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Flexible scaling and persistence of social vocal communication

Nature volume 593, pages 108–113 (2021)Cite this article

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Abstract

Innate vocal sounds such as laughing, screaming or crying convey one’s feelings to others. In many species, including humans, scaling the amplitude and duration of vocalizations is essential for effective social communication1,2,3. In mice, female scent triggers male mice to emit innate courtship ultrasonic vocalizations (USVs)4,5. However, whether mice flexibly scale their vocalizations and how neural circuits are structured to generate flexibility remain largely unknown. Here we identify mouse neurons from the lateral preoptic area (LPOA) that express oestrogen receptor 1 (LPOAESR1 neurons) and, when activated, elicit the complete repertoire of USV syllables emitted during natural courtship. Neural anatomy and functional data reveal a two-step, di-synaptic circuit motif in which primary long-range inhibitory LPOAESR1 neurons relieve a clamp of local periaqueductal grey (PAG) inhibition, enabling excitatory PAG USV-gating neurons to trigger vocalizations. We find that social context shapes a wide range of USV amplitudes and bout durations. This variability is absent when PAG neurons are stimulated directly; PAG-evoked vocalizations are time-locked to neural activity and stereotypically loud. By contrast, increasing the activity of LPOAESR1 neurons scales the amplitude of vocalizations, and delaying the recovery of the inhibition clamp prolongs USV bouts. Thus, the LPOA disinhibition motif contributes to flexible loudness and the duration and persistence of bouts, which are key aspects of effective vocal social communication.

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Fig. 1: Hypothalamic LPOAESR1 neurons underlie USV social calling.
Fig. 2: LPOAVGAT–PAGVGAT–PAGVGLUT2 di-synaptic disinhibition circuit triggers USVs.
Fig. 3: LPOAESR1 neuron activity scales USV amplitude and bout duration.
Fig. 4: Vocal persistence is generated through activity of local PAGVGAT neurons.

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Data availability

The data in this study are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

All analysis code are available on GitHub: https://github.com/stowerslab/USV_Analysis_Code.git.

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Acknowledgements

We thank the Stowers laboratory for support and advice; T. Holy and T. Barnes for early constructive comments and discussions; S. Tan, N. Koblesky and S. Simpson for exploratory behavioural tests; and L. Ye for aid with the fibre photometry experiments. The work was supported by the Dorris Neuroscience and Skaggs Scholarships (J.C.) the Anandamahidol Foundation Fellowship (V.L.), Career Award at the Scientific Interface from BWF (J.E.M.), and grants from NIH (R01NS097772 and R01DA049787 (B.K.L.); and R01NS108439 (L.S.)).

Author information

Authors and Affiliations

  1. Department of Neuroscience, Scripps Research, La Jolla, CA, USA

    Jingyi Chen, Sandra Taylor, Pete Sheurpukdi, Jennifer R. Jensen & Lisa Stowers

  2. Biomedical Sciences Graduate Program, Scripps Research, La Jolla, CA, USA

    Jingyi Chen

  3. Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    Jeffrey E. Markowitz & Sandeep Robert Datta

  4. Biological Sciences Graduate Program, University of California San Diego, La Jolla, CA, USA

    Varoth Lilascharoen

  5. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Jason A. Keller

  6. Neurobiology Section, Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA

    Byung Kook Lim

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Contributions

J.C. and L.S. designed the study and wrote the manuscript. J.E.M. and S.R.D. developed Voseq analysis; V.L. performed slice physiology; S.T. aided in histology, behavioural testing and cell counting; B.K.L., P.S. and J.A.K. aided in data analysis and MATLAB code. J.R.J. performed behavioural analysis. All other experiments were performed by J.C.

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Correspondence to Lisa Stowers.

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Peer review information Nature thanks Erich Jarvis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.

