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Chronic social isolation signals starvation and reduces sleep in Drosophila

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Abstract

Social isolation and loneliness have potent effects on public health1,2,3,4. Research in social psychology suggests that compromised sleep quality is a key factor that links persistent loneliness to adverse health conditions5,6. Although experimental manipulations have been widely applied to studying the control of sleep and wakefulness in animal models, how normal sleep is perturbed by social isolation is unknown. Here we report that chronic, but not acute, social isolation reduces sleep in Drosophila. We use quantitative behavioural analysis and transcriptome profiling to differentiate between brain states associated with acute and chronic social isolation. Although the flies had uninterrupted access to food, chronic social isolation altered the expression of metabolic genes and induced a brain state that signals starvation. Chronically isolated animals exhibit sleep loss accompanied by overconsumption of food, which resonates with anecdotal findings of loneliness-associated hyperphagia in humans. Chronic social isolation reduces sleep and promotes feeding through neural activities in the peptidergic fan-shaped body columnar neurons of the fly. Artificial activation of these neurons causes misperception of acute social isolation as chronic social isolation and thereby results in sleep loss and increased feeding. These results present a mechanistic link between chronic social isolation, metabolism, and sleep, addressing a long-standing call for animal models focused on loneliness7.

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Fig. 1: Sleep is reduced by chronic but not acute social isolation in Drosophila.
Fig. 2: Chronic social isolation induces a starvation gene expression program and results in excessive feeding.
Fig. 3: P2 neurons are required for sleep loss induced by chronic social isolation.
Fig. 4: Activation of P2 neurons during acute social isolation induces sleep loss and over-consumption of food.

Data availability

The RNA-seq data sets generated in this study have been deposited in NCBI’s Gene Expression Omnibus43 and are accessible through GEO series accession number GSE137498Source data are provided with this paper.

Code availability

Customized R script based on the R/rethomics package is available upon request.

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Acknowledgements

We thank G. Maimon for advice on [Ca2+] imaging experiments and quantitative analysis of the hemibrain connectome; J. Park, W. Ja, L. Shao, U. Heberlein, S. Park, S. Kim, A. Sehgal, M. Wu and H. Steller for sharing fly stocks, reagents, protocols, and equipment; B. McEwen, L. Vosshall, A. Patke, L. Zhao, N. Svetec, Y. Shuai, S. Axelrod and D. Top for comments on the manuscript; the Resource Center of Precision Instrumentation Technologies and the Resource Center of Genomics at the Rockefeller University for technical support; the Bloomington Drosophila Stock Center for fly stocks; and the Developmental Studies Hybridoma Bank for antibodies. This work was supported by NIH grants 5R37 NS053087 and 5R35 GM136237 to M.W.Y. W.L. was supported by fellowships from the Leon Levy Foundation, the Jane Coffin Childs Memorial Fund, and the Grass Foundation. S.S. was partially supported by NSF IOS no. 1656603. C.L. was supported by a seed grant from the Kavli Foundation.

Author information

Affiliations

Authors

Contributions

W.L. conceived the project, designed experiments, analysed data, and wrote the manuscript. W.L., Z.W., S.L., J.O., A.D.N. and I.F. performed experiments. C.L. performed [Ca2+] imaging experiments and analysed the data. S.S. supervised statistical analyses. M.W.Y supervised the project and wrote the manuscript. All authors discussed the results and contributed to the manuscript.

Corresponding authors

Correspondence to Wanhe Li or Michael W. Young.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Jason Rihel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Social isolation reduces sleep in Drosophila.

a, Schematic of social isolation paradigm. Adult fruit flies with social experience were subjected to social isolation or group enrichment for 7 days before sleep was measured using Drosophila activity monitors. Social isolation consists of housing one fly per vial. Group enrichment consists of housing 2, 5, 25 or 100 flies per vial. b, Sleep profiles (mean ± s.e.m. proportion of time spent sleeping in consecutive 30-min segments during a 24-h LD cycle) of flies after social isolation or group enrichment with different group sizes for 7 days. cg, Raster plots of sleep bouts of 20 individual animals after social isolation (c), group enrichment in a group of 2 animals (d), group enrichment in a group of 5 animals (e), group enrichment in a group of 25 animals (f) or group enrichment in a group of 100 animals (g). Each row is an individual fly, with each coloured bar representing a sleep bout in a 24-h LD cycle. hk, Quantification (mean ± s.e.m. with individual data points) of daily total sleep (h), daytime sleep (i), ZT0–4 sleep (j) and nighttime sleep (k) for flies after social isolation (Iso) or group enrichment (Grp) with different group sizes. For b and hk, n = 23–30 flies. Ordinary one-way ANOVA followed by Tukey’s multiple comparison tests; means sharing the same letter are not significantly different. For n and P values, see Source Data

Source data.

