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.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  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. Most organisms undergo predictable daily changes in gene expression, physiology, and behavior that are driven by endogenous circadian clocks . In animDrosophila 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  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 . The curDrosophila 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.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 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 . See TabDrosophila 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  are situated dorsally relative to the LNvs, residing in the lateral cell body rind . All sixIn addition to forming more synapses with more neurons than the PDF-positive s-LNvs, the LNds are markedly less uniform in their patterns of strong synaptic connections , 4F, G. Examining the cellular morphology of the LNds within the hemibrain volume reveals that LNds 4–6 extend both dorsal-medial and ventral projections, whereas LNds 1–3 extend only dorsal-medial projections . As showThough the Cry-positive and Cry-negative subgroups of the LNds appear to be two distinct connectomic subtypes, patterns of strong synaptic connectivity suggest that the LNds can be further divided based on their patterns of strong synaptic connections. Within the Cry-positive LNds, LNd4, and LNd5 share 27 strong synaptic inputs with one another, but only seven strong connections with LNd6 . SimilarThe LNds vary in their coupling patterns to the s-LNvs  in additThe Cry-positive LNds, therefore, appear to consist of two functional types; two LNds making up E1 and a single LNd corresponding to E2. The 5th LNv, which shares cellular and anatomical similarities with LNd6 , was assThe patterns of strong clock neuron connectivity within the hemibrain led us to hypothesize that the recognized functional subtypes, M (PDF-positive s-LNvs), E1 (LNd4 and LNd5), E2 (LNd6 and 5th LNv), and E3  display distinct patterns of synaptic connectivity. To test this hypothesis, we expanded our analysis to include both strong and medium strength synaptic connections, which together account for the vast majority of connections made by the identified clock neurons within the hemibrain dataset. To examine affinities between individual identified clock neurons in our expanded consideration of connectivity, we used the Jaccard index , a coeffThese functional LN divisions also have different numbers of synaptic connections and different ratios of input to output synapses. The M group displays the smallest number of synapses, E3 an intermediate number, and E1 and E2 form roughly twice the number of connections displayed by E3 . The M aIn animals, the brain’s endogenous central circadian clock drives myriad behavioral, physiological, and endocrine rhythms. Synaptic connections between the central clock network and neurons outside the timekeeping network are thought to mediate daily signals from the circadian system to the neural centers responsible for driving daily physiological and behavioral changes. Though specific output pathways linking the clock to specific endocrine and sleep control centers have been previously described in the fly  and much of their synaptic inputs are thought to be located in regions of the medulla that are not included in the hemibrain volume  are clear outliers for synaptic connectivity with other clock neurons, forming strong synaptic connections onto E1 LNds and DN1ps. E2 LNds also receive strong synaptic inputs from DN1as and DN1ps and form strong connections with one another . This stLike the strong shared synaptic targets of E1 LNds, the majority of E2 strong output targets do not synapse onto identified clock neurons. But, in contrast to E1, the minority of E2 targets that do form strong connections with identified clock neurons do so more broadly than clock recurrent E1 targets, forming strong synaptic connections onto the M group, E2, and the LPNs . Though The E3 LNds are distinct from the E1 and E2 groups, in that they form approximately half the number synaptic connections . While cThe E3 group is unique among the LN clock neurons by virtue of its lacking CRY and PdfR expression  and is tThe LN clock neuron classes are critical nodes of circadian timekeeping and are predicted to communicate directly with endocrine, sleep, and premotor centers to drive circadian outputs  and withAll four functional classes of LN  provide strong synaptic outputs onto neurons that, in turn, form strong synaptic connections onto identified clock neurons within the hemibrain volume —9. In thMost of the neurons forming strong but indirect connections between identified clock neurons bear no resemblance to known clock neurons and are unlikely therefore to express the molecular circadian clock e.g. . This fiMany central pattern generator networks consist of neurons both with and without endogenous pacemaking activity  while the retrieval of connectome data was done with the Neuprint python package , seaborn (https://seaborn.pydata.org/), and superVenn . Our analyses included the synapses to or from traced but unnamed fragments as well as select orphan bodies, in addition to synapses from full, traced, and named neurons. Fragments are partial or truncated neurons that mainly lie beyond or at the boundaries of the hemibrain section. Although fragments were often unidentified cell types, we felt their inclusion was warranted to obtain as accurate a picture as possible of the relative amounts of synaptic connectivity to and from clock neurons.Synaptic connectivity data were retrieved from the Janelia hemibrain dataset . Nodes are individual clock neurons and edges are labeled with the total synaptic connection weights from one neuron to another.The Sankey figures are based only on strong synaptic connections. Connectivity data were retrieved using the criteria for strong connections and exported into tables. The network structure of the clock connectome was graphed using the synaptic connectivity data retrieved from the hemibrain data and the networkX python package . The license can be found here: https://github.com/ClockConnectome/clock-connectome/blob/main/LICENSE.The data retrieval routines and the analyses used in this paper were done in the python coding language. We have made our scripts publicly available in a GitHub repository : https:/ 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.This paper analyzes the synaptic connections of two subsets of the  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Drosophila Lateral Neuron Clock Cells Reveals the Synaptic Basis of Functional Pacemaker Classes" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Claude Desplan as the Senior and Reviewing Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Charlotte Helfrich-Förster (Reviewer #1).Thank you for submitting your article "Connectomic Analysis of the The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The paper is already very dense and we do not want to add more work to it, although one reviewer would have liked to have some of the ideas that are proposed be tested. We ask you to take into consideration all the editorial suggestions from reviewer #1 and Reviewer #2 and make sure to help the reader with a complex paper.The list of comments is long and extensive but will improve the paper significantly and should be feasible in a few weeks without further experiments.Reviewer #1 (Recommendations for the authors):Although it is impossible to avoid abbreviations, these should be reduced to a minimum. For example, all words that are used less than 5 times should not be abbreviated . I also recommend talking about CRY-positive or CRY-negative clock neurons instead of CRY+ and CRY- clock neurons (the same applies for PDF-positive and -negative neurons).In many figures, the lettering is too small, and/or gray letters are used instead of black ones. This makes reading very difficult. Similarly, many drawings of individual neurons are too small, and lines are too faint or too thin. This needs general improvement. Below, I will only mention the most evident cases.Abstract