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Extended data figures and tables

Extended Data Fig. 1 ESR1-expressing subset of LPOA neurons express c-FOS during odour-evoked USV calling.

a, Example of c-FOS expression in the preoptic area after exposure to tonic water as a control odour (no USVs) or with female odour (USVs)25,36,37,38. Scale bar, 200 μm. Quantification of this experiment is shown in c and d. b, Immunostaining after exposure to female mouse odour shows that c-FOS+ in the LPOA largely overlaps with ESR1-ZsGreen cells. White squares delineate enlarged single z-stack sections shown in Fig. 1b. Scale bar, 100 μm. c, d, Expression of c-FOS and ESR1 in the LPOA following awake behaviour with control odour (black) or female odour (yellow). c, Rostrocaudal distribution of c-FOS+ cells in the LPOA. d, Rostrocaudal percentage of cells co-expressing c-FOS and ESR1 in the LPOA. Data are mean ± s.e.m. n = 4 mice.

Source data

Extended Data Fig. 2 LPOAESR1 neural activity correlates with USV calling, and chemogenetic inhibition of LPOAESR1 neurons reduce USV calling.

LPOAESR1+ GCaMP6s activity during natural social behaviour. a, Top, experimental design for LPOAESR1 fibre photometry recordings. Bottom, sample image showing fibre optic track and viral expression of GCaMP6s. Scale bar, 200 μm. n = 9 mice, >3 sections per mouse collected. In addition to evoking USV calling, the presence of a female mouse also markedly alters the behaviour of the male mouse (arousal, social sniffing, locomotion and sexual mounting), potentially confounding interpretation of the observed neural activity. We observed that after the removal of the female mice, male mice often generated intermittent USV calls, perhaps to lure the female mice back, without the behavioural noise of mounting or social sniffing. We leveraged this post-female period to observe increases in LPOAESR1/GCaMP6s activity with a rise shortly before the onset of post-female USVs, clearly suggesting that endogenous LPOAESR1 neural activity correlates with emission of USVs. b, Representative USV production and GCaMP6s fibre photometry of male LPOAESR1 neurons as he behaves alone (pre-female), with a behaving female mouse, and after the female mouse is removed (post-female). Dashed line indicates when the female was added and removed. Top, mean USV power. Blue dots indicate USV syllable detection. Bottom, ΔF/F of LPOAESR1 GCaMP6s signals was calculated by MATLAB GUI as previously described34. c, Dark green line denotes mean z-score of GCaMP6s signals before and after initiation of USV with behaving female phase, light green shading indicates 95% confidence interval. Grey shading denotes 95% confidence interval of the mean of the scrambled data (n = 9 mice). d, Mean average z-score of GCaMP6s signals during all USV syllables evoked with a behaving female compared to scrambled data points during pre-female behaviour. Data are mean ± s.e.m. n = 9 mice. ***P = 0.0003, unpaired t-test, two sided. e, Dark green line denotes mean z-score of GCaMP6s signals before and after initiation of USV during post-female stage, light green shading indicates 95% confidence interval. Grey shading denotes 95% confidence interval of the mean of the scrambled data. n = 9 mice. f, Mean average z-score of GCaMP6s signal of all USV syllables during post-female stage compared to scrambled data points during pre-female stages. Data are mean ± s.e.m. n = 9 mice. ***P = 2.2 × 10−4, unpaired t-test, two sided. g–i, To determine whether the increased hypothalamic activity is involved in odour-evoked USV calling, we targeted chemogenetic inhibition to the LPOA, which is a largely unstudied heterogeneous region that has been implicated in sleep, thirst and reward behaviour39,40,41,42,43, and quantified USV production during natural interactions with an awake female. g, Non-specific chemogenetics. Left, hM4Di viral injection in the LPOA of wild-type mice. Right, experimental assay; after expression of the hM4Di virus, male mice were injected intraperitoneally with CNO–saline–CNO–saline (every other day for 4 days in total) and allowed to interact with a freely moving female mouse to evoke USV calling. h, Number of USV syllables emitted after injection with CNO (purple) or saline (black). Data are mean ± s.e.m. n = 10 mice. N.S., P = 0.11, paired two-tailed Wilcoxon test. i, Number of USVs emitted across four sequential test days. Overall, the manipulation did not produce a significant effect on behaviour; however, half of this group (solid lines, n = 5 mice) did show a constant reduction in USVs, whereas the other half (dashed lines, n = 5 mice) continued to emit USVs in the presence of CNO. This experiment suggests the potential for a functional role of the hypothalamic neurons in social vocal communication and a need for a more specific viral labelling method. j, Average number of USVs between all saline and CNO injection days as shown in Fig. 1d. n = 6 mice. *P = 0.03, two-sided Wilcoxon test. k, Sample image of hM4Di expression in ESR1-ZsGreen mice. Scale bar, 500 μm. n = 6 mice, >3 sections per mouse collected. l, Quantification of total time performing social behaviours observed by LPOAESR1/hM4Di neurons from male mice during a 4-min interaction with live female mice on CNO and saline injection days. n = 6 mice. *P = 0.02, ***P < 0.001, paired two-sided t-test. Note that we observed an unexpected increase in the anti-social defensive behaviour of the female mouse (kicking, running away), which reduced the ability of the male mouse to direct sniffing to the anogenital region. This observation is consistent with male USVs serving to enhance female courtship behaviour44,45. m, Experiment design to express control AAV-tdTomato virus in LPOAESR1 cells. n, Number of USVs emitted with behaving female mice over five test days, with alternating injections of either CNO or saline. o, Average number of USVs between saline and CNO injection days. n = 5 mice. P > 0.05, two-sided Wilcoxon test.

