Flexible scaling and persistence of social vocal communication

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 communication^1-3. 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 (LPOA^ESR1 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 LPOA^ESR1 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 LPOA^ESR1 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.

Extended Data Figure 1.. Esr1 expressing subset of LPOA neurons express cFos during odor-evoked USV calling.

a, example of cFos expression in preoptic area following exposure to tonic water as a control odor (no USVs) or with female odor (USVs)^,–. BNST, Bed Nucleus of the Stria Terminalis; LPOA, Lateral Preoptic Area; MPOA, Medial Preoptic Area. Scale bar = 200μm. Quantification of this experiment is showed in panels c-d. b, immunostaining following female odor exposure shows cFos+ in LPOA largely overlaps with Esr1-Zsgreen cells. White squares delineate enlarged single z stack sections shown in main text Figure 1b. Scale bar = 100μm. c-d, expression of cFos and Esr1 in LPOA following awake behavior with control odor (black) or female odor (yellow). c) rostrocaudal distribution of cFos+ cells in LPOA. d) rostrocaudal percentage of cFos+/Esr1+ co-expressing LPOA cells. Mean ± s.e.m. N=4 animals.

Extended Data Figure 2.. LPOA^Esr1 neural activity correlates with USV calling; and chemogenetic inhibition of LPOA^Esr1 neurons reduce USV calling.

LPOA^Esr1+ GCaMP activity during natural social behavior. a, top: experimental design for LPOA^Esr1 fiber photometry recordings. Bottom: sample image showing fiber optic track and viral expression of GCaMP6s. Scale bar = 200μm. N=9 animals, >3 sections/animal collected. In addition to evoking USV calling, the presence of a female also dramatically alters male behavior (arousal, social sniffing, locomotion, sexual mounting) potentially confounding interpretation of the observed neural activity. We observed that following the removal of the female, males often generate intermittent USV calls, perhaps to lure her back, without the behavioral noise of mounting or social sniffing. We leveraged this post-female period to observe increases in LPOA^Esr1/GCaMP6s activity with a rise shortly before the onset of post-female USVs, clearly suggesting that endogenous LPOA^Esr1 neural activity correlates with emission of USVs. b, representative USV production and GCaMP fiber photometry of male LPOA^Esr1 neurons as he behaves alone (pre-female), with a behaving female, and after female 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: dF/F of LPOA^Esr1 GCaMP6s signals was calculated by MATLAB GUI described previously. c, dark green line: mean z-score of GCaMP6 signals before and after initiation of USV with behaving female phase, light green shading indicates 95% confidence interval. Grey shading: 95% confidence interval of the mean of the scrambled data (N=9 animals). d, mean average z-score of GCaMP signals during all USV syllables evoked with a behaving female compared to scrambled datapoints during pre-female behavior. Mean ± s.e.m. N=9 animals, unpaired t-test, two sided,*** p=0.0003. e, dark green line: 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: 95% confidence interval of the mean of the scrambled data. (N=9 animals) f, mean average z-score of GCaMP6s signal of all USV syllables during post-female stage compared to scrambled datapoints during pre-female stages. Mean ± s.e.m. N=9 animals, unpaired t test, two sided, *** p=2.2e⁻⁴. g-i, to determine whether the increased hypothalamic activity is involved in odor-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 behavior^–, and quantified USV production during natural interactions with an awake female. g, non-specific chemogenetics. left: AAV-hM4Di virus injection in LPOA of wildtype mice. Right: experimental assay; following expression of hM4Di virus, males were IP injected with CNO-saline-CNO-saline (every other day for 4 days total) and allowed to interact with a freely moving female to evoke USV calling. h, number of USV syllables emitted following injection with CNO (purple) or saline (black). Mean ± s.e.m., N=10 animals. Paired two-tailed Wilcoxon test, N.S.: p=0.11. i, number of USVs emitted across four sequential test days. Overall, the manipulation did not produce a significant effect on behavior, however half of this group (solid lines, N=5 animals) did show a constant reduction in USVs while the other half (dashed lines, N=5 animals) 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 injected days as showed in Fig 1d. N=6 animals, Wilcoxon test, two sided, * p=0.03. k, sample image of hM4Di expression in Esr1-Zsgreen animals. Scale bar = 500μm. N=6 animals, >3 sections/animal collected. l, quantification of total time performing social behaviors observed by male ^{LPOAEsr1/hM4Di} during a 4 min interaction with live females on CNO and Saline injection days. N=6 animals. Paired t test was performed, two-sided,*** p<0.001, * p=0.02. Note: we observed an unexpected increase in the female’s anti-social defensive behavior (kicking, running away) which reduced his ability to direct sniffing to her anogenital region. This observation is consistent with male USVs serving to enhance female courtship behavior^,.m, experiment design of expressing control AAV-TdTomato virus into LPOA^Esr1 cells. n, number of USVs emitted with behaving female over 5 test days, alternating injections of either CNO or saline. o, average number of USVs between Saline and CNO injected days. N=5 animals. Wilcoxon test, two sided, p>0.05.