Extended Data Fig. 2 Chronic social isolation does not alter nighttime sleep in wild-type Drosophila.

ad, Density plots for distribution of daytime sleep bouts for flies after 1, 3, 5 or 7 days of group enrichment or social isolation. All daytime sleep bouts collected from all animals in each condition were combined. e, Quantification (mean ± s.e.m. with individual data points) of daily nighttime sleep for wild-type flies after group enrichment or social isolation for 7 days. fi, Density plots for distribution of nighttime sleep bouts for flies after 1, 3, 5 or 7 days of group enrichment or social isolation. All nighttime sleep bouts collected from all animals in each condition were combined. jm, Plots of cumulative relative frequency for distributions of nighttime sleep bouts for flies after 1, 3, 5 or 7 days of group enrichment or social isolation. Kolmogorov–Smirnov tests were used to compare distributions. For e, n = 29–32 animals, two-sided unpaired t-test with Welch’s correction; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s., not significant. For n and P values, see Source Data

Source data.

Extended Data Fig. 3 Social isolation reduces Drosophila sleep in age-matched flies, in various isogenic strains, and in aged wild-type animals.

a, Schematics of measuring sleep using Drosophila activity monitors after 1 or 7 days of group enrichment or social isolation in age-matched flies. be, Sleep profile and quantification of daily total sleep, daytime sleep and ZT0–4 sleep after 1 day (b, c, n = 55–64 flies) or 7 days of group enrichment or social isolation (d, e, n = 61–64 flies). f, g, Sleep profile and quantification of daily total sleep, daytime sleep and ZT0–4 sleep of the Canton-S isogenic strain after social isolation or group enrichment with different group sizes for 7 days (n = 30–47 flies). h, A 7-day-long sleep profile of flies after group enrichment or social isolation for 7 days. i, j, Sleep profile and quantification of daily total sleep, daytime sleep and ZT0-4 sleep for Berlin-K flies after social isolation or group enrichment (25 flies in a group) for 7 days (n = 22–31 flies). k, l, Sleep profile and quantification of daily total sleep, daytime sleep and ZT0–4 sleep of aged wild-type flies after group enrichment or social isolation (25 flies in a group) for 7 days (n = 52–54 animals). m, n, Sleep profile and quantification of daily total sleep, daytime sleep and ZT0–4 sleep of male wild-type flies after group enrichment in a male-only group (30 flies in a group) or in a mixed-sex group (15 male and 15 female flies in a group) for 7 days (n = 32 animals). Sleep profiles are displayed as the average proportion of time spent sleeping in consecutive 30-min segments during a 24-h LD cycle (mean ± s.e.m.). Quantifications are displayed as mean ± s.e.m. with individual data points. For g, ordinary one-way ANOVA followed by Tukey’s multiple comparison test; means sharing the same letter are not significantly different. For c, e, j, l and n, two-sided unpaired t-tests with Welch’s correction; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant. For n and P values, see Source Data

Source data.

Extended Data Fig. 4 Social isolation reduces sleep in Drosophila SIP lines.

ad, Sleep profiles of long-sleeping flies: SIP-L1-3 (a), SIP-L1-4 (b), SIP-L2-1 (c), and SIP-L2-6 (d) after group enrichment or social isolation (25 flies in a group for group treatment) for 7 days. eh, Sleep profiles of short-sleeping flies: SIP-S1-1 (e), SIP-S1-9 (f), SIP-S2-3 (g), and SIP-S2-9 (h) after group enrichment or social isolation (25 flies in a group for group treatment) for 7 days. Sleep profiles are displayed as the mean ± s.e.m. proportion of time spent sleeping in consecutive 30-min segments during a 24-h LD cycle. The long-sleeping and short-sleeping fly lines were randomly selected from the SIP, a panel of inbred Drosophila melanogaster strains with extreme long or short sleep-duration phenotypes35,36.

Extended Data Fig. 5 RNA-seq reveals changes in gene expression during chronic social isolation.

a, b, Volcano plots of differential gene expression from RNA-seq results. a, Comparison between chronic isolation and group conditions (Iso_7D vs Grp). b, Comparison between chronic isolation and acute isolation conditions (Iso_7D vs Iso_1D). Red dots indicate genes with significant adjusted P values in both comparisons. Differential gene expression analyses were conducted using DESeq2, which uses a two-sided Wald test and Benjamini– Hochberg correction. c, Venn diagram showing the intersection of the above two comparisons. d, Heatmap of the 274 intersected genes showing significant differential gene expression changes in both comparisons. e, Gene ontology of the 214 candidate genes from categories II and IV in d. Black bar, counts of genes in each GO term; white bar, −log10P values for each GO term. See Methods and Supplementary Information for details on RNA-seq data analyses.