Source data

Extended Data Fig. 3 Optogenetic stimulation of LPOAESR1/ChR2 neurons triggers USV calling in both male and female mice.

a, Left, ChR2 virus injection in the LPOA region of ESR1-Cre mice. Right, sample image of co-expression of ChR2 and FOS after photostimulation. Scale bar, 50 μm. b, Viral expression in the LPOA region. Composite overlay of total sections at left: bregma 0.2 mm, and right: bregma 0.3 mm. Colour intensity scales with increasing expression. n = 12 mice. c, Example electrophysiology recording during photostimulation of LPOAESR1/ChR2 neurons in ex vivo slice. Blue bar denotes 10-Hz light stimulation. Neural response is time-locked to light pulses. d, Number of USVs detected during 10-Hz photostimulation of LPOAESR1/ChR2 cells from male mice (n = 23 mice) (left) and female mice (n = 11 mice) (right). Data are mean ± s.e.m. e, f, Average USV power across the 40–90kHz band evoked by 10-Hz photostimulation. Solid line indicates mean of all trials; shaded region indicates 95% confidence interval. The blue (e) and red (f) traces denote male and female trials, respectively14,46. Blue shaded bar denotes light stimulation at 5–10 s. g, Raster plot of complete dataset showing USV power evoked by photostimulation at 10 Hz (5-s duration, 15-ms pulses; between the blue lines) of LPOAESR1/ChR2 neurons from female mice (n = 11 mice, red) and male mice (n = 23 mice, blue). n = 242 trials, each row is a single trial. Colour intensity represents average USV power across the ultrasonic band (40–90 kHz). Note that the POA has been implicated in a variety of functions including homeostatic control of internal states such as thermoregulation and thirst, sexually dimorphic social behaviours including parenting and mating behaviour, as well as motivated behaviours39,47,48,49. It is likely that features of these neurons that enable them to generate USVs in the absence of social stimuli in the laboratory also enable them to participate in other neural computations that are currently unknown.