Extended Data Figure 3.. Optogenetic stimulation of LPOA^Esr1/ChR2 neurons triggers USV calling in both male and female mice.

a, Left; ChR2 virus injection in LPOA region of Esr1-Cre mice. Right; Sample image of ChR2 and cFos co-expression following photostimulation. Scale bar = 50μm. b, viral expression in LPOA region. Composite overlay of total sections at left; bregma-0.2mm and right: bregma-0.3mm. Color intensity scales with increasing expression. N=12 animals. c, example electrophysiology recording during photostimulation of LPOA^Esr1/ChR2 neurons in ex vivo slice. Blue bar = 10Hz light stimulation. Neural response is time-locked to light pulses. d, number of USVs detected during 10Hz photostimulation of LPOA^Esr1/ChR2 cells from left; males (N=23 animals) and right; females (N=11 animals) Mean ± s.e.m. e-f, average dB across 40-90kHz band evoked by 10Hz photostimulation. Solid line indicates mean of all trials; shaded region indicates 95% confidence interval. e) blue trace: male trials, f) red trace: female trials^,. Blue shading/bar = light stimulation at 5-10 seconds. g, raster plot of complete data set showing USV power evoked by 10Hz 5s 15ms photostimulation (between blue lines) from LPOA^Esr1/ChR2 females (N=11 animals, red) and males (N=23 animals, blue), N = 242 trials, each row is a single trial. Color intensity represents average dB across the ultrasonic band (40-90kHz). Discussion note: the POA has been implicated in a variety of functions including homeostatic control of internal states such as thermoregulation and thirst, sexually dimorphic social behaviors including parenting and mating behavior, as well as motivated behaviors^,–. It is likely that features of these neurons that enable them to generate USVs in the absence of social stimuli in the lab also enable them to participate in other neural computations that are currently unknown.

Extended Data Figure 4.. Activating LPOA^Esr1 neurons elicits a variety of USV syllables similar to natural USVs.

a, Evaluation of USV syllable types emitted by a wild-type male naturally interacting with a behaving female compared to; Orange: WT P7-P8 pup calls (N=18 animals) evoked by individually isolating from home cage; Grey: Wildtype adult male calls (N=20 animals) evoked by interaction with female urine, male urine, anesthetized male, or anesthetized female on successive days; Blue: left) calls from experimental males expressing ChR2 (LPOA^Esr1/ChR2, N=23 animals) evoked by interaction with either female urine or a live female with no ChR2 light stimulation to determine natural USV repertoire; Blue: right) (blue shading ‘light stimulation’) males expressing ChR2 (LPOA^Esr1/ChR2, N=23 animals) stimulated with light (10Hz, 25Hz and 50Hz) in the absence of a female (same males were used in no light and light stimulation experiments). Dot denotes the Pearson correlations for the top 5% of the most frequently used syllables, where the boxplot shows the mean and interquartile range of these correlations, and the plus sign (+) shows the correlation of the top 95% of the most frequency used syllables. One-way ANOVA test, P(F>83.4)= 6.12ê−115. Using MATLAB mulcompare function for group analyze compared to wildtype male USV triggered by live female interaction, ***=p<0.001, *=p<0.05. b, heatmap showing person’s correlation score among all 40 types of syllables detected across each condition compared to wildtype male USVs during interaction with live female. Results are grouped by types of sensory stimulation: female context (red); ChR2 stimulation (blue); male context (green) and pup (orange). Warmer colors in panel b indicates higher similarity, which is quantified in panel a. These data find that the repertoire of USV syllables evoked by photostimulation are rich and varied. When compared to natural USVs, they are quite similar to those produced by WT male mice as they interact with a live female; the natural male produced USVs evoked by male cues and pup USVs are less similar to the photostimulated USVs.