lines 27-30: "Moreover, we identify a small number of 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." I do not see why the small number of clock cell subtypes representing synaptically coupled nodes should suggest that neurons lacking molecular timekeeping play integral roles within the clock network. Something is missing here.Line 110: The author may wish to also cite the paper of Reinhard et al. (2022b) (https://doi.org/10.3389/fphys.2022.886432) that describes the dorsal clock neurons in more detail.Line 117: the lettering in Figure 1C is too small so that it is impossible to find the LNd6.Line 119: This is the wrong citation. Please cite the paper mentioned above.Line 142: The 5th LNv was first described by Kaneko et al. . Perhaps it is fair to cite this paper here, too.Lines 152-154: Schubert et al. did show that the cell body of the 5th s-LNv is significantly larger than those of the s-LNvs (it has exactly the same size as that of the LNd6). Furthermore, its soma is located more dorsally than that of the s-LNvs. Therefore, Schubert et al. suggest calling this cell not anymore 5th s-LNv, because it is not an s-LNv. Indeed, subsequent papers from this lab just called it 5th LN. Please think about changing the name of the cell to 5th LN.Line 197: "connectomic" should be replaced by "transcriptomic".Page 9 and 10: I find the description of the different LNds very puzzling and partly redundant. It may be helpful to enlarge Figure 3D  and not only include the synaptic connections between the 6 LNds in this figure but also add their neuropeptide/transmitter expression. In the text, it might help to combine the description of neuropeptide expression (lines 240-243) with neuropeptide/neurotransmitter expression (lines 253-258). Furthermore, it might help to mention PDF-receptor expression already earlier together with CRY-expression (lines 231-233).I also wonder, why ITP as neuropeptide of the 5th LN is only mentioned here (line 243) not already earlier when the 5th LN was initially described.Figure 3C: The green dots representing inter LNd contacts cannot be seen, even not when amplified to 200%. The same applies to several other figures.Figure 3D: The labeling of the LNd1-6 needs to be enlarged. Furthermore, the white labeling on the gray background is hard to see. Why do their names carry an additional "R"?Line 267: The second neuropeptide of the s-LNv should already be mentioned at the place, where these neurons were initially described (e.g. page 5). Furthermore, their fast neurotransmitter expression  should be mentioned there, too. This citation is currently completely lacking.Line 431: I miss a clear statement that neuropeptidergic signaling could represent an essential output from the clock network to descending neurons. This is only briefly mentioned in lines 501-504 with respect to the neuropeptide PDF. Since all clock neurons appear to be peptidergic and many clock neurons express even more than one neuropeptide, I suggest mentioning in the first paragraph of the discussion that not only electrical synapses likely contribute to circadian timekeeping but also neuromodulation through neuropeptides.Line 401-403: This sentence does not describe what the figure shows if I understand it correctly. In Figure 9E, the connectivity of the postsynaptic E3 neurons with other clock neurons is shown, not the connectivity of the E3 with other clock neurons.Line 433: Please mention the timepoint at which the hemibrain was generated also here and not only in M&M.Line 463: Please cite here additionally the paper of Schubert et al., because these authors were the first who showed the morphological similarity between these two neurons.Line 527: This statement might be supported by the study of Reinhard et al. (2022b) who found the same.Reviewer #2 (Recommendations for the authors):I think it is a very nice piece of work that clearly deserves publication and I only have minor comments.1. Some of the clock neurons, including LNds, send axons in the dorsal brain that go through the midline and end in the other hemisphere (see papers from C Helfrich-Forster). As indicated in the text, the Janelia farm dataset does not include this information about contralateral connections since it contains one hemisphere only. However, this important feature of some clock neurons is not mentioned in the manuscript and I think that the information that is available in the literature should be discussed when using axonal projections to define the different neuronal subsets.2. Page 7: "and all four receive strong or medium strength synaptic inputs from these three presynaptic cell types ."Figure 4D shows only two presynaptic cells, please clarify.3. Page8: "Among the inputs targeting only one s-LNv, and mediated by only three synapses, is the HB-eyelet, a surprising finding given the long-held model that this external photoreceptor provides direct excitatory input onto s-LNvs and contributes to light entrainment of circadian rhythms".The long-held model was already challenged by the finding that the absence of histamine transmission induces a loss of circadian entrainment .4. In Figures 2D, and E (inputs and outputs), please add the name of the non-LNv clock cells for the corresponding ID numbers.5. In figure 3E, F: please indicate for each LNd the number of synapses with strong partners as in figure 2D, E for the LNvs.Reviewer #3 (Recommendations for the authors):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. Reviewer #1 (Recommendations for the authors):Although it is impossible to avoid abbreviations, these should be reduced to a minimum. For example, all words that are used less than 5 times should not be abbreviated . I also recommend talking about CRY-positive or CRY-negative clock neurons instead of CRY+ and CRY- clock neurons (the same applies for PDF-positive and -negative neurons).We agree with the reviewer that these changes will make the text easier to read. “CPG” has been replaced with “central pattern generators” and “aMe” has been replaced with “accessory medulla”. All abbreviations that remain are used five times or more in the revised manuscript . “Cry+” and “Cry” have been replaced with “Cry-positive” and “Cry-negative”, respectively, throughout the manuscript.In many figures, the lettering is too small, and/or gray letters are used instead of black ones. This makes reading very difficult. Similarly, many drawings of individual neurons are too small, and lines are too faint or too thin. This needs general improvement. Below, I will only mention the most evident cases.The font size has been increased in all the figures, and changed from gray to black. The sizes of all images of individual neurons have increased, and we have improved the resolution when needed. There are times when the number of cell IDs are too numerous to make all the text in a figure significantly larger (like figure 3F). However, in these cases the major result in the figure is not dependent on the exact identification of each synaptic partner, but rather the number of synaptic partners shared by specific clock neurons. The exact identification of each synaptic partner can also be found in the supplementary tables (inputs and outputs).Abstract lines 27-30: "Moreover, we identify a small number of 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." I do not see why the small number of clock cell subtypes representing synaptically coupled nodes should suggest that neurons lacking molecular timekeeping play integral roles within the clock network. Something is missing here.The reviewer is correct, this was a typo. It should have said “non-clock”, and we have modified it in the abstract.Line 110: The author may wish to also cite the paper of Reinhard et al. (2022b) (https://doi.org/10.3389/fphys.2022.886432) that describes the dorsal clock neurons in more detail.This reference has been added (lines 128-129).Line 117: the lettering in Figure 1C is too small so that it is impossible to find the LNd6.This has been corrected in Figure 1C and in most other figures.Line 119: This is the wrong citation. Please cite the paper mentioned above.This citation has been replaced with Reinhard et al. (2022b).Line 142: The 5th LNv was first described by Kaneko et al. . Perhaps it is fair to cite this paper here, too.This citation has been added (line 158).Lines 152-154: Schubert et al. did show that the cell body of the 5th s-LNv is significantly larger than those of the s-LNvs (it has exactly the same size as that of the LNd6). Furthermore, its soma is located more dorsally than that of the s-LNvs. Therefore, Schubert et al. suggest calling this cell not anymore 5th s-LNv, because it is not an s-LNv. Indeed, subsequent papers from this lab just called it 5th LN. Please think about changing the name of the cell to 5th LN.We now introduce the “5th s-LNv” historically and then cite Schubert et al.’s re-naming of this neuron as the “5th LNv.” We refer to these neurons by this name for the remainder of the study. The figures and figure legends have also been modified to reflect this change.Line 197: "connectomic" should be replaced by "transcriptomic".In this case, we were referring to recent work from the Sprecher lab on the larval connectome, whose analysis included larval LNvs. We have clarified this by revising the sentence in question as follows:pdf+ neurons , which persist through metamorphosis to become the adult s-LNvs .”