Given that optogenetically activating Amg_C/M-PAG neurons suppresses USV production in male mice during courtship encounters with females, we reasoned that Amg_C/M-PAG neurons would be highly active during threatening contexts in which vocalization is potentially risky for the sender and thus likely to be suppressed. First, we tested how USV production during social encounters was affected by the synthetic fox urine odor 2-methyl-2-thiazoline (2MT), a derivate of 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) (Day et al., 2004; Lin et al., 2006; Root et al., 2014), a component of fox urine that is highly aversive to mice. In comparison to TMT, 2MT has been reported to induce freezing behavior more reliably (Bruzsik et al., 2021; Isosaka et al., 2015). We measured the number of USVs produced by male or previously isolated female (see following section) mice during a 5-min encounter with a novel female conspecific in the presence or absence of 2MT. We found that both male and female mice produced significantly fewer USVs when encounters with a novel female occurred in the presence of 2MT compared to controls (Figure 1A; Figure 1—video 1; Figure 1—video 2).

Next, we used an intersectional viral strategy to express the calcium indicator GCaMP8s in Amg_C/M-PAG neurons (Figure 1B) and used fiber photometry to measure calcium signals in these neurons in male and female mice as they investigated a dish containing either 2MT, a control odor (ethyl tiglate), a novel plastic ‘toy’, or the soiled bedding of male or female conspecifics (Figure 1C). The rationale for conducting fiber photometry experiments with only social partners, or novel objects, or 2MT (Figure 1C) as opposed to repeating the behavioral paradigm used in the vocalization behavioral test described above (Figure 1A) is that we sought to isolate Amg_C/M-PAG activity patterns that were evoked by one stimulus type or another, rather than a combinatorial signal that would be difficult to interpret in the absence of these single condition data. In both sexes, calcium signals in Amg_C/M-PAG neurons were significantly elevated above baseline when the animal approached and investigated dishes containing any of these items (Figure 1—figure supplement 2), but they were the highest when investigating the dish containing 2MT (Figure 1D–F). The overall pattern of responsiveness to these various stimuli was similar between male and female mice (Figure 1F; Figure 1—figure supplement 1A). Furthermore, the calcium increase evoked by 2MT was not linked to subsequent bouts of freezing, suggesting that activity in these neurons was driven by detection of the odorant rather than the subsequent behavioral response (Figure 1—figure supplement 1B). Therefore, an innately aversive predator odorant suppresses USV production and strongly activates USV-suppressing Amg_C/M-PAG neurons in both male and female mice.

We then tested how Amg_C/M-PAG neuron activity was affected during social interactions with other conspecifics that can either promote or suppress USV production. We used fiber photometry to measure calcium signals of Amg_C/M-PAG neurons in freely behaving male and female mice during 5-min encounters with novel male and female conspecifics. These encounters resulted in non-aggressive interactions when USV production was more likely to occur or aggressive interactions (i.e. fights between two males) when USV production is absent (we confirmed that males did not produce USVs during fights). In both sexes, we observed similar small increases in Amg_C/M-PAG neuronal activity when calcium signals were aligned to the onsets of non-aggressive social encounters with male or female conspecifics, or with a mouse’s investigation of a novel object (Figure 1G; Figure 1—figure supplement 1A; Figure 1—figure supplement 2A). However, calcium signals of Amg_C/M-PAG neurons did not change significantly at the onset or offset of USV bouts, suggesting that Amg_C/M-PAG neurons are not simply turning off USV bouts during non-aggressive social encounters (Figure 1H). In contrast, Amg_C/M-PAG neuronal calcium signals increased markedly during aggressive interactions between two males that included attacks (Figure 1I–J). Although many of these epochs included aggressive behavior from both males, Amg_C/M-PAG neuronal calcium signals were larger in the male being attacked (Figure 1—figure supplement 1C). In summary, Amg_C/M-PAG neurons exhibit large increases in activity during aggressive social encounters when USVs are not typically produced and much smaller increases in activity during affiliative social encounters in which USVs frequently occur. These observations support the idea that Amg_C/M-PAG neurons actively suppress USV production in the presence of threatening stimuli.

While we previously showed that optogenetic activation of Amg_C/M-PAG neurons transiently suppressed courtship USVs in male mice without disrupting non-vocal social behavior (Michael et al., 2020), it remained an open question as to whether these neurons also suppress USV production in female mice. To resolve this issue, we adopted a recently developed behavioral protocol that can reliably elicit robust USV production from female mice (Zhao et al., 2021). To elicit high numbers of USVs, female mice were socially isolated for at least three weeks and then, on the day of testing, a novel female conspecific was introduced to the socially isolated experimental animal’s home cage (Figure 2A). We observed that while group-housed female mice produce few USVs in response to a novel female conspecific, previously isolated female mice produced significantly more USVs per minute, more USV bouts per minute, and longer USV bouts, and in fact vocalized at rates comparable to isolated male mice during female presentation (Figure 2A). This finding confirms the recent study by Zhao et al., 2021, and provided us with a useful entry point for addressing whether Amg_C/M-PAG neurons suppress USV production in female mice.

