DOI: 10.5061/dryad.ht76hdrw5
title: Data from: Vocal repertoire expansion in singing mice by co-opting a conserved midbrain circuit node

description: How neural circuits generate diverse behaviors is a fundamental question
 in neuroscience. Distinct behavioral outputs may arise from either
 dedicated motor circuits or shared circuits operating in different
 functional states. While multifunctional circuits offer an efficient
 solution for behavioral flexibility and may drive rapid evolutionary
 adaptations, their neural mechanisms remain poorly understood, especially
 in mammals. Here, we leverage the rich vocal repertoire of the singing
 mouse (Scotinomys teguina) to investigate the organizational logic of
 multifunctional motor circuits. We developed a behavioral assay (PAIRId)
 that enables precise attribution of vocalizations during social
 interactions. This paradigm revealed two distinct vocal modes: soft,
 variable, ultrasonic vocalizations (USVs) ancestral to rodents used for
 short-range communication, and loud, rhythmic, human-audible songs unique
 to the singing mouse lineage used for long-range communication. Despite
 their substantial acoustic and contextual differences, we found that USVs
 and songs do not arise from parallel pathways. Instead, they share the
 same sound production mechanism, phonatory-respiratory coupling, and vocal
 gating from the midbrain caudolateral periaqueductal gray (clPAG). To
 understand the mechanism governing song production, we combined
 mathematical modeling of song rhythm with synaptic silencing of clPAG,
 which progressively reduced song amplitude and duration. We demonstrate
 that song duration decreases via a single parameter controlling its
 termination. Notably, this mechanism also accounts for sexual dimorphism
 in songs, identifying clPAG as a key locus for driving natural behavioral
 variability. Our findings reveal how parametric tuning of a central
 circuit node produces distinct vocal modes, providing a mechanistic basis
 for rapid behavioral evolution in mammals.
description: # Data from: Vocal repertoire expansion in singing mice by co-opting a
 conserved midbrain circuit node Dataset DOI:
 [10.5061/dryad.ht76hdrw5](https://doi.org/10.5061/dryad.ht76hdrw5) ##
 Description of data and file structure This dataset comprises all data
 required to reproduce the quantitative analyses and figures presented in
 the associated manuscript when processed using the accompanying analysis
 code. Data are organized into `.zip` archives corresponding to individual
 experiments. Each archive contains a metadata `.csv` file and one or more
 `.csv` files generated from audio analyses, which include note onset and
 offset times as well as computed acoustic features. Experiments that
 incorporate modalities beyond audio additionally include the corresponding
 processed data files. ### Dataset contents #### **solo_social.zip** Data
 from intact adult singing mice male–female close-range social interactions
 within the PAIRId paradigm. Two cohorts (each containing three males and
 two females) were recorded across ten sessions, each lasting five hours,
 encompassing all opposite-sex dyads (60 hours total). Included files
 comprise processed audio vocalization timestamps from the PAIRId
 assignment algorithm and manual curation, as well as tracked positions of
 both mice throughout each session in common world coordinates. The `.zip`
 contains two metadata files (`solosocial_sessions.csv`,
 `social_sessions_samples_info.csv`) and three main data directories: *
 `08_solosocial/`, `09_solosocial_02/` — *Scotinomys teguina* recordings
 from solo and social contexts. * `mmus/` — *Mus musculus* (laboratory
 mouse) dyad recordings * `cam_params/` — YAML files containing camera
 calibration and rig configuration parameters for video alignment and world
 coordinate reconstruction. Each *Scotinomys* session folder is named
 according to the format\ `sessionNumber_date_maleID_femaleID_condition`
 (e.g., `03_20230921_um006_uf004_mf`) and includes two subcontexts: *
 `01_solo/` — recordings from individual animals in acoustic isolated
 boxes, labelled `top` and `bottom`. * `02_social/` — recordings from when
 the mice were allowed to interact within PAIRId, with synchronized video
 tracking (`process_video)` and multi-microphone audio detections
 (`process_audio`) ##### File descriptions * `solosocial_sessions.csv` —
 Metadata linking each recording session to the corresponding subjects. |
 Column | Description | | :------------------- |
 :---------------------------------------------------------------------- |
 | `session` | Unique session identifier (date + subject IDs) | |