“A recent connectomic analysis of the larval brain by Larderet and colleagues reached similar conclusions regarding the uniformity of sensory inputs, anatomy, and local connections among the four larval Page 9 and 10: I find the description of the different LNds very puzzling and partly redundant. It may be helpful to enlarge Figure 3D  and not only include the synaptic connections between the 6 LNds in this figure but also add their neuropeptide/transmitter expression. In the text, it might help to combine the description of neuropeptide expression (lines 240-243) with neuropeptide/neurotransmitter expression (lines 253-258). Furthermore, it might help to mention PDF-receptor expression already earlier together with CRY-expression (lines 231-233).I also wonder, why ITP as neuropeptide of the 5th LN is only mentioned here (line 243) not already earlier when the 5th LN was initially described.We agree that our summary of the anatomical subclasses was redundant and not as clear as it could have been. We have edited this section of the manuscript to avoid redundancy and have revised Figure 3D to make the anatomical and functional subclasses of the LNds clearer. For the sake of consistency, we have also revised Figure 2D and now show not only synaptic connections between the 4 sLNvs but also their neuropeptide expression. We have also, as suggested, described PdfR expression earlier in the text, and now introduce it with our initial description of Cry expression. In addition, we have mentioned that the 5th LNv expresses both ITP and CHAT when we first introduce these neurons in the section introducing the LNv classes.Figure 3C: The green dots representing inter LNd contacts cannot be seen, even not when amplified to 200%. The same applies to several other figures.We have replaced the images in 2C and 3C and now use orange instead of green for inter-group contacts, which makes them more visible. We have also substantially increased the size of panels 2A-C, 2E-F, and all other panels with neuron morphology in the main figures.Figure 3D: The labeling of the LNd1-6 needs to be enlarged. Furthermore, the white labeling on the gray background is hard to see. Why do their names carry an additional "R"?Our new panel 3D has larger labeling and does not include the “R”. This was a typo, left-over from the NeuPrint nomenclature for the individual annotated neurons.Line 267: The second neuropeptide of the s-LNv should already be mentioned at the place, where these neurons were initially described (e.g. page 5). Furthermore, their fast neurotransmitter expression  should be mentioned there, too. This citation is currently completely lacking.Frenkel et al., 2017 reference (lines 148-150).We agree. We now describe sNPF and glycine expression by the sLNvs earlier in the Results section. We have also incorporated the Line 431: I miss a clear statement that neuropeptidergic signaling could represent an essential output from the clock network to descending neurons. This is only briefly mentioned in lines 501-504 with respect to the neuropeptide PDF. Since all clock neurons appear to be peptidergic and many clock neurons express even more than one neuropeptide, I suggest mentioning in the first paragraph of the discussion that not only electrical synapses likely contribute to circadian timekeeping but also neuromodulation through neuropeptides.We agree that such paracrine/peptide connections are likely important for timekeeping and should be mentioned when discussing the limitations of connectomics focused on chemical synapses. We have added two sentences to the first paragraph of the discussion, as suggested:“In addition, neuropeptides can be released non-synaptically ), act over long distances, and such paracrine signaling likely contributes to circadian timekeeping in the brain ). The connectomic analysis presented here does not include such peptidergic connectivity.”Line 401-403: This sentence does not describe what the figure shows if I understand it correctly. In Figure 9E, the connectivity of the postsynaptic E3 neurons with other clock neurons is shown, not the connectivity of the E3 with other clock neurons.The reviewer is correct. We have clarified this (lines 429-431), and this section now reads:“With regard to its shared strong outputs, the E3 subgroup forms strong shared synaptic connections onto only 16 neurons , five of which form synapses onto identified clock neurons within the hemibrain . The synapses that those five neurons form back onto clock neurons are limited to LNds and LPNs, with most connections forming back onto E3 LNds themselves .”Line 433: Please mention the timepoint at which the hemibrain was generated also here and not only in M&M.This information has been incorporated (line 471).Line 463: Please cite here additionally the paper of Schubert et al., because these authors were the first who showed the morphological similarity between these two neurons.This reference has been added (line 506).Line 527: This statement might be supported by the study of Reinhard et al. (2022b) who found the same.This reference has been added (line 640).Reviewer #2 (Recommendations for the authors):I think it is a very nice piece of work that clearly deserves publication and I only have minor comments.1. Some of the clock neurons, including LNds, send axons in the dorsal brain that go through the midline and end in the other hemisphere (see papers from C Helfrich-Forster). As indicated in the text, the Janelia farm dataset does not include this information about contralateral connections since it contains one hemisphere only. However, this important feature of some clock neurons is not mentioned in the manuscript and I think that the information that is available in the literature should be discussed when using axonal projections to define the different neuronal subsets.We agree that this is an important consideration for our study and that we could have said more about which of the annotated clock neuron classes within the hemibrain are known to project contralaterally. We have therefore added the following to the opening paragraph of the discussion:“Several classes of clock neurons, including the large LNvs, the 5th LNv, the LNds, and DN1ps, project across the midline and form connections in the contralateral hemisphere . Many of these connections are expected to reside outside of the hemibrain volume. Our estimates of inter-clock connectivity will therefore not include these contralateral connections.”2. Page 7: "and all four receive strong or medium strength synaptic inputs from these three presynaptic cell types ."Figure 4D shows only two presynaptic cells, please clarify.We have clarified this point (lines 198-200):“Only three neurons in the hemibrain provide ten or more synapses onto at least one of these cells , and only two of those three neurons form strong connections onto all four sLNvs .”3. Page8: "Among the inputs targeting only one s-LNv, and mediated by only three synapses, is the HB-eyelet, a surprising finding given the long-held model that this external photoreceptor provides direct excitatory input onto s-LNvs and contributes to light entrainment of circadian rhythms".The long-held model was already challenged by the finding that the absence of histamine transmission induces a loss of circadian entrainment .We have added the following sentence and reference (lines 219-221):The sparse connectivity between the eyelet and the s-LNVs found within the hemibrain is consistent with recent work suggesting that the eyelet makes negligible contributions to light entrainment .4. In Figures 2D, and E (inputs and outputs), please add the name of the non-LNv clock cells for the corresponding ID numbers.We have added this information to Figure 2, and also to Figure 3 for consistency.5. In figure 3E, F: please indicate for each LNd the number of synapses with strong partners as in figure 2D, E for the LNvs.We have incorporated this information to Figure 3E and F.


