Fig 2: Activation of Esr1 +AmgCC/M neurons suppresses vocalizations.(A) In situ hybridization data for Esr1 mRNA in wild-type male mouse. (Allen Institute for Brain Science. Allen Mouse Brain Atlas, available from: http://mouse.brain-map.org/experiment/show/79591677). (B) (Left) Quantification showing overlap of GFP-labeled AmgC/M-PAG neurons with Esr1 (N=913 cells from 7 hemispheres from 7 mice, on average seven 80 µm coronal sections or 130 cells per animal; 3 males and 4 females). Pie chart shows 450/913 GFP-labeled cells (49%) are Esr1+. Bar graph shows distribution of %Esr1+/GFP+ overlap for the seven mice examined (mean = 47.73%, with S.E. of 4.08%). (Right) Representative confocal image of immunofluorescent Esr1 protein antibody staining performed on AmgC/M-PAG neurons (labeled with GFP, shown in green), showing overlap with expression of Esr1 (magenta). Blue is NeuroTrace. Gray arrow points to an example Esr1- AmgC/M-PAG neuron; yellow arrow points to an example Esr1+ AmgC/M-PAG neuron. (C) Axonal projections of Esr1+ amygdala cells (labeled by tdTomato) in caudolateral PAG, the region that contains PAG-USV neurons. (D) Viral strategy used to express ChR2 in Esr1+ AmgC/M neurons. (E) Example spectrogram showing a representative set of trials in which activation of AmgC/M-PAG neurons suppresses ongoing USV production. (F) Example traces of the USV rate during optogenetic stimulation vs a light-control condition for all trials (N=174 optogenetic trials and 62 light-only control trials) for a single representative Esr1-Cre-ChR2 animal. Error shading above and below the mean represents S.E.M. (G) (Left) Average USV rate traces during optogenetic stimulation and a light-only control for all Esr1-Cre-ChR2 animals (N=9 mice, 5 males and 4 females). Error shading above and below the mean represents S.E.M. (Right) Group averages of the mean USV rate 1 s before laser stimulation, during a 1 s laser stimulation, and 1 s after laser stimulation (blue) compared to light-only control trials (black) and to the natural decay of vocal bouts with no stimulation (gray) for all optogenetic stimulation animals (N=9 mice, 5 males and 4 females). Data for each mouse were normalized by dividing the mean USV rate pre, during, and post laser stimulation by the mean USV rate for the pre-laser period (***p<0.001 for differences between optogenetic condition and both control conditions during the laser stimulation period, two-way ANOVA followed by post-hoc pairwise Tukey’s HSD tests). Error bars represent S.E.M. (H) Summary plots show USV rate normalized to pre-laser baseline during optogenetic stimulation (purple: females; blue: males) when stimulating (1) Amgc/m-PAG neurons in males (data were from Michael et al., 2020; Figure 4) vs. (2) Amgc/m-PAG neurons in females (Figure 2) vs. (3) Esr1+ Amgc/m neurons in males and (4) Esr1+ Amgc/m neurons in females (Figure 3). Gray bars include two controls: GFP controls and natural vocal decay controls (light gray: females; dark gray: males) (F=17.37, df = 7, p=1.105e-09, one-way ANOVA between all groups, with post-hoc Tukey’s HSD tests showing that each experimental condition was significantly different from control conditions at **p<0.01, while no significant difference was found between any two experimental conditions: p1,2=0.9538, p1,3=0.9801, p1,4=0.9954). Figure 3—source data 1.Source data for Figure 3.