Extended Data Fig. 6 Chronic social isolation results in reduced sleep and excessive feeding, whereas food consumption is not altered in sleep mutants or after acute social isolation.

a, Sleep profiles and matching feeding profiles of three representative individual flies after seven days of group enrichment. b, Sleep profiles and matching feeding profiles of three representative individual flies after seven days of social isolation. Sleep profile is presented as sleep amount (min) in consecutive 30-min segments during a 24-h LD cycle. Matching feeding profile is presented as food consumption (μl) in consecutive 30-min segments during a 24-h LD cycle. c, Quantification of daily total food consumption, daytime food consumption, nighttime food consumption and ZT0–4 food consumption for wild-type and sleep mutant inc1 flies (n = 25–29 flies). d, Quantification of daily total food consumption, daytime food consumption, nighttime food consumption and ZT0–4 food consumption for wild-type and sleep mutant fmn flies (n = 25–29 animals). e, Quantification of daily total food consumption, daytime food consumption, nighttime food consumption and ZT0–4 food consumption for wild-type and sleep mutant wakeD2 flies (n = 23–30 flies). f, Feeding profile measured by ARC assay in flies following 1 day of group enrichment or social isolation (mean ± s.e.m.). g, Quantification of daily total food consumption, daytime food consumption, nighttime food consumption and ZT0–4 food consumption for flies after 1 day of group enrichment or social isolation (f, g, n = 49–50 flies). All quantifications are displayed as mean ± s.e.m. with individual data points. Unpaired t-tests with Welch’s correction. For n and P values, see Source Data

Source data.

Extended Data Fig. 7 Limostatin transcripts are detected in fly head RNA-seq sample libraries; NPF-GAL4 expression pattern; feeding profile of flies in experiments silencing P2 neurons; and silencing P2 neurons with UAS-shibirets1 during social isolation is insufficient to block chronic social isolation-induced sleep loss.

a, Reads from RNA-seq sample libraries (Grp, Iso_1D, and Iso_7D) align to the gene region of Lst (CG8317). b, Akh and Lst are known to be co-expressed in the corpora cardiaca13. No reads were detected or aligned to the gene region of Akh, suggesting that the RNA-seq samples were free of corpora cardiaca materials and that the measured Lst transcripts come from sources in the brain. c, Expression pattern of NPF-GAL4-labelled neurons revealed by UAS-myr::GFP and NPF antibody staining. NPF+ cells overlap with GFP+ cells. P1, P2, DM, L1-l (or LNd)22, s-LNv44 and NPFM (ref. 45) neurons are labelled. d, An additional brain imaged from the posterior end to show NPF+ and GFP+ cell bodies of P2 neurons (dashed circle). Magenta, NPF; green, GFP; blue, N-cadherin; scale bars, 50 μm. eg, Feeding profiles measured by ARC assay for parental control flies (e, f) and flies expressing UAS-Kir2.1 with P2-GAL4 (g) following 7 days of group enrichment or social isolation (mean ± s.e.m.; n = 27–30 flies). hm, Sleep profiles for parental control flies (hk) and flies expressing UAS-shibirets with P2-GAL4 (l, m) following 7 days of group enrichment or social isolation at 22 °C (h, j, l) or 29 °C (i, k, m). All sleep behaviour was tested at 22 °C. n, Quantification (mean ± s.e.m. with individual data points) of daily total sleep, daytime sleep and ZT0–4 sleep for parental control flies and flies expressing UAS-shibirets with P2-GAL4 following 7 days of group enrichment or social isolation at 22 °C or 29 °C. Sleep profiles are displayed as the mean ± s.e.m. proportion of time spent sleeping in consecutive 30-min segments during a 24-h LD cycle. For hn, n = 31–32 flies; two-way ANOVAs were used for detecting interactions between temperature treatment and group/isolation status; Šidák multiple comparisons tests were used for post hoc analyses between group-treated and isolated animals of the same genotype and temperature treatment; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For n and P values, see Source Data

Source data.

Extended Data Fig. 8 P2 neurons show similar activity patterns after chronic social isolation or group enrichment and P2 neurons synapse onto cell types labelled by R23E20-GAL4.

a, Tethered, walking, [Ca2+]-imaging setup with an infrared-sensitive camera that tracks the rotation of the ball. b, The anatomy of hDeltaK cells from neuPrint26 (left), compared with time-averaged z-projection of GCaMP7f signals driven by P2-GAL4 (right). Both images show two separate layers (higher layer and lower layer) of fan-shaped body neuropils. c, Cross-correlation analysis of the lower-layer GCaMP7f activity and the fly’s forward walking velocity. Thin lines, individual fly data; thick lines, population means. d, Cross-correlation analysis of the higher-layer GCaMP7f activity and the fly’s forward walking velocity. Thin lines, individual fly data; thick lines, population means. e, Quantification (mean ± s.e.m. with individual data points) of GCaMP7f activity during standing moments of flies after 7 days of group enrichment or social isolation. Identical two-photon acquisition parameters were used in all experiments (ce, n = 5–6 flies). f, The anatomy of FB6A cells from neuPrint. FB6A has been identified as one of the few cell types labelled by R23E10-GAL427. g, Synapse-number matrix for detected synapses from P2 neurons (named hDeltaK cells in neuPrint) to FB6A cells. Connectivity data and cell-type names are based on those in neuPrint, hemibrain: v1.126.