Does vaccine effectiveness against hospitalization and mortality due to the SARS-CoV-2 Omicron variant change over time?In this case-control study with 164 151 participants, the CoronaVac and BNT162b2 vaccines were generally estimated to be effective against severe outcomes due to SARS-CoV-2 Omicron infection, but protection among older individuals was more likely to wane 6 months after the second dose.These findings suggest that a booster dose should be recommended to older individuals to restore immunity, and this is especially urgent for a setting like Hong Kong, where third-dose coverage is still insufficient among older residents. This case-control study examines change over time in estimated vaccine effectiveness against hospitalization and mortality caused by the SARS-CoV-2 Omicron variant among adults in Hong Kong. Few studies have evaluated the waning of vaccine effectiveness against severe outcomes caused by SARS-CoV-2 Omicron infection. Hong Kong is providing inactivated and mRNA vaccines, but the population had limited protection from natural infections before the Omicron variant emerged.To examine the change in vaccine effectiveness against hospitalization and mortality due to the Omicron variant over time.This case-control study included adults with SARS-CoV-2 Omicron variant infection who died or were hospitalized in Hong Kong from January 1 to June 5, 2022 , and adults with SARS-CoV-2 Omicron, sampled from the public health registry during the study period , who were matched to case participants by propensity score.Vaccination status of the individuals.Estimated vaccine effectiveness against death, death or hospitalization, and death among hospitalized patients. Vaccine effectiveness was calculated as 1 − adjusted odds ratio obtained by conditional logistic regression adjusted with covariates for each period following vaccination.There were 32 823 case participants  and 131 328 control participants  in the sample analyzed for the death or hospitalization outcome. Vaccine effectiveness against death or hospitalization was maintained for at least 6 months after the second dose of both CoronaVac  and BNT162b2  vaccines. Vaccine effectiveness against death in those aged 18 to 49 years was 86.4%  and 92.9%  for those receiving 2 doses of CoronaVac and BNT162b2, respectively, while for patients aged 80 years or older, it dropped to 61.4%  and 52.7%  for CoronaVac and BNT162b2, respectively. Nevertheless, overall vaccine effectiveness against death at 4 to 6 months after the third dose was greater than 90% for CoronaVac, BNT162b2, and the mixed vaccine schedule .While vaccines were generally estimated to be effective against severe outcomes caused by SARS-CoV-2 Omicron infection, this analysis found that protection in older patients was more likely to wane 6 months after the second dose. Hence, a booster dose is recommended for older patients to restore immunity. This is especially critical in a setting like Hong Kong, where third-dose coverage is still insufficient among older residents. The daily COVID-19 death rate was among the highest ever recorded.10 In early 2021, the Hong Kong government authorized the free distribution of COVID-19 vaccines BNT162b2 mRNA vaccine (Pfizer-BioNTech) and CoronaVac inactivated vaccine (Sinovac) to the resident population. Approximately 85% of the population had received the second dose of the COVID-19 vaccine by June 5, 2022.11Hong Kong had successfully contained the Delta variant spread, with almost no COVID-19 local cases for about 4 months before the Omicron variant emerged.To date, few studies have assessed the waning of protection against severe outcomes caused by SARS-CoV-2 Omicron infection. In this study, we used the official linked data to estimate how vaccine effectiveness (VE) against hospitalization and mortality changed over time after vaccination in patients with SARS-CoV-2 Omicron in Hong Kong, where the population had minimal protection against infections before the Omicron variant emerged.STROBE) reporting guidelines for case-control studies.This is a case-control study using the linked administrative data. The Department of Health (DH) of the Government of the Hong Kong Special Administrative Region (HKSAR) permitted us to use the database in this investigation. All study data were completely anonymized. Ethics approval was obtained from the Survey and Behavioral Research Ethics Committee, The Chinese University of Hong Kong. Because this study was a retrospective analysis using secondary data with no personal information, the requirement for obtaining informed consent was waived. This study followed Strengthening the Reporting of Observational Studies in Epidemiology  test or rapid antigen test (RAT) were included. Before mid-February 2022, the Omicron status of patients with PCR-confirmed COVID-19 was determined by genomic sequencing based on the defining mutations (N501Y and E484A). Due to laboratory capacity being overwhelmed by the rapid and massive increase in cases from no infections to more than 50 000 cases daily, the DH launched a self-registration system for PCR test and RAT results in mid-February 2022, a period dominated by the SARS-CoV-2 Omicron variant. While the variant status of all patients was not confirmed by genomic sequencing, subsampling revealed more than 99% of the specimens sequenced carried the mutated strain of the Omicron variant. Imported cases were excluded from the analysis. Aside from all registered patients, we included all patients aged 18 years or older who were hospitalized in public hospitals and were diagnosed with a COVID-19 infection by PCR test as a secondary study population.12 The number of days from the last dose (second or third dose) to the hospitalization or death date was used to calculate the time from vaccination to hospitalization or death. For the analyses of the hospitalized population, only COVID-19 death was used as the study outcome.We used hospitalization or COVID-19 death, defined as the death episode with a positive test result, as a proxy for severe outcomes in the primary study population. The mortality data were corroborated by the birth and death records of HKSAR government, representing a complete coverage of deaths in Hong Kong. In the analysis of the hospitalization outcome, we excluded patients with COVID-19 who became infected during their hospital stay by excluding those whose PCR confirmation date was more than 3 days after admission. The data for this analysis did not include those admitted before week 6 in 2022; during that period, all confirmed cases were hospitalized regardless of clinical status following the government’s COVID-19 patient management policy. After the Omicron outbreak, the admission policy was changed, and only patients in severe or critical condition were admitted to hospitals.In the analysis of all the registered patients, case participants were the patients with COVID-19 who had the defined study outcomes , and control participants were patients infected with SARS-CoV-2 who did not experience the study outcomes before the cutoff date. Because of the scarcity of individual-level data available for the community control population, case and control participants were only matched by age, sex, and the calendar date of COVID-19 infection.In the