Extended Data Figure 5.. LPOA^Esr1 projections to USV motor center (ambiguous) are sparse and unable to be functionally validated while LPOA^Esr1 projections to PAG produces robust USV production.

a, strategy to test whether LPOA^Esr1 neurons are anatomically or functionally connected to either the PAG or the Amb which are known to evoke USV calling in the mouse^,,,. b-f, retrograde tracing experiment from either PAG or AMB to label LPOA^Esr1 cell projections by injecting a Cre-dependent FLP-expressing pseudotyped equine infectious anemia virus (RG-EIAV-DIO-Flp) in either the PAG or Amb, and a FLP-dependent AAV expressing eGFP in the LPOA of Esr1-Cre mice^,. We confirmed the specificity of viral expression by multiplex fluroscent in situ hybridization (Extended Data Figs. 6a–b). We observed sparse labeling of LPOA cells that directly project to the Amb, and a larger population centered in the LPOA region of cFos/Esr1 overlap that directly project to the region of the PAG USV-gate neurons. b) example image of PAG-projecting (top) or Amb-projecting (bottom) eGFP positive cells in LPOA as described and quantified in c-f. Scale bar = 200 μm. N=5 animals, 5 sections/animal collected. c-d, anatomic tracing from PAG to LPOA resulted in robust labeling. c) experimental design to express DIO-FLP virus in PAG and fDIO-eGFP in LPOA of Esr1-Cre mice. d) restrocaudal distribution of total number of PAG-projecting eGFP positive cells in the LPOA. Mean ± s.e.m. N=5 animals. e-f, anatomic tracing from Amb to LPOA resulted in sparse labeling. e) experimental design to express DIO-FLP virus in Amb and fDIO-eGFP in LPOA of Esr1-Cre mice. f) restrocaudal distribution of total number of Amb-projecting eGFP positive cells in the LPOA. Mean ± s.e.m. N=5 animals. To test if 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 Amb. g, sample image of optical fiber position for stimulation of LPOA^Esr1/ChR2 terminals in PAG show in panel j. h, experimental design for stimulation of LPOA^Esr1/ChR2 terminals in Amb. i, sample image of optical fiber position for terminal stimulation at Amb. Scale bar = 200μm. N=5 animals, 4 sections/animal collected. j, average USV power across 40-90kHz band of recording during PAG terminal stimulation, solid line indicates the mean and shaded region indicate 95% confident interval. (blue shading, N=13 male animals. Red shading, N=4 female animals. 4 trials/animal.) k, average USV power across 40-90kHz band of recording during Amb terminal stimulation, solid line indicates the mean and shaded region indicate 95% confident interval. (Blue shading, N=5 male animals. 4 trials/animal.) l-m, average USV power during single trials of the same male stimulated with 25Hz 5s from either l) LPOA^Esr1/ChR2 cell somas, or m) LPOA^Esr1/ChR2 axon terminals in the PAG.

Extended Data Figure 6.. LPOA-PAG projecting cells are largely inhibitory and stimulation of LPOA^vGat cells elicits USVs.

a-c, RNAScope multiplex in situ hybridization histology of LPOA sections following injection of retro travelling Cre-dependent FLP expressing virus in the PAG and a FLP dependent eGFP (AAV-fDIO-dGFP) in the LPOA of Esr1-Cre mice (see Extended data Figure 4c-d) reveals overlap of a) eGFP (green), b) Esr1 (red), and c) vGat (magenta) probes. Yellow traces are eGFP positive used as ROIs and applied to Esr1 and vGat channels for analysis. Scale bar = 50μm. N=3 animals, >3 sections/animal 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 and e) IPSC and EPSC recording while photostimulating (blue bars) LPOA^Esr1/ChR2 terminals. f) IPSC and EPSC during single light pulse. g) peak conductance (calculated by amplitude/driving force) of EPSC (red) and IPSC (blue) recorded. Mean ± s.e.m. Bottom, percentage of observed cells with monosynaptic IPSC (blue), EPSC (red), or both (grey). N=5 mice, 29 cells. h-i, strategy to test if LPOA excitatory neurons elicit USVs. h) top: experimental design to express ChR2 in the LPOA of vGluT2-Cre mice. Bottom: ChR2% overlap with vGLuT2-Zsgreen marker or cFos staining. I) sample images showing overlap between ChR2 with vGluT2-Zsgreen marker and cFos staining. Scale bar = 50μm. N=4 animals, 7 images per animal used for cell quantification. j, number of USVs emitted during light stimulation of LPOA^vGluT2/ChR2 neurons. N=4, 16 trials per condition. k, sample image indicating fiber position (white square) and ChR2 expression in LPOA of vGat-Zsgreen mice. Scale bar = 200μm. N=9 animals, 4 sections/animal collected. l, experimental design to express ChR2 in LPOA of vGat-Cre mice and number of USV syllables emitted during light stimulation of LPOA^vGst/ChR2 neurons. Mean ± s.e.m. N=9 animals, 91 trials per condition. m, composition of cFos expression (after odor evoked USVs) in PAG of vGat-mice. cFos positive cells (red) are largely vGat negative (consistent with PAG USV gate neurons being excitatory) while vGat neurons are largely clustered in the ventrolateral PAG. N=3 animals, overlay of 100um thick sections roughly at Bregma – 4.4mm.