Fig 3: POAAmg cells are Esr1+, project to the PAG, promote USV production, and make inhibitory synapses onto AmgC/M-PAG neurons.(A) Viral strategy for labeling AmgC/M-projecting POA neurons. (B) (Left) Confocal images showing GFP-labelled AmgC/M-projecting POA neurons and Esr1 staining. Gray arrow points to an example Esr1- POAAmg neuron; yellow arrow points to an example Esr1+ POAAmg neuron. (Right) Quantification of Esr1+ POAAmg neurons (N=362 cells from 2 hemispheres from 2 mice: 173 cells from 6 80 µm coronal sections from one mouse; 189 cells from 6 80 µm coronal sections from the other mouse). Pie chart shows 298/362 GFP-labeled cells are Esr1+ (82.32%). Bar graph shows distribution of %Esr1+ /GFP+ overlap in the two mice examined (mean = 82.32% with S.E. of 1.89%). (C) Axon terminals of GFP-labelled AmgC/M-projecting POA neurons in PAG. (D) Viral strategy for optogenetic activation of AmgC/M-projecting POA neurons in male mice (N=4). (E) Top two plots show USV syllables (black line) elicited in two representative mice following 2s-long, 10 Hz optogenetic activation (blue) of ChR2-expressing POAAMG cells. Stimulation was performed on an animal alone in his home cage. Bottom plot shows an absence of USVs during light control, where the 2s-long, 10 Hz laser (blue) shined above the mouse’s head but was not connected to the ferrule. (F) Raster plots show USVs elicited from all trials in the two representative mice shown in (E). (G) (Left) Bar plots compare the mean number of USVs elicited per stimulation (Top), and the success rate of eliciting USVs (**p<0.01; paired t test) (Bottom) during optogenetic stimulation and light control for N=4 male mice (***p<0.001; paired t test). (Right) Mean USV rate plotted for N=4 males following optogenetic stimulation of ChR2-expressing POAAMG cells in the absence of social cues (alone in home cage). Gray shading above and below the mean represents S.E.M. (H) Summary plots show comparison among four experimental approaches for eliciting USVs. Vocalizations elicited by optogenetically stimulating POAAmg neurons (current study; dark blue) did not differ significantly from those elicited by optogenetically stimulating (1) POAPAG neurons, (2) Esr1+ POA neurons or (3) Esr1+ POA terminals at PAG in terms of mean USV rate (Kruskal-Wallis test, Chi-sq=6.36, df = 3, p=0.0954), success rate (Kruskal-Wallis test, Chi-sq=5.1, df = 3, p=0.1645), and mean latency (Kruskal-Wallis test, Chi-sq=7.12, df = 3, p=0.0681). The four experimental approaches include POAAMG neurons (dark blue, N=4 males from current study), POAPAG neurons (light blue, N=9 males from Michael et al., 2020), Esr1+POA neurons (light blue, N=7 males from Michael et al., 2020), Esr1+ POA axon terminals within the PAG (light blue, N=5 males from Michael et al., 2020). For left and middle panels, the following controls were also shown for reference: light control (N=4 males from current study), GFP-expressing Esr1+ POA neurons (N=5 males from Michael et al., 2020), VGLUT2+ POA neurons (N=3 males from Michael et al., 2020), VMHPAG neurons (N=3 males from Michael et al., 2020), and Esr1+ POA axon terminals within the ventral tegmental area (VTA) (N=4 males from Michael et al., 2020). Error bars show S.D. (I) Viral strategy for in vitro whole-cell voltage clamp recordings from visually identified AmgC/M-PAG neurons while optogenetically activating POAPAG axon terminals in brain slices containing the amygdala. (J) (Left) Light-evoked IPSCs recorded in TTX/4AP (observed in N=9 of 13 td-Tomato-tagged AmgC/M-PAG neurons from N=5 males) were abolished by application of gabazine (N=4 cells also recorded in gabazine). IPSC amplitude refers to the peak of the light-evoked current at 0 mV holding potential. (Right) (Top) Mean IPSC amplitude recorded in TTX/4AP (N=9; one cell with an IPSC amplitude 757.79 pA not shown on the plot) and SR-95531 (N=4). (Bottom) Comparison of IPSC amplitude for the four cells recorded in both TTX/4AP and SR-95531, normalized to IPSC amplitude in TTX/4AP (***p=6.32e-06; paired t test,). Error bars represent S.E.M. Figure 6—source data 1.Source data for Figure 6.