analysis of the hospitalized patients, case participants were the patients with COVID-19 who had a positive PCR test on admission and died while hospitalized. Patients with SARS-CoV-2 who were hospitalized and alive before the cutoff date served as control participants. Given that clinical information could be linked to the hospitalization database, case and control participants were matched by the covariates of age, sex, chronic conditions or cancer, and use of oral COVID-19 treatment drugs (molnupiravir or paxlovid) or other coronavirus medications.The propensity score method was performed for all case-control matches in a 1:4 ratio by using the nearest neighbor method without replacement, and the scores were determined by the logit link model regressing on the matching variables. We tested the robustness of the results by setting the matching ratio to 1:1 or 1:2.Only individuals who received the BNT162b2 or CoronaVac vaccines were included in the analysis. Given the latency between vaccine ingestion and full development of immune responses, only a dose taken within 14 days of infection was considered a completed dose in the study.The baseline characteristics of the case participants and matched control participants in the 2 study populations were descriptively compared. The adjusted odds ratio (AOR) for the association between vaccination and death was estimated using conditional logistic regression controlling for covariates. VE against the severe outcome was calculated as 1 − AOR. The 95% CIs of VE estimates were constructed by bootstrapping with 1000 resampled data sets with replacement. VE was compared across age groups , vaccine types, and number of doses. To assess the change in effectiveness over time, we estimated the VE of each vaccine for each period following vaccination  using conditional logistic regression.Vaccine protection over time, defined as the time from the last dose (second or third) to hospitalization or death for vaccinated individuals, was examined using the conditional Cox proportional hazard model adjusted for the covariates and is presented using the adjusted survival curves with an estimate of the median survival time of protection. Time from the last dose to infection was adjusted in the Cox model to control the temporal variation of coverage of the 2 vaccine types. A cutoff date of June 19, 2022, was used as the right censoring of the death outcome. R version 3.6  was used for all the statistical analyses. Because this study used a large official administrative database with a high study power, no hypothesis testing was carried out.In all registered patients, 9362 deaths and 37 488 matched controls were included in the analysis of death outcome, whereas 32 832 case participants with death or hospitalization and 131 328 matched controls were included in the analysis of the death or hospitalization outcome . In the The characteristics of the case and control participants were generally balanced within each of the matching groups . In the In the registered population, most case participants were unvaccinated , while only 13 838 control participants (37.0%) in the death outcome and 28 124 (21.4%) in the death or hospitalization outcome were unvaccinated. Two doses of CoronaVac and BNT162b2 had similar VE estimates . The VEsAcross age groups, both types of vaccines were less effective in older groups  against severe and critical conditions remained greater than 90%.18 The inclusion of patients with the Delta variant and receiving a different vaccine type in their study affects the compatibility of their findings with ours.Our study assessed VE in the hospitalized population. In a cohort study of hospitalized older patients with COVID-19 infections during the Omicron wave in Israel, 3 doses of BNT162b2 did not provide a significant protection from death or mechanical ventilation compared with no vaccination.20 our study suggested that both CoronaVac and BNT162b2 provide protection against severe outcomes caused by the Omicron variant and the VE waned consistently after the second dose. In fact, a national study in Chile found that CoronaVac was 86% effective against death in early 2021, when the Gamma and Alpha variants were circulating.21 In comparison with the antibody titer developed after 2 doses of BNT162b2, which decreased by 4.7-fold within 5 to 6 months, the antibody titer developed after CoronaVac reduced by 6- to 10-fold  within 6 months,23 potentially indicating a lower efficacy in protection.24 Nonetheless, vaccine recipients retained cellular immunity that protected against progression to severe COVID-19 even after neutralizing antibodies waned.2While several studies have found that CoronaVac was less effective against severe outcomes than BNT162b2,25 very few empirical studies have investigated boosting in CoronaVac recipients, including those who received heterologous boosting (2 doses of CoronaVac followed by 1 BNT162b2 booster).26 While neutralization tests revealed that binding and neutralizing antibodies were higher after heterologous boosters than after homologous boosters,27 we were unable to demonstrate that a heterologous booster was more effective than 3 doses of CoronaVac. Nonetheless, our findings were consistent with those of a Chilean cohort study suggesting that either 3 doses of CoronaVac or a heterologous booster after a primary series of CoronaVac could result in a VE greater than 85% against death.28According to our findings, a booster dose offered durable protection against severe outcomes caused by the Omicron variant over time. While several studies have documented the benefit of the BNT162b2 booster,29 It also echoed an early Hong Kong study that found VE was lower in older patients, even when they were fully vaccinated.30 Nevertheless, our findings suggest that the booster dose was capable of restoring VE and maintaining protection over time. Indeed, neutralization tests among individuals who received BNT162b2 revealed that a third dose of the vaccine increased antibody titer by more than 7-fold compared with the level after the second dose in individuals aged 65 to 85 years.22 The third dose of CoronaVac administered 8 months after the primary series completion increased titers by 3- to 5-fold compared with the level after 2 doses. Antibodies increased in both age groups younger and older than 60 years, with titers slightly higher in the older group.23Our research found that VE was lower in older patients, and protection was more likely to wane after 6 months following the second dose. This finding echoed a study in Finland showing that VE against hospitalization 6 months after 2 doses of BNT162b2 was only 61% in older patients during the Omicron-dominant period.31 Because our study was a retrospective secondary analysis, immunocompromise status was unavailable in the linked administrative database, which may have affected VE estimates. Fourth, because we had limited information on other covariates in the self-report system, we were unable to adjust for confounders other than age and sex. The analysis could also have been confounded by behavioral factors  and lifestyle factors . Nevertheless, we were able to link hospitalization records to account for chronic conditions and inpatient treatments in the analyses of hospitalized population.Our study has several major limitations. First, although local public health officials launched a self-registration system for reporting RAT results during the rapidly rising phase of the Omicron epidemic, the database would have missed a certain number of cases with mild symptoms or asymptomatic cases due to underreporting and other factors. Second, because critical care facilities were overcrowded during the peak of infections, many patients were unable to receive intensive care support; thus, the number of patients receiving care in intensive care units is an unreliable proxy for disease severity. Therefore, we excluded this indicator when defining disease severity, which inevitably resulted in inconsistency when generalizing the findings to other studies. Third, other studies found that seroconversion rates and antibody titers following COVID-19 vaccines were low in both immunocompromised and immunocompetent patients.In this case-control study, we found that vaccines had high estimated VE against severe outcomes in patients with SARS-CoV-2 Omicron, but that protection in older patients was more likely to wane 6 months after the second dose. Therefore, a booster dose is recommended for older individuals to restore immunity. This is especially critical in a setting like Hong Kong, where coverage of the third dose of the vaccine is still insufficient among older residents.