Extended Data Figure 7.. Local PAG^vGat neurons inhibit PAG USV-gate cells; photostimulation inhibits natural USVs.

The majority of immediate neurons ‘upstream’ of the PAG USV-gate cells, are vGluT2-negative neurons in the PAG, ipsilateral to the PAG^vGluT2 neurons. a, experimental design for rabies viral-tracing from PAG in vGluT2-Cre mice. b, sample PAG image from rabies tracing. Red cells on left are the TVA+G helper virus infected cells that overlap with vGluT2-Zsgreen (starter cells). White box on right side is enlarged and showed in (c). Scale bar = 250μm. c, 76 out of 87 rabies-tdt labeled cells (87%) observed in vlPAG do not overlap with vGluT2-Zsgreen cells, supporting that they are likely expressing vGat. Scale bar = 100μm. N=5 animals, total 32 sections counted. To functionally test if the PAG USV-gate neurons are subjected to local inhibition, we engineered males to express ChR2 in the local PAG inhibitory cells (PAG^vGat/ChR2) and injected retroAAV-eGFP in the Amb to specifically identify PAG USV-gate neurons. d, experimental design for retrograde labeling from Amb to PAG USV-gate neurons and optical manipulation of PAG^vGat/ChR2 cells for behavior and physiology. e, sample image of PAG section showing Amb-projecting cells in lPAG (green) and vGat/ChR2 expressing cells (red) in vlPAG. Scale bar = 100μm. Aq = Aqueduct, fourth ventricle. Ex vivo whole-cell recordings and cell attached recordings revealed all tested PAG^GFP neurons (USV-gating neurons) were inhibited within 5ms after photostimulation of PAG^vGat/ChR2 neurons consistent with monosynaptic inhibitory inputs. N=2 animals, 2 section/animal collected. f-h, ex vivo slice electrophysiology recordings of PAG USV-gate cells while photostimulating of PAG^vGat/ChR2 neurons. f) IPSC and EPSC during single light pulse. g) photostimulation of PAG^vGat/ChR2 neurons generate monosynaptic iPSCs in all cells investigated. N=2 animals, 14 cells total recorded. h) cell attached physiology of USV-gate neurons (PAG^GFP). Blue shading indicates photstimulation period. Each line showed as individual cell recorded. I-j, stimulating local PAG^vGat neurons inhibit socially evoked USVs. i) increasing frequency or duration of photostimulation (5s of 1Hz, 5Hz, 10Hz ,50Hz and 10s of 25Hz) of PAG^vGat/ChR2 males during interaction with awake behaving females to evoke natural USVs. Blue bar/shading = light stimulation. Number of USVs are calculated in 10s time bins. Mean ± s.e.m. N=3 animals, 5-6 trials/animal/condition. j) raster plot of USVs emitted before, during and after photostimulation of 25Hz 5s stimulation as showed in main text Fig 2e. Blue light indicates light stimulation period. Each row is a single trial. Mean ± s.e.m. N=3 animals, 19 trials.

