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The neuropeptide Pth2 dynamically senses others via mechanosensation

Abstract

Species that depend on membership in social groups for survival exhibit changes in neuronal gene expression and behaviour when they face restricted social interactions or isolation1,2,3. Here we show that, across the lifespan of zebrafish (Danio rerio), social isolation specifically decreased the level of transcription of pth2, the gene that encodes the vertebrate-specific neuropeptide Pth2. However, 30 minutes of exposure to conspecifics was sufficient to initiate a significant rescue of pth2 transcript levels in previously isolated zebrafish. Transcription of pth2 exhibited bidirectional dynamics; following the acute isolation of socially reared fish, a rapid reduction in the levels of pth2 was observed. The expression of pth2 tracked not only the presence of other fish but also the density of the group. The sensory modality that controls the expression of pth2 was neither visual nor chemosensory in origin but instead was mechanical, induced by the movements of neighbouring fish. Chemical ablation of the mechanosensitive neuromast cells within the lateral line of fish prevented the rescue of pth2 levels that was induced by the social environment. In addition, mechanical perturbation of the water at frequencies similar to the movements of the zebrafish tail was sufficient to rescue the levels of pth2 in previously isolated fish. These data indicate a previously underappreciated role for the relatively unexplored neuropeptide Pth2 in both tracking and responding to the population density of the social environment of an animal.

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Fig. 1: Transcriptional response to social isolation.
Fig. 2: Transcriptional dynamics of pth2.
Fig. 3: Sensory perception of conspecifics.
Fig. 4: Specific mechanosensation triggers transcription of pth2.

Data availability

All data are provided in this paper or the accompanying Supplementary Information files. All sequencing data have been made available under the Sequence Read Archive BioProject PRJNA627056. Gene annotation was performed using the publicly available ENSEMBL database (https://www.ensembl.org/Danio_rerio/Info/Index). The Z-Brain atlas can be downloaded from https://github.com/owenrandlett/Z-Brain60. Gene expression data from Raj et al.23 are available under the Gene Expression Omnibus accession number GSE105010.

Code availability

All relevant code is available under the public repository https://github.com/Anneser/SensingOthers/.

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Acknowledgements

We thank S. Chanda, J. Glüsing, F. Vollrath and C. Polisseni for their contribution to the experiments; A.-Y. Loos for her help with fish husbandry and discussions; and G. Laurent for discussions. S.R. and E.M.S. are funded by DFG CRC 1193: Neurobiology of Resilience. E.M.S. is supported by the Max Planck Society.

Author information

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Authors

Contributions

L.A. and E.M.S. conceived the project. L.A., A.G., S.R. and E.M.S. designed the experiments. L.A., I.C.A., A.G. and K.M. conducted the experiments. L.A. wrote the code for data acquisition, analysis and modelling. L.A. and E.M.S. wrote the manuscript.

Corresponding author

Correspondence to Erin M. Schuman.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Iain Couzin, Allen Mensinger and Ted Usdin for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Extended analysis of transcriptional responses to social isolation.

a, Experimental scheme. Beginning at 2 dpf, larval zebrafish were raised in an isolated or social (with four conspecifics) environment for 5, 8, 14 or 21 days. After the specified periods, RNA was obtained from whole brains and next-generation sequencing (NGS) was conducted. 6 biological replicates were obtained for 5 and 8 dpf, 5 replicates for 14 dpf, and 4 replicates for 21 dpf. b, Box plots depicting the fold change of gene expression between isolated and social groups (5 dpf) for all members of the pth-family in zebrafish. Only pth2 showed a significant change. Values are expressed relative to the isolated condition (n = 10 for all experiments, paired t-test, one-sided, Benjamini-Hochberg-corrected: pth2, pt = 9.82 < 0.001, pthla, pt = -0.52 = 0.62, pthlb, pth1a, pt = -2.55 = 0.09, pth1b, pt = 1.55 = 0.31, pt = -0.92 = 0.57, pth4, pt = 0.51 = 0.62). c, Gene expression data of previously described stress-responsive genes, StAR: steroidogenic acute regulatory protein; nr3c1: nuclear receptor subfamily 3, group C, member 1; avp: arginine vasopressin; pomca: proopiomelanocortin a; pomcb: proopiomelanocortin b; and oxt: oxytocin (n = 6 for all genes, FDR-corrected quasi-likelihood F-test implemented in edgeR, StAR: P = 0.99, nr3c1: P = 0.99, avp: P = 0.99, pomca: 0.99, pomcb: P = 0.99, oxt: P = 0.99). d, Validation of gene expression analysis by qPCR for StAR and nr3c1 after short exposure of previously isolated fish to conspecifics for 3 h. Box plots show expression relative to mean levels of isolated animals. Paired, one-sided t-tests, n = 6 for both genes. For StAR, pt = 0.37 = 0.71, for nr3c1, pt = -0.45 = 0.66. Box plots in b and d represent the median (black line), the lower and upper quartile (box) with the whiskers indicating at most 1.5 × the interquartile range. Significance is reported as follows: ***P < 0.001.