US and Northern European studies have found a higher prevalence of alcohol-related problems among men who have sex with men (MSM) than among the general population of men (GPM). However, most of them relied on traditional sampling methods, not profiting from MSM dating apps and websites for recruitment. Besides, analogous comparisons in Southern Europe are lacking.This study aimed to compare several indicators of excessive drinking between MSM and GPM in Spain.Overall, 5862 MSM were recruited through dating apps or websites for the Méthysos Project, and 10,349 GPM were recruited using probability sampling via the Household Survey on Alcohol and Drugs in Spain from 2018 to 2020. The outcomes were the prevalence of hazardous or harmful drinking (Alcohol Use Disorders Identification Test [AUDIT] ≥8), hazardous drinking (AUDIT-Consumption ≥4), harmful drinking (AUDIT-Problem ≥4), regular hazardous drinking (>14 standard drinks per week), and monthly binge drinking. The prevalence of excessive drinking indicators was calculated for MSM and GPM and compared using the adjusted prevalence ratio (aPR). Two different aPRs and their 95% CIs were estimated using Poisson regression models with robust variance. The first was adjusted for sociodemographic characteristics, and the second was adjusted for the aforementioned covariates plus other drug use.The prevalence of hazardous or harmful drinking was 15.6% (913/5862) among MSM versus 7.7%  among GPM. After adjusting for sociodemographic covariates, the risk was higher in MSM than in GPM for harmful or hazardous drinking , harmful drinking , and binge drinking ; the same in both populations for hazardous drinking ; and higher in GPM than in MSM for regular hazardous drinking . The relative excess risk of harmful drinking and binge drinking among MSM tended to increase with increasing education level and size of the place of residence, and the opposite was true for the deficit risk in regular hazardous drinking. Additional adjustment for other drug use greatly buffered the relative excess risk in harmful drinking and binge drinking in MSM, while it deepened its deficit risk in regular hazardous drinking.The use of web-based resources allowed recruiting a large sample of MSM. The risk of hazardous or harmful drinking was 80% greater in MSM than in GPM, which was mainly because of the higher risk of harmful drinking and binge drinking among MSM. Nearly 1 in 6 MSM would benefit from early brief alcohol intervention procedures. The subgroup with harmful or binge drinking combined with other drug use is an important contributor to excess MSM risk in hazardous or harmful drinking and must be a priority target for harm reduction interventions. In recent years, concerns have been raised about health disparities due to sexual orientation -5. SurveTo our knowledge, the only studies in Europe that compared alcohol use between MSM and GPM have been conducted in Northern or Central European countries ,17-21. OKnowledge of alcohol use among MSM living in southern European countries is important given that historical cultural norms regarding alcohol use differ between these countries and those where research has been conducted . The MedIn contrast, most of the former studies comparing MSM and GPM are based on representative population samples ,9,12,16.A common problem when comparing drinking indicators between MSM and GPM is the existence of important differences between both groups in sociodemographic characteristics  that can distort the comparison -28. To aThe Alcohol Use Disorders Identification Test (AUDIT) is a 10-item scale that screens for alcohol use disorders to identify and assist individuals at high risk . It inclTo the best of our knowledge, only 2 published studies have compared AUDIT scores between MSM and GPM ,41. The In this study, we took advantage of dating apps and websites commonly used by MSM to recruit a large sample of MSM and compare various indicators of excessive drinking, including hazardous and harmful drinking, between MSM and GPM in Spain. We used the AUDIT to assess MSM who would potentially benefit from early alcohol interventions. In addition, we aimed to discern the differences between the 2 dimensions of hazardous drinking (distinguishing regular hazardous drinking from binge drinking) and harmful drinking.We conducted a cross-sectional comparison of several measures of excessive drinking between MSM and GPM in Spain. Data were obtained from the results of the AUDIT questionnaire on 2 different samples for MSM and GPM.Participants, MSM Sample) between May and July 2020. The GPM sample was recruited between February and April 2018 within the framework of the Household Survey on Alcohol and Drugs in Spain  [The MSM sample included participants from the Méthysos Project, which aimed to investigate their health status. Participants were recruited through different web-based resources (see edition) .MSM were invited to participate through 3 types of web-based resources:A total of 7 MSM dating apps using promotional banners , personal messages (GROWLr), or both  contributed 70.5% (4655/6602) of the participants.Furthermore, 3 influencers largely followed by the MSM community (Gabriel José Martin–with a video on YouTube available throughout the recruitment period–and @frewaskachannel and @tigrilloig–with 24-hours available stories launched twice (@frewaskachannel) or once (@tigrilloig) during the recruitment period), which contributed 26.3% (1741/6602) of the participants.Finally, a message encouraging the diffusion of the study among friends and acquaintances, placed at the end of the questionnaire, and distribution lists from 3 organizations , which contributed with 3.1% (206/6602) of the participants.Individuals who decided to participate were addressed to an initial screen where they were informed about the aim and content of the survey. Before starting, they were obliged to check on the “I am of legal age for sexual intercourse and want to participate in the study.” All participants were required to meet the inclusion criteria of being male, at least 16 years old, and having ever had anal intercourse with a male. We provided unique links to each recruitment site to identify the recruitment site for each participant. To avoid multiple participation, the initial screen also included a request to complete the questionnaire only once if the invitation was received in various ways. In addition, to further limit the possibility of multiple responses from one individual, we used the option provided by the software that only allowed the completion of one questionnaire per electronic device. No incentive was offered for participation, limiting the chances of multiple participation. The questionnaire was self-administered, computer- or app-based, and treated for sexual behavior and drug and alcohol use (including AUDIT). The full questionnaire is available in the In studies using this type of methodology, it is impossible to calculate or estimate the response rate. The apps identify the number of displayed banners and sometimes the number of clicks on the banner, but not the number of different people who see or click on them to obtain some information. Something similar happens with influencers. They may know the number of views on a promotion, but not the number of people, or whether those people meet the criteria to participate. Of the 6602 MSM who began the questionnaire, 740 abandoned it before completing the AUDIT questions; thus, the final sample comprised 5862 MSM.The GPM sample was obtained from the anonymous database of EDADES, 2017 edition provided by the National Plan on Drugs. The EDADES is a biennial national survey of a representative sample of the population aged 15-64 years living in