Extended Data Figure 8.. LPOA^vGat cell population connect to PAG^cGat 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 = 100μm. Aq = Aqueduct. b, sample image of LPOA section for experiment described in main Figure 2f–g showing overlap of Rabies-tdt positive cells (red) with vGat-Zsgreen. (272/320 cells counted, N=3 mice, total of 17 sections. Scale bar = 200μm. c, example image of RNAScope multiplex in situ hybridization in LPOA to complement main Figure 2c. Sections are stained with eGFP (green), vGat (blue) and Esr1(red) probes. Scale bar = 25μm. The majority of LPOA neurons that projected to PAG^vGat co-express Esr1. N=2 animals, total of 16 sections (20um thick) collected. Top right panel: quantification of rabies positive cells overlapping with vGat+ or Esr1+ using RNAScope multiplex in situ hybridization. N=2 animals, total 24 rabies positive cells quantified. d, PAG^vGat cells receive monosynaptic IPSC from LPOA^vGat/ChR2 neurons. IPSC and EPSC evoked by single light pulse, N=5 animals, 16 cells recorded. e-h,To study the in vivo effects of LPOA^Esr1 activity we expressed both ChR2 in the LPOA and GCaMP6s in the PAG of vGat-Cre mice (LPOA^vGat/ChR2;PAG^vGat/GCaMP6s). Photostimulation in the LPOA of awake behaving mice resulted in a decrease in fiber photometry measured GCaMP6s fluorescence in the local PAG inhibitory neurons and an accompanying initiation of USV production. E) representative image of fiber track and viral expression of GCaMP6s in PAG. Scale bar = 200μm. N=4 animals, 3 sections/animals collected. F) number of USV syllables emitted following light stimulation of LPOA^vGat/ChR2 neurons while recording of PAG^vGat/GCaMP6s signals. Mean ± s.e.m. N=4, 16 trials per condition. g) mean z-score of fiber photometry signal from PAG^vGat/GCaMP with 25Hz 10s photostimulation of LPOA^vGat/ChR2 cells. h) mean z-score of fiber photometry signal from PAG^vGat/GCaMP with 1Hz 5s photostimulation of LPOA^vGat/ChR2 cells, where the photostimulation is below the threshold to produce USVs. Solid line indicates mean of signals and shaded region indicates 95% confident intervals. N=4 animals, 3 trials/animal. i-k, to study the extend of USV syllables flexibility during natural social behavior, we collected wild-type male mice USV during difference social context. Wild-type male USVs were recorded during 2 minute interactions with a variety socially relevant sensory contexts including awake female (in dark or light), female urine, anesthetized female, male urine, or an anesthetized male. Flexibility of social vocalization is underscored by the longer and louder USV-bouts triggered by awake behaving females compared to the USVs evoked by anesthetized females 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 (IVIs) of USVs evoked by different sensory stimuli. Red bar = female context (live female in either the dark with red light or bright light, female urine, anesthetized female); green bar, male context (male urine, anesthetized male). Mean ± s.e.m. N=20 animals. k) raster plot of USVs emitted while interacting with different sensory contexts. Each line is a single wildtype male. Average USV power is calculated by mean dB in 40-90kHz band from raw recording.

Extended Data Figure 9.. Increasing LPOA^Esr1 activity generates more USV syllables without altering syllable identity.

a, two sonograms (12 seconds long each) analyzed for USV production. White bars (top of each 3 second line) indicate production and duration of USV syllables automatically identified. In upper sonogram there was little additional acoustic noise so USV syllables are easy to identify; in lower sonogram there is abundant low frequency noise (from self-movement or from interactions with another individual during USV recording) however, white bars robustly identify USV syllables. b.to determine if USV bout length is fixed or variable, we analyzed the inter-USV vocalization intervals (IVI) and found a natural threshold of 2 second pauses as a basis to define the end of a USV-bout as determined by the distribution plot of inter-vocalization-intervals reveals a peak of duration. c, number of USVs emitted at 25 or 50Hz of photostimulation on LPOA^Esr1/ChR2 cells from 1-20 seconds stimulation duration. Mean ± s.e.m. N=16 animals. d, syllables maintain their identity and structure from 1-50 Hz photostimulation of LPOA^Esr1/ChR2 neurons. e, Jensen-Shannon divergence score of USVs produced by 1-50 Hz photostimulation of LPOA^Esr1/ChR2 cells compared to natural USVs during interaction with female urine or live female. Box plot: min-max, 5%-95% percentile, grey shading, control data’s 95% confident interval. For computing p-values, we random choose mice with no stim and mice with ChR2 stimulation and computer the JSD, this step was repeated 1000 times to build a null distribution. Then we computer the probability that each bootstrap exceeds the observed median at each stim frequency. We found only 5Hz stimulation frequency results in a significant p value: * p=0.036, N=26 animals.

