synaptic output that forms strong connections with all six LNds (Figure and Supplementary file 1, Supplementary file 2). The number of LNds that receive strong synaptic inputs from the presynaptic neuron is three (Figure The number of LNds that form strong synaptic outputs onto the neuronal targets is also three (Figure Based on these patterns of shared strong there appear to be two connectomic within the each consisting of three LNds and LNds LNds within these two are more similar in their patterns of strong synaptic connectivity to each other than to LNds of the other group (Figures and F and and These two to a in the number of their synaptic connections, with the LNd group forming approximately the number of synaptic connections (Figure 4C; Figure supplement 1 and Supplementary file 1, Supplementary file 2). the morphology of the LNds within the hemibrain volume reveals that LNds extend both and ventral LNds extend only (Figure supplement 1). As shown by Schubert et al., this indicates that LNds are the expressing while LNds to the LNds (Figure and LNds in their expression of receptor with the LNds expressing and the LNds lacking receptor expression et al., suggesting that these two of LNd are to PDF from the l-LNvs and s-LNvs. Though the and of the LNds appear to be two distinct connectomic patterns of strong synaptic connectivity that the LNds can be further based on their patterns of strong synaptic connections. the and share strong synaptic inputs with one but only strong connections with (Figure and share strong synaptic targets with one another but only or fewer with (Figure Thus, the LNds appear to of at least two connectomic and This connectomic is with differences in neuropeptide expression within the with two LNds expressing short neuropeptide F (sNPF) and the expressing ion transport peptide a it shares with the expressing 5th LNv (Johard et al., 2009). A similar can be made for the and in of patterns of strong synaptic connections (Figure and F). Based on these patterns of strong synaptic connections, the LNd class to of four distinct connectomic types. This number the number of classes of LNds recently revealed by single cell et al., 2021). The four connectomic of LNd clock neurons to previously identified and functional clock subsets The LNds in their patterns to the s-LNvs and Shafer, in addition to differences in their expression (Figure The two positive which express and coupled to the s-LNvs the circadian clock in the latter neurons is and Shafer, 2014). The expressing LNds were based on this temporal to the s-LNvs and Shafer, 2014), which also express (Johard et al., 2009). The expressing to PDF from the LNvs, not display to the s-LNv clocks and was with the 5th s-LNv as and Shafer, 2014). The which of the were as which are PDF coupled to s-LNvs with clock and Shafer, 2014). The therefore, appear to of two functional two LNds making and a single LNd corresponding to Schubert et al., 2018 the anatomical of these two with the single LNd more extensive ventral
Source: eLife
Language: English
Subfield: Endocrine and Autonomic Systems
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
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