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^16,17,50,51. 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^32,33. 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.