Extended Data Figure 10.. Circuit and intrinsic properties of PAG^vGat neurons support USV persistence.

a-b, USVs emitted during photostimulation of LPOA^vGAT/ChR2/ PAG^vGat/hM4Di following injection of saline (grey) or CNO (purple) on alternate days. USV number during a) 1Hz stimulation. b) raster plot of USVs emitted during and following photostimulation (blue bars). Each row is 30” of a 230” trial aligned to light stimuli applied every 40”. Mean ± s.e.m. N=3, 6 trials/animal per test day. Paired Wilcoxon test, two sided, ****p<0.0001. ns: p>0.05. c, representative image of co-expression of hM4Di-tdt with vGat-Zsgreen cells in PAG. Scale bar = 200μm. N=2 animals, 2 sections/animal collected. d, number of USVs emitted during increasing frequency of photostimulation of LPOA in LPOA^vGAT/ChR2/ PAG^vGat/hM4Di males with CNO or saline. N=3, 36 trials per condition. Mean ± s.e.m. Wilcoxon test,two sided. **** = p<0.0001. e, experimental design to express control virus (tdt) in PAG of vGat-Cre mice (to control Main text Fig. 4e–f). f, number of USVs emitted by photostimulating LPOA^vGat cells either under CNO or saline conditions in animals expressing FLEX-tdt (control). N=3, 18 trials/condition. Mean ± s.e.m. Wilcoxon test, two sided. ns= p>0.05. g, raster plot of USV bouts emitted during either CNO or saline test days under different stimulation frequency. Note: drosophila courtship songs similarly show 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 which persists beyond stimulation (as with the LPOA^Esr1 neurons). This suggests diverse social species utilize general circuit strategies to maintain persistent auditory responses outlasting the detection of sensory information.

Figure 1.. Hypothalamic LPOA^Esr1 neurons underly USV social calling.

a, top; raster plot and bottom; mean number of USVs emitted by naive males during interaction with tonic water control odor (grey) or with female urine odor (yellow). Mean ± s.e.m. N=12 animals. b, expression of cFos and Esr1 in LPOA following awake behavior with control odor (black) or female odor (yellow). Scale bar = 50μm. c, experimental design for LPOA^Esr1 chemoinhibition experiment. d, number of USVs in presence of behaving female after male received either CNO or saline on alternate days over 5 test days. N=6 animals. e-h, LPOA^Esr1/ChR2 neurons evoke USVs. e) ChR2 virus injection in LPOA region of Esr1-Cre mice. f) average USV power evoked by 5s, 15ms, 10Hz photostimulation of LPOA^Esr1/ChR2 animals. Solid line indicates mean of all trials; shaded region indicates 95% confidence interval. g) number of USVs detected during photostimulation of LPOA^Esr1+ cells. Mean ± s.e.m. (both sex, N=34 animals). h) sample USV sonograms evoked during photostimulation of male or female mice.

Figure 2.. LPOA^vGat -> PAG^vGat -> PAG^vGluT2 di-synaptic disinhibition circuit triggers USVs.

a-b, LPOA^Esr1/ChR2 axon terminal photostimulation in the PAG evokes USV calls. a) experimental design, b) number of USVs detected in the Amb (Males, N=5 animals) and the PAG (Males N=13 animals, Females N=4 animals) Mean ± s.e.m. c, sample images of LPOA^Esr1+ co-expression with vGat-Zsgreen (upper, N=4 animals, 3 sections/animal counted) and vGluT2-Zsgreen (lower, N=3 animals, 3 sections/animal counted). Scale bar = 50μm. d-e, photostimulation (blue bar) of male inhibitory PAG^vGat/ChR2 neurons during social interaction with an awake female blocks natural USVs. d) experimental design to express ChR2 in local inhibitory PAG neurons and e) quantification of evoked USV syllables. Mean ± s.e.m. N=3 animals, 19 trials. f, experimental design to assess direct anatomy to LPOA by retrograde tracing from PAG^vGat cells. g, percentage of tdt expression (rabies) in LPOA vGat+ cells. N=3 animals, 320 cells counted. h, experimental design to express ChR2 in LPOA^vGat and GCaMP6s or tdt or hM3Dq in PAG^vGat. i, mean z-score of fiber photometry signal from PAG^vGat/GCaMP with 25Hz photostimulation (blue bar) on LPOA^vGat/ChR2 cells. Solid line indicates mean of all trials; shaded region indicates 95% confidence interval. N=4 animals, 4 trials/animal. j, USVs are silenced during LPOA^vGat/ChR2 photostimulation and PAG^vGat/hM3Dq CNO. Mean ± s.e.m. N=3 animals, 36 trials (hM3Dq), 18 trials (tdt control). Paired Wilcoxon test, two sided, ***=0.005.