Extended Data Fig. 2 Registration and morphology of pth2+ cells.

a, Maximum-intensity projection (dorsal view) of the averaged Elavl3-H2BRFP stack from the z-brain atlas overlaid with the manually annotated position of pth2+ cells. b, Lateral view of the same stack. c, Number of pth2+ cells in each of the bilateral clusters. Stacks from 7 different 5 dpf larvae were counted. In the left cluster, 9.4 ± 2.4 cells were found, in the right one 10.9 ± 1.7 (mean ± standard deviation). Box plots represent the median (black line), the lower and upper quartile (box) with the whiskers indicating at most 1.5 × the interquartile range. d, Depth-encoded (0 – 184 μm) Z-projection of whole-mount immunostaining against pth2 (5 dpf). White box shows area magnified in eg. e, Single frame on the level of the pth2+ cell bodies. f, Magnification of a single frame on the level of the posterior projections. g, Single frame on the depth of the anterior projections of the pth2+ cells, forming a dense neuropil in the telencephalon. Scale bar indicates 100 μm.

Extended Data Fig. 3 Localization of pth2+ cells.

a, pth2+ cell bodies form bilateral clusters at the edge of the otpa domain in the diencephalon. More ventrally, pth2+ cell projections were closely apposed to the otpa domain and enter the telencephalon. 18 animals were imaged across 4 different experiments. All scale bars indicate 100 μm. b, The pth2+ cells were found dorsolateral to the main TH+ cell clusters in the diencephalon. The rostral projections of the pth2 clusters were found caudal to the telencephalic dopaminergic neurites. 14 animals were imaged across 3 different experiments. c, pth2+ cell bodies were slightly more dorsally located than the more rostrally situated OXT+ neurons. Their telencephalic projections formed a dense neuropil structure rostral to the neurosecretory hypothalamic preoptic area, where OXT+ neurons were found. 4 different animals were imaged.

Extended Data Fig. 4 Evidence for widespread expression of the Pth2 receptor, pth2r.

a, Clustering of cell types reproduced from a previous study65. Data were used as provided under Gene Expression Omnibus accession number GSE105010 and analysed using the Seurat pipeline. b, Distribution of pth2r-expressing cells. Grey points correspond to cells in which pth2r was not detected, the intensity of blue indicates how many reads were detected. c, Distribution of oxytocin receptor (oxtr)-expressing cells. d, Distribution of avpr2aa-expressing cells. Four receptors of avp were detected in the data set, we show the one that was most widely expressed. e, Barplots show the percentage of pth2r+ cells within each cluster (“measured”). For each cluster, we also provide a bootstrapped estimation of what percentage would be expected by random sampling of cells, with numbers of bootstrapped cells being equal to cells belonging to the given cluster. In addition, we show the percentage of pth2r+ cells across the entire population (“all”) f, Barplots show percentage of avpr2aa+ cells within each cluster, same as in e. For oxtr+ cells, an insufficient number of cells were identified in this data set to perform this kind of comparison.

Extended Data Fig. 5 The effect of sex and density on pth2 expression in adult zebrafish.

a, Male and female adult zebrafish (3 mpf) were sampled from the same tank. Box plots show pth2 levels in relation to the mean level of male gene expression. Unpaired, one-sided t-test, p(n = 4, t = 0.40) = 0.69. b, Box plots show pth2 levels in adult zebrafish that were kept at densities of 5 and 35 per 3.5 l for one week, respectively. Expression levels were normalized to the mean level at the lower density. Unpaired, one-sided t-test, p(n = 6, t = 2.44) = 3.4E-2. Box plots in a and b represent the median (black line), the lower and upper quartile (box) with the whiskers indicating at most 1.5 × the interquartile range. Significance is reported as follows: *P < 0.05.

Extended Data Fig. 6 The effect of genotype on pth2 transcription.

a, Box plot shows the difference in pth2 levels between socially reared and isolated Nacre (mitfa−/−) larvae (5 dpf). 6 replicates were obtained, a paired, one-sided t-test showed that pt = 7.98 = 2.1E-4. b, Same experiment as shown in Fig. 3d. Here, pth2 levels of KN larvae were assessed and Nacre (mitfa−/−) animals used as stooges. The impact of visual and physical access as well as the interaction of both was evaluated using a 2-way ANOVA. For all groups, n = 6. For visual access, pF = 0.83 = 0.37, physical access, pF = 144.96 = 1.3E-10, visual-physical interaction, pF = 2.41 = 0.14. Box plots in a and b represent the median (black line), the lower and upper quartile (box) with the whiskers indicating at most 1.5 × the interquartile range. Significance is reported as follows: ***P < 0.001.