 `basepath` | Filepath | | `mouse_1`, `mouse_2` | Identifiers of the
 recorded individuals (male and female, respectively) | *
 `social_sessions_samples_info.csv` — Summary of total recording frame
 counts per session, used to relate acoustic and video sample indices |
 Column | Description | | :------------------- |
 :--------------------------------------------------------------- | | 
 
 | Sequential index | | `session_id` | Session identifier matching entries
 in `solosocial_sessions.csv` | | `audio_total_frames` | Total number of
 frames in the processed audio data | | `video_total_frames` | Total number
 of frames in the synchronized video data | *
 `all_notes_corrected_features.csv` — Note-level acoustic feature tables
 located within `./process_audio/all_notes` then the audio file number
 folder (`0000###/`). | Column | Description | |
 :----------------------------- |
 :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | # | Sequential note index | | `start`, `end` | Start and end time of the note within the session (seconds) | | `duration` | Duration of the note (seconds) | | `maxfreq`, `min` | Placeholder numeric values displayed in the detection GUI; **these do not represent measured frequencies and are not biologically meaningful** | | `type` | Automatically assigned call type label (not curated) | | `code` | For social data recorded in PAIRId, indication of assigned source (`0` = left, `1` = right, `6` = unknown). This column missing and not applicable for solo data. | | `max_amp_high`, `max_amp_song` | Peak amplitude of the note within the 10–120 kHz frequency range as calculated on high-gain audio (`high`) or low-gain audio (`song`) | | `note_rate` | Instantaneous note rate, calculated as the reciprocal of the interval between the start times of two consecutive notes (1 / Δstart). Reflects the local tempo of vocal production. | | `isi` | Intervening silence interval, defined as the time difference between one note’s end and the next note’s start. Represents the silent gap between consecutive notes. | * `common_coords_00###.npz` — mouse positions estimated using a SLEAP model (stored within `./process_video/common_coords` . The final 5 digit number of each filename indicates the video index. | Variable | Description | | :------------------------------------------- | :---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | `l_coordinates_world`, `r_coordinates_world` | For every video frame, world-coordinate (x, y) positions for the six SLEAP pose-estimation nodes for each mouse (`l_` = left mouse, `r_` = right mouse). Nodes include, in order: right ear, left ear, nose, mid-head, mid-back, and tail base. | #### **heliox.zip** Data from four unique singing mice male–female dyads recorded while vocalizing first in normal atmospheric conditions and subsequently with heliox gas introduced into the enclosure. Included files contain the fundamental frequency measurements of selected note classes for each pair, encompassing ultrasonic vocalizations (USVs), song notes, and squeaks ##### File descriptions * `heliox_sessions.csv` — metadata table summarizing each recording session. | Column | Description | | :------------------------------ | :---------------------------------------------------- | | `session` | Unique session identifier | | `date` | Recording date | | `male_id, female_id` | Subject identifiers for each dyad | | `atm_num_song_notes_quantified` | Number of song notes analyzed in air | | `atm_num_usvs_quantified` | Number of ultrasonic vocalizations analyzed in air | | `atm_num_squeaks_quantified` | Number of squeaks analyzed in air | | `hel_num_song_notes_quantified` | Number of song notes analyzed in heliox | | `hel_num_usvs_quantified` | Number of ultrasonic vocalizations analyzed in heliox | | `hel_num_squeaks_quantified` | Number of squeaks analyzed in heliox | * `helium_manual_calls.csv`, `helium_manual_songs.csv`, `helium_manual_squaks.csv` — Quantified acoustic features for vocalization modes. | Column | Description | | :---------------------- | :----------------------------------------------------------------------------------------- | | `trial` | Unique identifier for the recording trial | | `path` | File path | | `file_call` | Name of source audio file containing the call | | `file_det` | Name of source detection file containing the call | | `air` | Recording atmosphere (`atm` = air, `hel` = heliox) | | `class` | Vocalization class (`downsweep` or `trill` = USV subtype, `song` = song note, or `squeak`) | | `callStart`, `callStop` | Onset and offset time of the call within the file (seconds) | | `f0_hi`, `f0_lo` | Highest and lowest measured portion of the fundamental