Source data

Extended Data Fig. 4 Activating LPOAESR1 neurons elicit a variety of USV syllables similar to natural USVs.

a, Evaluation of USV syllable types emitted by a wild-type male mouse naturally interacting with a behaving female mouse, compared to wild-type P7–P8 pup calls (n = 18 mice) (orange) evoked by individually isolating mice from the home cage; wild-type adult male calls (n = 20 mice) evoked by interaction with female or male urine, anaesthetized male or female mice on successive days (grey); or calls from experimental male mice expressing ChR2 (LPOAESR1/ChR2, n = 23 mice) evoked by interaction with either female urine or a live female mouse with no ChR2 light stimulation to determine the natural USV repertoire (blue, left); or male mice expressing ChR2 (LPOAESR1/ChR2, n = 23 mice) stimulated with light (10 Hz, 25 Hz and 50 Hz) in the absence of a female mouse (blue, right; blue shading ‘light stimulation’); the same male mice were used in the no-light and light stimulation experiments. Dot denotes the Pearson correlations for the top 5% of the most frequently used syllables, in which the box plot shows the mean and interquartile range of these correlations, and the plus symbol (‘+’) shows the correlation of the top 95% of the most frequency used syllables. P = 6.12 × 10−115 (F > 83.4), *P < 0.05, ***P < 0.001, one-way ANOVA test. MATLAB ‘mulcompare’ function was used for group statistical analysis comparing all other groups to wild-type male USV triggered by interaction with a live female mouse. b, Heat map showing Pearson’s correlation score among all 40 types of syllable detected across each condition compared to wild-type male USVs during interaction with live female mouse. Results are grouped by types of sensory stimulation: female context (red); ChR2 stimulation (blue); male context (green) and pup (orange). Warmer colours indicate higher similarity, which is quantified in a. These data show that the repertoire of USV syllables evoked by photostimulation are rich and varied. When compared to natural USVs, they are similar to those produced by wild-type male mice as they interact with live female mice; and less similar to USVs evoked by male cues and pup USVs.

Source data

Extended Data Fig. 5 LPOAESR1 projections to USV motor centre (ambiguous) are sparse and unable to be functionally validated, whereas LPOAESR1 projections to PAG produces robust USV production.

a, Strategy to test whether LPOAESR1 neurons are anatomically or functionally connected to either the PAG or the nucleus ambiguous, which are known to evoke USV calling in the mouse16,17,50,51. b–f, Retrograde tracing experiment from either PAG or nucleus ambiguous to label LPOAESR1 cell projections by injecting a Cre-dependent FLP-expressing pseudotyped equine infectious anaemia virus (RG-EIAV-FLEX-Flp) in either the PAG or nucleus ambiguous, and a FLP-dependent AAV expressing eGFP in the LPOA of ESR1-Cre mice32,33. We confirmed the specificity of viral expression by multiplex fluorescent in situ hybridization (Extended Data Fig. 6a, b). We observed sparse labelling of LPOA cells that directly project to the nucleus ambiguous, and a larger population centred in the LPOA region (previously showed to be co-labelled by c-FOS and ESR1) that directly project to the region of the PAG USV-gate neurons. b, Example image of PAG-projecting (top) or nucleus ambiguous-projecting (bottom) eGFP+ cells in the LPOA as described and quantified in c–f. Scale bar, 200 μm. n = 5 mice; 5 sections per mouse. c, d, Anatomical tracing from the PAG to the LPOA resulted in robust labelling. c, Experimental design to express the FLEX-FLP virus in the PAG and fFLEX-eGFP in the LPOA of ESR1-Cre mice. d, Restrocaudal distribution of the total number of PAG-projecting eGFP+ cells in the LPOA. Data are mean ± s.e.m. n = 5 mice. e, f, Anatomical tracing from the nucleus ambiguous to the LPOA resulted in sparse labelling. e, Experimental design to express FLEX-FLP virus in the nucleus ambiguous and fFLEX-eGFP in the LPOA of ESR1-Cre mice. f, Restrocaudal distribution of the total number of nucleus ambiguous-projecting eGFP+ cells in the LPOA. Data are mean ± s.e.m. n = 5 mice. To test whether either of these projections function to evoke USV calling, we expressed ChR2 in the LPOA of ESR1-Cre mice and photostimulated from axon terminals in either the PAG or the nucleus ambiguous. g, Sample image of optical fibre position for stimulation of LPOAESR1/ChR2 terminals in the PAG show in j. h, Experimental design for stimulation of LPOAESR1/ChR2 terminals in the nucleus ambiguous. i, Sample image of optical fibre position for terminal stimulation in the nucleus ambiguous. Scale bar, 200 μm. n = 5 mice, 4 sections per mouse. j, Average USV power across the 40–90-kHz band of recording during PAG terminal stimulation. Solid line indicates the mean and shaded region indicate 95% confident interval (blue shading, n = 13 male mice; red shading, n = 4 female mice; 4 trials per mouse). k, Average USV power across 40–90-kHz band of recording during terminal stimulation of the nucleus ambiguous. Solid line indicates the mean and shaded region indicate 95% confident interval (blue shading, n = 5 male mice, 4 trials per mouse). l, m, Average USV power during single trials of the same male stimulated with 25 Hz for 5 s from either LPOAESR1/ChR2 cell somas (l) or LPOAESR1/ChR2 axon terminals in the PAG (m).