Extended Data Fig. 9 Sleep profiles and feeding profiles of flies in which P2 neurons were thermally activated by expressing UAS-dTPRA1 during acute (1 day) group enrichment or social isolation; parental and temperature controls are included.

a, Schematics of activating P2 neurons for 1 day of group enrichment or social isolation. Treatment at 22 °C (no thermoactivation) was used as control. Flies in group enrichment or social isolation were kept at 28 °C for 1 day to thermally activate P2 neurons. After 1 day of thermal activation (or no activation), sleep behaviour was measured at 22 °C. b, c, Sleep profiles of UAS-dTRPA1/+ heterozygous control flies after group enrichment or social isolation at 22 °C for 1 day or at 28 °C for 1 day. d, e, Sleep profiles of P2-GAL4/+ heterozygous control flies after group enrichment or social isolation at 22 °C for 1 day or at 28 °C for 1 day. f, g, Sleep profiles of flies expressing UAS-dTRPA1 under the control of P2-GAL4 after group enrichment or social isolation at 22 °C for 1 day or at 28 °C for 1 day. h, Schematics of activating P2 neurons for 1 day of group enrichment or social isolation. Treatment at 22 °C (no thermoactivation) was used as control. Flies in group enrichment or social isolation were kept at 28 °C for 1 day to thermally activate P2 neurons. After 1 day of thermal activation (or no activation), feeding behaviour was measured at 22 °C. i, j, Feeding profiles of UAS-dTRPA1/+ heterozygous control flies after group enrichment or social isolation at 22 °C for 7 days or at 28 °C for 7 days. k, l, Feeding profiles of P2-GAL4/+ heterozygous control flies after group enrichment or social isolation at 22 °C for 7 days or at 28 °C for 7 days. m, n, Feeding profiles of flies expressing UAS-dTRPA1 under the control of P2-GAL4 after group enrichment or social isolation at 22 °C for 7 days or at 28 °C for 7 days. Sleep profiles are displayed as the mean ± s.e.m. proportion of time spent sleeping in consecutive 30-min segments during a 24-h LD cycle. Feeding profiles are presented as mean ± s.e.m. food consumption (μl) in consecutive 30-min segments during a 24-h LD cycle. be, in, n = 28–32 flies.

Extended Data Fig. 10 Sleep profiles of flies in which P2 neurons were thermally activated by expressing UAS-dTPRA1 during chronic (7 days) group enrichment or social isolation; parental and temperature controls are included.

a, Schematics of activating P2 neurons for 7 days of group enrichment or social isolation. Treatment at 22 °C (no thermoactivation) was used as control. Flies in group enrichment or social isolation were kept at 28 °C for 7 days to thermally activate P2 neurons. After 7 days of thermal activation (or no activation), sleep behaviour was measured at 22 °C. b, c, Sleep profiles of UAS-dTRPA1/+ heterozygous control flies after group enrichment or social isolation at 22 °C for 7 days or at 28 °C for 7 days. d, e, Sleep profiles of P2-GAL4/+ heterozygous control flies after group enrichment or social isolation at 22 °C for 7 days or at 28 °C for 7 days. f, g, Sleep profiles of flies expressing UAS-dTRPA1 under the control of P2-GAL4 after group enrichment or social isolation at 22 °C for 7 days or at 28 °C for 7 days. h, Quantification (mean ± s.e.m. with individual data points) of daily total sleep, daytime sleep and ZT0–4 sleep for experimental and heterozygous control flies grouped or isolated for 7 days with (28 °C) or without (22 °C) thermal activation of P2-GAL4-labelled neurons. Sleep profiles are displayed as the mean ± s.e.m. proportion of time spent sleeping in consecutive 30-min segments during a 24-h LD cycle. For h, two-way ANOVAs were used for detecting interactions between temperature treatment and group/isolation status. Šidák multiple comparisons tests were used for post hoc analyses between group-treated and isolated animals of the same genotype and temperature treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; bh, n = 29–32 animals. For n and P values, see Source Data

Source data.

Supplementary information

Supplementary Information

This file contains Supplementary Sections 1-6 and Supplementary References.

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Li, W., Wang, Z., Syed, S. et al. Chronic social isolation signals starvation and reduces sleep in Drosophila. Nature 597, 239–244 (2021). https://doi.org/10.1038/s41586-021-03837-0

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