Figure 3.. LPOA^Esr1 activity scales USV amplitude and bout-duration.

a-c, wildtype males flexibly adjust USV syllable number, amplitude, and bout-length within and across sensory/social contexts. a) Number of USVs produced. b) raster plot of USV bout-length (strings of syllables separated by >2” pauses) emitted by wild-type males in different sensory/social contexts sorted by bout-length. c) high resolution of single USV bouts (* and orange line in panel b showing variable bout-length and amplitude (heat map in b) emitted by wildtype males during sensory/social interaction. Red: female sensation, green: male sensation. Mean ± s.e.m. N=20 mice. d, distribution of mean USV power produced by males exposed to either male or female urine, and by pups isolated from the nest. e, increasing frequency of photostimulation of LPOA^Esr1+/ChR2 neurons scales number of USV syllables. Mean ± s.e.m. N=5 for control, N=23 for ChR2. f, upper; experimental design to express ChR2 in PAG of vGluT2-cre mice. Lower; average USV power during photostimulation of PAG^vGluT2/ChR2 neurons. g, number of USV syllables emitted during photostimulation of PAG^vGluT2/ChR2 neurons. Mean ± s.e.m. N=3 animals, 32 trials. h, representative sonograms (upper) and raster plot of USV amplitude and bout-lengths (lower) emitted by LPOA^Esr1/ChR2 males interacting with left, female odor or live female to evoke natural USVs; and right, in absence of female with increasing frequency of 5 second light stimulation. N=26 animals, sorted by bout-length. Sonograms occur in the range of 40-90kHz. i, representative sonograms (upper) and raster plot of USV amplitude and bout-lengths (lower) emitted during 5 or 10Hz, 5 second photostimulation of PAG^vGluT2/ChR2 neurons. N=3 animals. j, average USV bout length and k, average amplitude of USV during photostimulation of either LPOA^Esr1/ChR2 (N=26 animals) or PAG^vGluT2/ChR2 neurons (N=3 animals).Box plot: min-max, 5%-95% percentile. l, distribution of mean USV power during 10Hz photostimulation in either PAG^vGluT2/ChR2 or LPOA^Esr1/ChR2.

Figure 4.. Vocal persistence is generated through activity of local PAG^vGat neurons.

a, representative USVs emitted during scaling (ascending/descending frequency) of photostimulation in LPOA^Esr1/ChR2 male demonstrate USVs persist after photostimulation ceases (blue bar). b, raster plot of USV syllables emitted during 10Hz photostimulation of PAG^vGluT2/ChR2 neurons. USVs are locked to light stimulus (blue bars). Each row is a single trial. N=3 animals. 32 trials total. c, Number of syllables from photostimulation of either LPOA^Esr1/ChR2 (blue, N=26 animals, 102 trials) or PAG^vGluT2/ChR2 neurons (orange, N=3 animals, 32 trials) during light on (top) or light off (bottom). ND = not done. d, experimental design to simultaneously express ChR2 in LPOA^vGat and hM4Di in PAG^vGat cells. e-f, USVs emitted during optostimulation of LPOA^vGAT/ChR2/PAG^vGat/hM4Di following injection of saline (grey) or CNO (purple) on alternate days. USV number during e) 25Hz stimulation. f) raster plot of USVs emitted during and following photostimulation (blue bars). Each row is 30” of a 230” trial aligned to light stimuli applied every 40”. Mean ± s.e.m. N=3, 6 trials/animal per test day. Paired Wilcoxon test, two sided, ****p<0.0001. ns: p>0.05. g, Ex vivo cell-attached recording of PAG^vGat neurons during photostimulating (blue shading) of LPOA^vGat/ChR2 axon terminals. N=4 animals, total 23 cells. Left bars group the same cell during multiple trials. h, model of disinhibition circuitry for USV calling. LPOA disinhibition to scale USV power and bout duration enables flexible response to social/sensory context.