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Extended Data Fig. 6 LPOA-PAG projecting cells are largely inhibitory and stimulation of LPOAVGAT cells elicits USVs.

a–c, RNAScope multiplex in situ hybridization histology of LPOA sections after injection of retro travelling Cre-dependent FLP-expressing virus in the PAG and a FLP-dependent eGFP (AAV-fFLEX-dGFP) in the LPOA of ESR1-Cre mice (see Extended Data Fig. 4c, d) reveals overlap of eGFP (green) (a), ESR1 (red) (b) and VGAT (magenta) (c) probes. Yellow traces are eGFP+ cells used as regions of interest and applied to ESR1 and VGAT channels for analysis. Scale bar, 50 μm. n = 3 mice, >3 sections per mouse with RNAScope staining. d–g, Electrophysiology recording of PAG neurons in ex vivo slice shows functional inhibition. d, Sample trace showing cell-attached recording of a PAG cell. e, IPSC and EPSC recordings while photostimulating (blue bars) LPOAESR1/ChR2 terminals. f, IPSC and EPSC recordings during single light pulse. g, Peak conductance (calculated by amplitude or driving force) of EPSC (red) and IPSC (blue) recordings. Data are mean ± s.e.m. Bottom, percentage of observed cells with monosynaptic IPSCs (blue), EPSCs (red), or both (grey). n = 5 mice, 29 cells. h, i, Strategy to test whether LPOA excitatory neurons elicit USVs. h, Top, experimental design to express ChR2 in the LPOA of VGLUT2-Cre knock-in mice. Bottom, percentage of ChR2 overlap with either the VGLUT2-ZsGreen marker or c-FOS staining. i, Sample images showing overlap between ChR2 with VGLUT2-ZsGreen or c-FOS staining. Scale bars, 50 μm. n = 4 mice, 7 images per mouse used for cell quantification. j, Number of USVs emitted during light stimulation of LPOAVGLUT2/ChR2 neurons. n = 4, 16 trials per condition. k, Sample image indicating fibre position (white square) and ChR2 expression in the LPOA of VGAT-ZsGreen mice. Scale bars, 200 μm. n = 9 mice, 4 sections per mouse collected. l, Experimental design to express ChR2 in LPOA of VGAT-Cre mice and the number of USV syllables emitted during light stimulation of LPOAVGAT/ChR2 neurons. Data are mean ± s.e.m. n = 9 mice, 91 trials per condition. m, Composition of c-FOS expression (after odour-evoked USVs) in the PAG of VGAT-Cre mice. c-FOS+ cells (red) are largely VGAT− (consistent with PAG USV gate neurons being excitatory), whereas VGAT neurons are largely clustered in the ventrolateral PAG52. n = 3 mice, overlay of 100-μm thick sections roughly at bregma −4.4 mm.

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Extended Data Fig. 7 Local PAGVGAT neurons inhibit PAG USV-gate cells, and photostimulation inhibits natural USVs.

