GoKawiilMouse husbandry
All experiments were approved and conducted in accordance with the Cold Spring Harbor Laboratory Institutional Animal Care and Use Committee. All animals used were adult (>3 months) male and female mice. Laboratory mice were acquired from The Jackson Laboratory (C57BL/6J). Colonies of laboratory and singing mice were maintained at 20–22 °C, 30–70% humidity and a 12–12 h light–dark cycle.
Behavioural recordings and analysis
A female of each species was muted by synaptic silencing of the caudolateral PAG using tetanus-toxin light chain7,50 and allowed 2 weeks for protein expression. For five sessions per species, the mute female was placed with a conspecific male in a clean cage for interaction (Thoren Systems 8; 30.8 × 40.6 × 22.2 cm) lined with Alpha-pad cotton paper (Shepherd Specialty Papers). Audio of each dyad was recorded for 1–2 h using two Avisoft CM16/CMPA microphones positioned above the cage with high and low gain settings and sampling at 250 kHz (digitized with Avisoft UltraSoundGate 116H). Vocalizations were segmented from the audio using USVSEG69 software for MATLAB (v.09r2), and parameters were optimized to the sounds emitted by each species. For singing mice, songs and their individual notes were also detected using custom code in Python. Segmented sounds were manually curated in a customized spectrogram browser derived from the open-source MATLAB graphical user interface DeepSqueak70. Curation consisted of correcting any vocalization boundary errors and removal of abiotic false positives. Spectrogram inspection confirmed that stimulus females were mute, with no overlapping vocalizations observed; female singing mice also ceased singing during continuous post-surgery monitoring. Songs were operationally defined as sequences of loud notes with a minimum duration of 1.5 s and gaps of no greater than 0.5 s. Song notes were defined as part of the songs, whereas the remaining quiet notes were classified as USVs50. For each curated note, we calculated the maximum amplitude in a fixed frequency range (10–100 kHz). We calculated the instantaneous note rate as the inverse of the interval between two consecutive note start times. For note pitch, the minimum of the fundamental frequency of each note was calculated in Python, using USVSEG-based preprocessing to enhance vocal signals and to suppress background noise69. Kernel densities for note acoustic parameters were calculated using the kdeplot function in the Python package seaborn71, specifying either two or five levels.
Stereotaxic viral injections
Before surgery, subcutaneous meloxicam was delivered at a dose of 2 mg kg–1. Surgeries were performed on a stereotaxic apparatus under 1–2% isoflurane in oxygen. The OMC was localized on the basis of published data (laboratory mice, refs. 35,36; singing mice, ref. 32), which identifies the OMC as the area in the motor cortex that, when stimulated, results in orofacial muscle contraction (coordinates relative to bregma: anterior–posterior (AP), 2.25 mm; medial–lateral (ML), 2.25 mm). These coordinates were used for all OMC injections unless otherwise noted. To mute female stimulus mice for behavioural recordings, we bilaterally expressed tetanus-toxin light chain7,50 in the caudolateral PAG using AAVs. A female singing mouse and a laboratory mouse were bilaterally injected in the caudolateral PAG with 80 nl of a 1:1 mixture of AAV2/DJ-hSyn-flex-TeLC-eYFP (custom packaged by WZ Biosciences) and CamKII-Cre. The following coordinates were used: singing mouse, AP, –4.2 mm; ML: ±0.6 mm; dorsal–ventral (DV), –2.3 mm (ref. 50); laboratory mouse, AP, −4.7 mm; ML, ±0.7 mm; DV, −1.75 (ref. 7).
Histology of these mice were also used in anterograde trans-synaptic tracing of the OMC to the PAG.
For mapping of bulk neuronal projections, neurons were targeted to express tdTomato using a 1:1 mixture of CaMKII-Cre and FLEX-tdTomato viruses (Supplementary Table 1). Next, 30 nl of this viral mixture was injected into the OMC at two depths: 500 and 750 µm ventral to the brain surface.
For MAPseq viral injections, Sindbis virus carrying the barcode library72 was diluted 1:3 in sterile saline. The OMC was injected with 50 nl of diluted Sindbis virus at 300, 600 and 900 µm ventral to the brain surface at two locations: AP, 2.0 and 2.5 mm; and ML, 2.25 mm.
For visualizing synaptic boutons, a 1:1 mixture of CaMKII-Cre and Flex-mGFP-2A-Synaptophysin-mRuby (Supplementary Table 1) was injected into the OMC using the coordinates listed above. Specifically, 60 nl of this viral mixture was injected at two depths: 500 and 750 µm ventral to the brain surface.
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