frequency (Hz) | #### **thermistor.zip** Data from a male singing mouse implanted with an intranasal thermistor used as a proxy for respiration. Included files contain detected vocalization timestamps recorded when the focal male was either alone or paired with a mute female, along with the corresponding analog thermistor signals acquired using an Intan recording controller. The `.zip` contains a metadata file (`thermistor_sessions.csv`) and three session folders named according to the format\ `YYMMDD_mouseID_sessionNumber`. Each session folder includes two components: * `process_audio/` — processed acoustic detections and note-level acoustic features. * `process_intan/` — synchronized thermistor voltage traces recorded via an Intan acquisition system (`.mat` format). ##### File descriptions * `thermistor_sessions.csv` — Metadata linking each session to the recording base path and subject ID. | Column | Description | | :-------------- | :---------------------------------------------------------------------------------------------- | | `session` | Unique session identifier (date + ID code) | | `basepath` | Relative file path for the session folder | | `mouse_id` | Identifier of the implanted subject | | `therm_clipped` | Logical indicator for whether thermistor signal contained clipping artifacts (`TRUE` / `FALSE`) | * `all_notes_corrected_features.csv` — Per-note acoustic feature table generated from processed audio. | Column | Description | | :-------------- | :--------------------------------------------------------------------------------------------------------------------------------------------- | | `#` | Sequential note index | | `start`, `end` | Start and end time of the note with the session (seconds) | | `duration` | Duration of note (seconds) | | `maxfeq`, `min` | Placeholder numeric values displayed in the detection GUI; **these do not represent measured frequencies and are not biologically meaningful** | | `type` | Automatically assigned call type label (not curated) | | `max_amp_high` | Peak amplitude of the note within the 10–120 kHz frequency range | * `therm_*_*.mat` -- Each `.mat` file in `process_intan/` contains synchronized analog recordings acquired using an Intan system. Files are named according to the session date and start time (e.g., `therm_231017_152230.mat`) and include four variables: | Variable | Description | | :-------------- | :---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | `cam_trigs_exp` | Digital trigger channel indicating camera frame times used as synchronization events. | | `ch1_audio_exp` | Audio channel 1 waveform recorded from one microphone input | | `ch2_audio_exp` | Continuous analog voltage trace from the intranasal thermistor sensor, used as a proxy for respiratory airflow. This is the primary signal of interest for respiration–vocal coupling analyses. | | `d2_exp` | Placeholder variable representing an unused digital input channel in this dataset (values are all 0). | 
 
 #### **pag_opto.zip** Data from four male singing mice subjected to
 unilateral optogenetic stimulation of the caudolateral periaqueductal gray
 (clPAG) using excitatory opsins. Included files contain detected
 vocalization timestamps along with corresponding timestamps for
 optogenetic stimuli of one, two, or four seconds in duration.\ The `.zip`
 contains a metadata file (`pag_opto_usv_sessions.csv`) and multiple
 session folders named according to the format\
 `YYMMDD_mouseID_sessionNumber`. Each session folder includes two
 components: * `process_audio/` — processed acoustic detections and
 note-level acoustic features. * `process_intan/` — synchronized
 optogenetic stimulation timestamps (`.csv` format). ##### File
 descriptions * `pag_opto_usv_sessions.csv` — Metadata summarizing all
 optogenetic recording sessions. | Column | Description | |
 :---------------- |
 :----------------------------------------------------------------------- |
 | `session` | Unique session identifier (date + ID code) | | `Animal` |
 Subject identifier | | `Duration` | Duration of optogenetic stimuli used
 in the session (1, 2, or 4 seconds) | | `h_audio_file_sr` | audio file
 sampling rate | | `h_audio_real_sr` | estimated audio sampling rate for
 synchronization | * `h_T*_corrected.csv` — Per-note acoustic feature table
 generated from processed audio. | Column | Description | |
 :---------------------------- |