Most immediate neurons ‘upstream’ of the PAG USV-gate cells are VGLUT2− neurons in the PAG, ipsilateral to the PAGVGLUT2 neurons. a, Experimental design for rabies viral tracing from the PAG of VGLUT2-Cre knock-in mice. b, Sample PAG image from rabies tracing. Red cells on the left are cells infected with the TVA+G helper virus that overlaps with VGLUT2-ZsGreen (starter cells). The white box on the right is enlarged and showed in c. Scale bars, 250 μm. c, In total, 76 out of 87 cells labelled with rabies-tdTomato (87%) observed in ventrolateral PAG do not overlap with VGLUT2-ZsGreen cells, which suggests that they probably express VGAT. Scale bar, 100 μm. n = 5 mice, total 32 sections counted. To functionally test whether the PAG USV-gate neurons are subjected to local inhibition, we engineered male mice to express ChR2 in the local PAG inhibitory cells (PAGVGAT/ChR2) and injected retroAAV-eGFP in the nucleus ambiguous to specifically identify PAG USV-gate neurons. d, Experimental design for retrograde labelling from the nucleus ambiguous to PAG USV-gate neurons, and optical manipulation of PAGVGAT/ChR2 cells for behaviour and physiology. e, Sample image of PAG section showing nucleus ambiguous-projecting cells in lateral PAG (green) and VGAT/ChR2-expressing cells (red) in ventrolateral PAG. Scale bar, 100 μm. Aq, aqueduct, fourth ventricle. Ex vivo whole-cell recordings and cell-attached recordings showed that all tested PAGGFP neurons (USV-gating neurons) were inhibited within 5 ms after photostimulation of PAGVGAT/ChR2 neurons, consistent with monosynaptic inhibitory inputs. n = 2 mice, 2 sections per mouse collected. f–h, Ex vivo slice electrophysiology recordings of PAG USV-gate cells while photostimulating PAGVGAT/ChR2 neurons. f, IPSC and EPSC recordings during a single light pulse. g, Photostimulation of PAGVGAT/ChR2 neurons generate monosynaptic iPSCs in all cells investigated. n = 2 mice, 14 cells recorded. h, Cell-attached physiology of USV-gate neurons (PAGGFP). Blue shading indicates photostimulation period. Each line showed as individual cell recorded. i, j, Stimulating local PAGVGAT neurons inhibit socially evoked USVs. i, Increasing the frequency or duration of photostimulation (5 s of 1 Hz, 5 Hz, 10 Hz, 50 Hz and 10 s of 25 Hz) of PAGVGAT/ChR2 neurons from male mice during interaction with awake behaving female mice to evoke natural USVs. Blue bar/shading denotes light stimulation. Number of USVs are calculated in 10-s time bins. Data are mean ± s.e.m. n = 3 mice, 5–6 trials per mouse per condition. j, Raster plot of USVs emitted before, during and after photostimulation at 25 Hz for 5 s, as showed in Fig. 2e. Blue light indicates light stimulation period. Each row is a single trial. Data are mean ± s.e.m. n = 3 mice, 19 trials.

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Extended Data Fig. 8 LPOAVGAT cell population connect to PAGVGAT cell population both anatomically and functionally, and the number of USV syllables and latency flexibly varies with social context.