Spain. It uses a 3-stage random sampling design . Census tracts and subjects within households were selected using random probabilistic methods. The sample was stratified by age (15-34/35-64) and living region (19 categories). In this edition, people aged 15 to 34 years and living in small regions were oversampled, so analyses were weighted to account for strata imbalance compared with the universe. The global response rate was 50.6%, and the main causes of nonresponse were not opening the door, preventing interviews, and the absence of all household members or the selected person. Before classifying a household or person as absent and selecting another household, the fieldworker should visit the household that was initially selected at least three times on different days and times.We selected all men aged 16-64 years who participated in the survey (women and men aged 15 years were excluded). EDADES does not have any variables to identify sexual identity or behavior; thus, we could not estimate the proportion of MSM among the group of men participating in the survey, nor excluding them in our analysis. Therefore, we refer to this group as GPM. However, we can infer that this sample is principally composed of heterosexual men, relying on the most recent estimation of the Spanish population on sexual orientation, in which 93.9% (2791/2972) of people declare themselves heterosexual . The EDAOnly the participants who had consumed alcohol in the past 12 months answered the AUDIT questionnaire. For the remaining participants, the AUDIT score was imputed as 0. Questionnaire skips were automatically included in the resources used by the MSM sample but had to be manually included in the GPM sample. This study uses labels and concepts from the recently published 11th revision of the International Classification of Diseases  to referRegarding consumption levels, we differentiated a variable of regular hazardous drinking  from binge drinking to identify differences in drinking patterns. Although weekly alcohol consumption above a certain threshold entails health-related consequences , these aQ1. How often do you have a drink containing alcohol?0. Never (skip to Qs 9-10)1. Monthly or less2. 2-4 times a month3. 2-3 times a week4. 4 or more times a weekQ2. How many drinks containing alcohol do you have on a typical day when you are drinking?0. 1 or 21. 3 or 42. 5 or 63. 7, 8, or 94. 10 or moreQ3. How often do you have six or more drinks on one occasion?0. Never1. Less than monthly2. Monthly3. Weekly4. Daily or almost dailySkip to questions 9 and 10 if total score for questions 2 and 3=0Q4. How often during the last year have you found that you were not able to stop drinking once you had started?0. Never1. Less than monthly2. Monthly3. Weekly4. Daily or almost dailyQ5. How often during the last year have you failed to do what was normally expected from you because of drinking?0. Never1. Less than monthly2. Monthly3. Weekly4. Daily or almost dailyQ6. How often during the last year have you needed a first drink in the morning to get yourself going after a heavy drinking session?0. Never1. Less than monthly2. Monthly3. Weekly4. Daily or almost dailyQ7. How often during the last year have you had a feeling of guilt or remorse after drinking?0. Never1. Less than monthly2. Monthly3. Weekly4. Daily or almost dailyQ8. How often during the last year have you been unable to remember what happened the night before because you had been drinking?0. Never1. Less than monthly2. Monthly3. Weekly4. Daily or almost dailyQ9. Have you or someone else been injured because of your drinking?0. No2. Yes, but not in the last year4. Yes, during the last year.Q10. Has a relative or friend or a doctor or another health worker been concerned about your drinking or suggested you cut down?0. No2. Yes, but not in the last year4. Yes, during the last year.Scores for the full-scale AUDIT (hazardous or harmful drinking), AUDIT-C (hazardous drinking), and AUDIT-P  instruments were calculated by adding the scores in Q1-10, Q1-Q3, and Q4-Q10, respectively. The weekly average number of standard drinks was used as an indicator of the intensity of regular alcohol consumption. It was estimated by multiplying the answers for questions on drinking frequency (Q1) and quantity (Q2). To this end, the median values of each response category of Q1 and Q2 were used, except for the extreme categories “monthly or less” in Q1, where 0.25 drinking days per week was assigned, and “ten or more were assigned to Q2, where 10 standard drinks per day were assigned. A similar method was used previously . The occ2χ test. The main outcomes were the prevalence of harmful or hazardous drinking (AUDIT score≥8), hazardous drinking (AUDIT-C score≥4), harmful drinking or alcohol-related problems (AUDIT-P score≥4), regular hazardous drinking , and monthly binge drinking (occurrence of binge drinking at least monthly in a year). The AUDIT cutoff was originally established by the World Health Organization [For the GPM sample, to account for strata imbalance compared with the universe, data were weighted by region (19 categories), size of place of residence (7 categories), age (7 categories), and sex (2 categories), resulting in 619 different values for the ponderation factor (from 0.04 to 3.06). For the MSM sample, as the global universe of MSM residing in Spain was unknown, all participants were assigned a ponderation factor of 1. An initial descriptive analysis of the general characteristics of the MSM and GPM samples was performed, and the statistical significance of the differences was assessed using the Pearson nization , and thenization ,42. As tnization  considernization ,29. We snization -55.P-values were <.05. The analysis was not preregistered, and the results should be considered exploratory. Statistical analyses were performed using Stata version 15 (StataCorp).The prevalence of the different drinking indicators was calculated in both MSM and GPM and compared between the groups using the adjusted prevalence ratio (aPR). aPR is a measure of relative inequality without units, which indicates the number of times the risk is higher or lower in MSM compared with GPM. Two different aPRs and their corresponding 95% CI were estimated using Poisson regression models with robust variance . The firThis study was approved by the Research Ethics Committee of the Instituto de Salud Carlos III . Concerning the MSM sample, individuals approved their conformity to participate by clicking on “On my age, I am legally authorized to have sexual relations and I want to participate in the study.” This message appeared before starting the questionnaire, as can be observed in the . PrivacyParticipants were more frequently aged 25-34 years (MSM) and 35-44 years (GPM). Compared with the GPM, they had a higher education level, lived more frequently in urban settings, and declared a better economic status. The prevalence of alcohol use in the past 12 months was similar between MSM and GPM, but that of other drugs was much higher among MSM .The prevalence of hazardous or harmful drinking among MSM was 15.6% (913/5862) compared with 7.7%  in the GPM group, leading to an aPR of 1.8 (95% CI 1.6-2.0). Significant MSM-GPM disparities in prevalence in the same direction were observed in all subgroups, except for those who did not consume other drugs in the past 12 months . The preThe prevalence of regular hazardous drinking (>14 drinks per week) was lower among MSM  than among GPM , with an aPR of 0.7 (95% CI 0.6-0.9). A statistically significant MSM-GPM disparity in such prevalence was not observed among individuals aged between 16 and 34 years, with university education level, or living in places with >500,000 inhabitants .The prevalence of monthly binge drinking was higher among MSM  than among GPM , with an aPR of 1.7 (95% CI 1.5-1.9). Significant MSM-GPM disparities in prevalence in the same direction were observed in all subgroups, except for other drug users .Finally, the prevalence of harmful drinking was higher among MSM  than among GPM , with an aPR of 2.3 (95% CI 2.0-2.7) Significant MSM-GPM disparities in such prevalence in the same direction were observed in all subgroups .The aPRs of Model II after further