Extended Data Fig. 7 The effect of experimental conditions on locomotion.

a, Box plot shows the average velocity of 5 dpf animals that were raised either in isolation or with conspecifics. Unpaired, one-sided t-test, n = 14 for isolation-reared fish, n = 11 for socially-reared fish, pt = 0.28 = 0.78. b, Box plot depicts average velocity of isolation-reared animals when swimming alone or together with 14 conspecifics. Unpaired, one-sided t-test, n = 8 for both conditions, pt = 0.32 = 0.75. c, Scheme of the open field used in d. Animals were placed in a rectangular dish, which contained an adjacent compartment separated by a transparent barrier (indicated by dashed line), where (in some experiments) conspecifics were placed. d, Box plot shows the average velocity of animals at different developmental stages after rearing in isolation or with conspecifics. Visual access indicates whether conspecifics were placed in the adjacent compartment. An ANOVA revealed that visual access did not lead to differences between the groups (pF = 0.21 = 0.65). Speed increased with age (pF = 333.62 = 4.1E-47), and was influenced by raising condition (pF = 16.81 = 5.7E-5), although one-sided post hoc t-tests corrected with Holm’s method indicated no consistent influence on locomotion for the different age groups. Interaction effects were not observed. At 7 dpf without visual access (v.a.) and socially reared: N = 15, isolation-reared: N = 17, pt = 4.71 = 2.1E-4; N = 16 with v.a. and socially reared; N = 18 isolation-reared, pt = 2.38 = 6.9E-2; at 14 dpf without v.a., socially reared: N = 17 and isolation-reared: N = 16, pt = 1.55 = 0.19; with v.a. and socially reared N = 18 and isolation-reared N = 15, pt = 1.79 = 0.17; at 21 dpf, without v.a., socially reared: N = 18, isolation-reared: N = 17, pt = 3.47 = 5.2E-3; with v.a. and socially reared N = 16 and isolation-reared N = 13, pt = 0.06 = 0.82; at 28 dpf, isolation-reared: N = 9 for both cases, without v.a., socially reared: N = 15, pt = 0.23 = 0.82; with v.a. N = 18, pt = 2.25 = 8.3E-2. Box plots in a, b and d represent the median (black line), the lower and upper quartile (box) with the whiskers indicating at most 1.5 × the interquartile range. e, Graph depicts the bout onset of larvae in response to piezo stimulation as explained in Fig. 4d, e. Bout onsets are displayed as relative frequencies, data from individual fish are shown in grey with the mean (smoothed with a rolling window of length 0.05 s) overlaid in purple.

Extended Data Fig. 8 Artificial mechanical stimulation.

a, Experimental scheme for artificial mechanical perturbation. A randomized series of stimulation and pause periods was drawn from two distributions using a custom-written Matlab script. Periods of activity were propagated via an Arduino board to gate a frequency generator. The output signal was amplified before activating a piezo actuator transferring specific frequencies to a well in a 12-well dish with an isolated fish. b, Scatter plot indicating pth2 levels relative to isolated animals (dashed line) after 3 h of different stimulation paradigms (either continuous stimulation or short bouts of stimulation interrupted by pauses of the same length). Paired, one-sided t-tests with BH-correction were used. Single piezo element: for continuous stimulation, p(n = 6, t = -0.65) = 0.54, for continuous stimulation in the presence of kin-imbued water, n = 2. Two piezo elements: for periodic stimulation at 300 ms intervals, p(n = 3, t = -0.18) = 0.87, at 5 s intervals, n = 2. Two piezo elements with appendages: for periodic stimulation at 300 ms intervals, p(n = 3, t = -0.68) = 0.57, at 500 ms intervals, n = 2, at 700 ms, p(n = 3, t = -0.82) = 0.50, at 800 ms intervals, p(n = 5, t = -0.85) = 0.44. c, Larval zebrafish (5 dpf) were recorded for 10 min during a free exploration in 10 cm diameter dishes. Trajectories were used to extract behavioural features. d, Locomotion of larval fish occurs in discrete bouts, facilitating the extraction of interbout-intervals (IBIs). In total, we extracted 16.326 IBIs from 25 fish. e, Distribution of all IBIs is shown as a histogram, overlaid with a log-logarithmic distribution fit to the data. f, Table displays all distribution types that were fitted to the IBI data set and shows the corresponding Akaike information criterion.

Supplementary information

Reporting Summary

Supplementary Table 1

Table listing all genes identified as differentially expressed at the different developmental stages tested. Provided are ENSEMBL gene ID, external gene name, entrezgene ID, Log-fold change, logCPM, P-value, FDR. P-values are derived using a quasi-likelihood F-test.

Supplementary Table 2

Table listing all Δ Ct values obtained in our qPCR experiments for all figures.

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Anneser, L., Alcantara, I.C., Gemmer, A. et al. The neuropeptide Pth2 dynamically senses others via mechanosensation. Nature 588, 653–657 (2020). https://doi.org/10.1038/s41586-020-2988-z

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