a, Example image of PAG section for experiment described in Fig. 2f, g. Starter cells (magenta) overlap with VGAT-ZsGreen. Scale bar, 100 μm. b, Sample image of LPOA section for experiment described in Fig. 2f, g showing overlap of rabies-tdTomato+ cells (red) with VGAT-ZsGreen (272 out of 320 cells counted, n = 3 mice, total of 17 sections). Scale bar, 200 μm. c, Example image of RNAScope multiplex in situ hybridization in LPOA to complement Fig. 2f. Sections are stained with eGFP (green), VGAT (blue) and ESR1(red) probes. Scale bars, 25 μm. Most LPOA neurons that projected to PAGVGAT neurons co-express ESR1. n = 2 mice, 16 sections (20-μm thick) collected. Top right, quantification of rabies-positive cells overlapping with VGAT+ or ESR1+ using RNAScope multiplex in situ hybridization (n = 2 mice, total 24 rabies-positive cells quantified. d, PAGVGAT cells receive monosynaptic IPSC from LPOAVGAT/ChR2 neurons. IPSCs and EPSCs evoked by single light pulse (n = 5 mice, 16 cells recorded). e–h, To study the in vivo effects of LPOAESR1 neuron activity, we expressed ChR2 in the LPOA and GCaMP6s in the PAG of VGAT-Cre mice (LPOAVGAT/ChR2;PAGVGAT/GCaMP6s). Photostimulation in the LPOA of awake behaving mice resulted in a decrease in fibre photometry as measured GCaMP6s fluorescence in the local PAG inhibitory neurons and an accompanying initiation of USV production. e, Representative image of fibre track and viral expression of GCaMP6s in the PAG. Scale bar, 200 μm. n = 4 mice, 3 sections per mouse. f, Number of USV syllables emitted following light stimulation of LPOAVGAT/ChR2 neurons while recording of PAGVGAT/GCaMP6s signals. Data are mean ± s.e.m. n = 4, 16 trials per condition. g, Mean z-score of fibre photometry signal from PAGVGAT/GCaMPs with photostimulation at 25 Hz for 10 s of LPOAVGAT/ChR2 cells. h, Mean z-score of fibre photometry signal from PAGVGAT/GCaMPs neurons with photostimulation at 1 Hz for 5 s of LPOAVGAT/ChR2 cells, in which the photostimulation is below the threshold to produce USVs. Solid line indicates mean of signals and shaded region indicates 95% confident intervals. n = 4 mice, 3 trials per mouse. i–k, To study the extent of USV syllable flexibility during natural social behaviour, we collected USVs from wild-type male mice during different social contexts. Wild-type male mouse USVs were recorded during 2-min interactions with a variety of socially relevant sensory contexts including the presence of an awake female mouse (in the dark or light), female urine, an anaesthetized female mouse, male urine, or an anaesthetized male mouse. The flexibility of social vocalization is underscored by the longer and louder USV bouts triggered by awake behaving female mice compared to the USVs evoked by anaesthetized female mice even though much of the contextual sensory cues are similar. i, Latency to first USV evoked by different sensory contexts. j, Average inter vocalization intervals of USVs evoked by different sensory stimuli. Red bar denotes female context (live female mouse in either the dark with red light or bright light, female urine, anaesthetized female mouse); green bar denotes the male context (male urine or anaesthetized male mouse). Data are mean ± s.e.m. n = 20 mice. k, Raster plot of USVs emitted while interacting with different sensory contexts. Each line is a single wild-type male. Average USV power is calculated by mean decibels in the 40–90-kHz band from raw recording.

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Extended Data Fig. 9 Increasing LPOAESR1 neuron activity generates more USV syllables without altering syllable identity.

a, Two sonograms (12-s long each) analysed for USV production. White bars (top of each 3-s line) indicate the production and duration of USV syllables automatically identified. In the top sonogram, there was little additional acoustic noise so USV syllables are easy to identify; in the bottom sonogram, although there is abundant low-frequency noise (from self-movement or from interactions with another individual during the USV recording), the white bars robustly identify USV syllables. b, To determine whether USV bout length is fixed or variable, we analysed the inter-USV vocalization intervals, and found a natural threshold of 2-s pauses as a basis to define the end of a USV bout23. c, Number of USVs emitted after photostimulation of LPOAESR1/ChR2 cells at 25 Hz or 50 Hz and 1–20-s duration. Data are mean ± s.e.m. n = 16 mice. d, Syllables maintain their identity and structure from 1–50-Hz photostimulation of LPOAESR1/ChR2 neurons. e, Jensen–Shannon divergence score of USVs produced by 1–50-Hz photostimulation of LPOAESR1/ChR2 cells compared to natural USVs during interaction with female urine or live female mice. Box plot denotes the minimum and maximum values and quantile (0.25, 0.75). Grey shading denotes the 95% confident interval of the control data. To compute P values, we randomly choose mice with no stimulation and mice with ChR2 stimulation and computed the Jensen–Shannon divergence score. This step was repeated 1,000 times to build a null distribution. We then computed the probability that each bootstrap exceeded the observed median at each stimulation frequency. We found that a stimulation frequency of only 5 Hz resulted in a significant P value. *P = 0.036, n = 26 mice.