 :--------------------------------------------------------------------------------------------------------------------------------------------- | | `#` | Sequential note index | | `start`, `end` | Start and end time of the note within the session (seconds) | | `duration` | Duration of the note (seconds) | | `maxfreq`, `min` | Placeholder numeric values displayed in the detection GUI; **these do not represent measured frequencies and are not biologically meaningful** | | `type` | Automatically assigned call type label (not curated) | | `max_amp_low`, `max_amp_high` | Peak amplitude of the note within the 10–120 kHz frequency range as calculated on low-gain audio (`low`) or high-gain audio (`high`) | * `opto_stims.csv` — Optogenetic stimulation timestamps. | Column | Description | | :-------------------------- | :---------------------------------------------------- | | `stim_starts`, `stim_stops` | Start and end times of optogenetic stimulus (seconds) | #### **pag_telc.zip** Data from five male singing mice subjected to bilateral synaptic silencing of neurons in the caudolateral periaqueductal gray (clPAG) via expression of tetanus toxin light chain (TeLC). Included files comprise processed audio vocalization timestamps generated by the PAIRId assignment algorithm and manual curation from recordings collected the day before surgery and during the five to six days following surgery, as the mice produced progressively deteriorating vocalizations before becoming mute. The `.zip` contains metadata files (`pag_telc_sessions.csv`, `pag_telc_curation.csv`) and multiple session folders named according to the format\ `YYMMDD_mouseID_sessionNumber`. Each session folder includes one or more subdirectories containing the processed acoustic data: * `process_audio/` or `original_audio/` — folders containing extracted note-level acoustic features in `all_notes_corrected_features.csv` format, organized by audio file number (`0000###`). ##### File descriptions * `pag_telc_sessions.csv` — Metadata linking each session to the recording path, experimental condition, and post-surgery time point. | Column | Description | | :------------------- | :------------------------------------------------------------------ | | `session` | Unique session identifier (date + subject IDs) | | `basepath` | Filepath | | `mouse_1`, `mouse_2` | Identifiers of the recorded individuals | | `condition` | Experimental condition (`pre` = pre-surgery, `post` = post-surgery) | | `processed` | date on which the data were processed (`YYMMDD`) | | `anchor_file_idx` | the initial audio file index | | `num_post_days` | number of days recorded | | `num_vocal_days` | Number of days in which vocalizations were detected | * `pag_telc_curation.csv` — Manual curation log corresponding to PAIRId output and post-processing review. | Condition | Description | | :------------------- | :----------------------------------------------------------------------------------------------------------------------------------------------------- | | `exptitle` | Experiment title | | `session` | Session identifier | | `basepath` | Relative file path for the session folder | | `mouse_1`, `mouse_2` | Identifiers of the recorded individuals | | `condition` | Experimental condition (`pre` = pre-surgery, `post` = post-surgery) | | `idx` | sequential position of curated file | | `hr` | audio file number corresponding to curated file | | `lights_turn_on` | Logical indicator for whether this was a file where the light turned on for the mice (`1`=TRUE / `0` = FALSE) | | `truepos_usvs` | Count of manually verified ultrasonic vocalizations (true positives); provided as partial internal annotation notes rather than a comprehensive count. | * `all_notes_corrected_features.csv` — Per-note acoustic feature table generated from processed and manually curated recordings. | Column | Description | | :----------------------------- | :--------------------------------------------------------------------------------------------------------------------------------------------- | | 
 
 | Sequential note index | | `start`, `end` | Start and end time of the
 note within the session (seconds) | | `duration` | Duration of the note
 (seconds) | | `maxfreq`, `min` | Placeholder numeric values displayed in
 the detection GUI; **these do not represent measured frequencies and are
 not biologically meaningful** | | `type` | Automatically assigned call
 type label (not curated) | | `code` | Indication of assigned source (`0` =
 left, `1` = right, `6` = unknown) | | `max_amp_high`, `max_amp_song` |
 Peak amplitude of the note within the 10–120 kHz frequency range as
 calculated on high-gain audio (`high`) or low-gain audio (`song`) | ##
 Code/software The code necessary to reproduce the results in the
 associated publication are available on GitHub.
 [https://github.com/singingmicelab/zheng-harpole-2025-vocalmodes-pag](https://github.com/singingmicelab/zheng-harpole-2025-vocalmodes-pag)