adjustment for the use of other drugs in the past 12 months are shown in Tables S1 and S2 in Compared with GPM, we found a relative excess risk of hazardous or harmful drinking  among MSM living in Spain. Nearly 1 in 6 MSM had hazardous or harmful drinking and would benefit from early brief alcohol intervention procedures  . The MSMThe relative excess risk in the GPM of hazardous drinking and regular hazardous drinking tended to increase among older, less educated, and rural-dwelling individuals. In contrast, the relative excess risk of harmful and binge drinking in MSM tended to be larger among individuals aged 16-24 years (only in the case of harmful drinking) or with university education level and living in big cities (in both cases).Additional adjustment for other drug use greatly buffered the considerable relative excess risk in MSM compared to that in GPM for harmful drinking and binge drinking while deepening the risk deficit in MSM for regular hazardous drinking. This suggests that drinking behaviors and other drug use were strongly associated in the studied populations. Therefore, the considerable excess of risk in MSM compared with GPM for binge drinking and harmful drinking is mainly because of the subgroup having both these drinking patterns and the use of other drugs. This subgroup represents a much higher proportion of the total in MSM than in GPM, and must clearly be a priority target for harm reduction interventions.This is the first study to directly compare excessive drinking indicators (hazardous and harmful drinking) between MSM and GPM in a southern European country. Moreover, this is the first study ever published that differentiates between these 2 dimensions using a validated instrument (AUDIT). Moreover, because of the use of new technologies in the recruitment process, namely apps and websites, it includes a larger sample of MSM than that in most previous studies inside and outside Europe -18,41,58Only one study has compared hazardous or harmful drinking between these 2 populations using full-scale AUDIT , and theThere are 3 studies ,12,17 thOne study  comparedNo other study has analyzed harmful drinking using AUDIT-P; however, our findings of a high aPR MSM or GPM are similar to those previously described in studies using Diagnostic and Statistical Manual of Mental Disorders criteria -8.Considering these findings, we can affirm that there is a slight difference in hazardous drinking between MSM and GPM, but a remarkable increase in harmful drinking among MSM also exists in Mediterranean countries such as Spain. However, the reasons for this discrepancy are not well understood. In the case of Spain, it could be that MSM adopted Northern European drinking patterns more quickly, characterized by a higher frequency of binge drinking, which is more likely to produce harmful consequences . As an iIn contrast, minority stress theory has also been proposed to explain the increase in harmful and hazardous drinking among MSM ,60. AccoOther factors may have influenced the results. It is well known that certain potentially alcohol-linked phenomena, such as sex work , chemsexIn summary, it could be that there are differences in consumption patterns between MSM and GPM that have not been properly analyzed, or, more probably, that MSM are more susceptible to the harmful effects of alcohol use or that they have a greater ability to identify or disclose them. Further research is needed to clarify this last point, which requires the use of large samples for both comparison groups. Studies based on general population surveys, which represent the majority to date, do not permit discerning of these particular issues and often include a small number of MSM.Finally, although not an objective of this study, our findings suggest that AUDIT measures 2 different domains . It alsoThe use of 2 different sampling methods for MSM and GPM is a limitation of this study. However, using dating apps and websites for the recruitment of MSM was a way to overcome 2 handicaps: the small sample of MSM frequently included in studies based on representative samples of the general population and the fact that the national drug survey in Spain does not include questions about sexual orientation. Although this sampling method may have overrepresented hazardous drinkers among MSM, it provided a significantly larger MSM sample than that in most studies with probabilistic sampling methods, in which the inclusion of a large number of MSM was hindered by the low percentage of men who reported having sex with other men. In our study, the MSM questionnaire was self-administered, which is not always guaranteed in general population surveys. This might have reduced the probability of reporting bias for nonnormative behaviors, such as homosexual behavior or alcohol use.Another limitation related to recruitment is the different contexts in which alcohol was more frequently consumed in these 2 groups. Even though dating apps as well as other Internet-based resources were used to recruit MSM, a skew toward “party-goers” may have taken place. As some questions in AUDIT  are related to nightlife drinking, this point may have had an influence. Unfortunately, we did not have a variable to measure the context of consumption. On the contrary, we do not think that recruiting MSM, but not GPM, during the COVID-19 pandemic (between May and July 2020) may have increased the differences favoring MSM. It has been observed that hazardous drinking increased during lockdowns -68, but Another limitation is related to AUDIT thresholds. Cutoffs are needed for screening; however, when different cutoff points are used, epidemiological research is hindered. We offset this limitation by estimating new results by applying different cutoffs used in other studies. In addition, problems related to thresholds were not exclusive to AUDIT; they also appeared when establishing the recommended levels of weekly alcohol consumption. The final limitation relates to the use of only 2 AUDIT questions (quantity and frequency) to estimate alcohol consumption. Although this was not the first study to measure this variable using these 2 questions (or similar questions) , a more This is the first study to compare indicators of hazardous and harmful drinking (problems or consequences) between MSM and GPM living in southern Europe. The use of different Internet-based resources in recruitment (mainly apps and influencers) allowed us to obtain information on alcohol use from a large and diverse sample of MSM. Using a validated brief screening tool (AUDIT), we found an 80% excess risk of hazardous or harmful drinking among MSM, which was mainly owing to the higher risk of harmful drinking and binge drinking among MSM.From the perspective of the implications for clinical practice, it is important to emphasize that the periodic use of brief screening tools such as AUDIT would seem to be highly recommended in health consultations of MSM. This would result in a significant proportion of MSM benefiting from early brief alcohol harm reduction interventions and from referral to specialized services when needed. Harm reduction strategies should focus on binge drinking. Nursing professionals in primary care and sexual transmitted infections or HIV clinics (which take care of a large number of MSM) may provide appropriate services for the implementation of these screening instruments.From the perspective of future research, it is desirable to enrich these findings by conducting studies based on a single population-based survey. To achieve this goal, surveys in Spain (and in other countries) should include a variable on sexual behavior or gender identity, as it has been common in the United States for a decade. Second, we want to highlight that there are currently no clear explanations for the disparity in the findings in the 2 domains of AUDIT. Neither the tendency toward incorporation of non-Mediterranean consumption patterns among MSM nor the minority stress theory satisfactorily explains these results. Therefore, further studies are needed to analyze whether differential consumption patterns exist or whether there are different levels of susceptibility to alcohol use and its harmful consequences.