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Extended Data Fig. 10 Circuit and intrinsic properties of PAGVGAT neurons support USV persistence.

a, b, USVs emitted during photostimulation of LPOAVGAT/ChR2 cells with injection of saline as control (grey) or CNO (purple) to inhibit PAGVGAT/hM4Di neurons on alternative days. a, Number of USVs emitted with 1 Hz 5s light stimulation. b, Raster plot of USVs emitted during and after photostimulation (blue bars). Each row is 30 s of a 230-s trial aligned to light stimuli applied every 40 s. Data are mean ± s.e.m. n = 3, 6 trials per mouse per test day. ****P < 0.0001, paired Wilcoxon test, two sided. ns, not significant (P > 0.05). c, Representative image of co-expression of hM4Di-tdTomato with VGAT-ZsGreen cells in the PAG. Scale bar, 200 μm. n = 2 mice, 2 sections per mouse collected. d, Number of USVs emitted during increased frequency photostimulation in LPOA of male mice expressing LPOAVGAT/ChR2 and PAGVGAT/hM4Di and injected with CNO or saline on alternative days. n = 3, 36 trials per condition. Data are mean ± s.e.m. ****P < 0.0001, Wilcoxon test, two sided. e, Experimental design to express control virus (tdTomato) in the PAG of VGAT-Cre mice (Fig. 4e, f). f, Number of USVs emitted by photostimulating LPOAVGAT cells under either CNO or saline conditions in mice expressing FLEX-tdTomato viral control (PAGVGAT/tdTomato) (control). n = 3, 18 trials per condition. Data are mean ± s.e.m. ns, not significant (P > 0.05), Wilcoxon test, two sided. g, Raster plot of USV bouts emitted during either CNO or saline test days under different stimulation frequency. Note that Drosophila courtship songs show similar feature separation across the circuit with songs evoked by pIP10 neurons tightly locked to stimulation (like the PAG USV-gate neurons) compared to calling generated by P1 neurons that persists beyond stimulation (as with the LPOAESR1 neurons)53. This suggests diverse social species use general circuit strategies to maintain persistent auditory responses that outlast the detection of sensory information.

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Reporting Summary

Video 1

Female odour evokes USVs from males. Female odour (female urine) triggers the productions of USVs from a sexually naïve male. Upper: Video of male interacting with odour source. Lower: USV sonogram representing frequency, syllable type, and amplitude of USVs. USVs were transposed to audible range through MATLAB. Video and audio played in 3X speed.

Video 2

Photoactivation of LPOAEsr1 neurons triggers USVs. Sample video and USV sonogram during photostimulation LPOAEsr1 neurons. Same individual was given 5s 15ms light stimulation at 1Hz (No USVs), 5Hz, 10Hz (moderate USVs) and 50Hz (Strong USVs).

Video 3

Chemoinhibition of local PAGvGat neurons decreases the threshold and prolongs USV bouts triggered by LPOAvGat photostimulation. Sonograms of USVs produced by a single individual receiving 5s photo stimulation at 1Hz/25Hz on LPOAvGat neurons and chemoinhibition of PAGvGat neurons with CNO (upper) or control saline (lower).

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Chen, J., Markowitz, J.E., Lilascharoen, V. et al. Flexible scaling and persistence of social vocal communication. Nature 593, 108–113 (2021). https://doi.org/10.1038/s41586-021-03403-8

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