To this end, we used an intersectional viral strategy to selectively express channelrhodopsin (ChR2) in Amg_C/M-PAG neurons of female mice (Figure 2B). We found that this strategy labels neurons in the Amg_C/M but not the CeA of female mice, similar to the pattern previously observed with this strategy in male mice (Figure 2B; see also Figure 4A in Michael et al., 2020). Using the social isolation protocol, we were able to elicit high numbers of USVs routinely in 5 of 7 Amg_C/M-PAG-ChR2 female mice as they investigated a novel female partner. Optogenetic stimulation of Amg_C/M-PAG neurons in freely vocalizing female mice immediately suppressed USV production, and this suppression persisted throughout the duration of the laser pulse (Figure 2C–D, N=5 of 5 female mice). The optogenetically evoked decrease in USV rate was significant when compared to a light-only control condition in which the same mice were connected to a dummy ferrule that shone blue light above their heads, a control condition in which the same mice did not receive laser stimulation and USV rates decreased naturally, and to a separate group of female mice in which GFP was expressed in Amg_C/M-PAG neurons and that were subjected to optical fiber illumination in the Amg (Figure 2D; Figure 2—video 1; N=5 female mice in the optogenetic condition, light control condition, and natural bout decay condition and N=5 female mice in the GFP control condition, p<0.01 for differences between ChR2 group vs. control groups during laser time; two-way ANOVA with repeated measures on one factor, p<0.01 for interaction between group and time, followed by post-hoc pairwise Tukey’s HSD tests). After laser offset, the USV rate in the Amg_C/M-PAG-ChR2 female mice rebounded to a level comparable to that in all control conditions (p>0.05 for differences between groups in post-laser period; Figure 2C–D). When we compared these vocal suppression results to those reported in Michael et al., 2020, we found that vocal suppression by stimulating Amg_c/m-PAG neurons in females was comparable to the previously reported vocal suppression by stimulating Amg_c/m-PAG neurons in males (Michael et al., 2020; Figure 3H).

In male mice, USV-suppressing Amg_C/M-PAG neurons are predominantly GABAergic (Michael et al., 2020). We used in situ hybridization to establish that 100% of Amg_C/M-PAG cells in female mice express VGAT (Slc32a1), suggesting that they mediate USV suppression by directly inhibiting PAG-USV neurons (N=167/167 neurons in 2 female mice were VGAT⁺ (Slc32a1), and 0/167 were VGLUT2⁺ (Slc17a6); Figure 2E), as described in males (Michael et al., 2020). In summary, Amg_C/M-PAG neurons are GABAergic and function to suppress vocalizations in female mice during social encounters with other females, similar to the manner in which these neurons suppress courtship USVs in male mice. Therefore, although female and male mice produce USVs in different social and sexual contexts, Amg_C/M-PAG neurons in both sexes function similarly to suppress USV production.

Neurons that express estrogen receptor alpha (Esr1) have been implicated in a wide range of sexual and social behaviors including aggression, social recognition, courtship, and vocalization (Chen et al., 2021; Hashikawa et al., 2017; Kudwa et al., 2006; Lee et al., 2014; Michael et al., 2020; Moran et al., 2020). Indeed, several recent studies showed that optogenetically activating Esr1⁺ neurons in the POA was sufficient to elicit long-lasting bouts of USVs in both male and female mice (Chen et al., 2021; Karigo et al., 2021; Michael et al., 2020). Given these observations, we wondered whether Esr1 might also be expressed in Amg_C/M-PAG neurons. Consistent with this idea, in situ hybridization data from the Allen Brain Atlas shows expression of Esr1 mRNA in the same region of the amygdala as Amg_C/M-PAG neurons are found in male mice (Figure 3A). We therefore tested the hypothesis that Amg_C/M-PAG neurons express Esr1 using immunofluorescent Esr1 protein antibody staining. We found that nearly half of GFP-labeled Amg_C/M-PAG neurons express Esr1 (Figure 3B,~49%, N=913 cells from 7 mice, 3 males and 4 females; Amg_C/M-PAG neurons labeled by injecting PAG with AAV-retro-Cre and amygdala with AAV-flex-GFP, similar to the strategy shown in Figure 2B). By injecting the amygdala of Esr1-cre mice with flex-tdTomato we further found that Esr1⁺ amygdala cells send axonal projections to the region of caudolateral PAG, the region that contains PAG-USV neurons, and consistent with our observation that a large subset of Amg_C/M-PAG neurons are Esr1⁺ (Figure 3C).

To test whether activation of Esr1⁺ Amg_C/M cells is sufficient to suppress USV production, we injected the Amg_C/M of Esr1-Cre male and female mice with a Cre-dependent AAV driving ChR2 expression (Figure 3D). All mice were single-housed post-surgery. Optogenetic activation of Esr1⁺ Amg_C/M cells immediately and reversibly suppressed USV production (N=9 mice; 5 males and 4 females; Figure 3E–G). As with stimulation of Amg_C/M-PAG cells, this suppression was specific to the time period of optogenetic stimulation and did not occur in light-only control conditions or in control conditions in which USV bouts were allowed to decay naturally (Figure 3G; Figure 3—video 1; 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). Moreover, stimulating either Esr1⁺ Amg_C/M neurons or Amg_C/M-PAG neurons reduced the USV rate (normalized to pre-laser baseline) to a comparable level (Figure 3H). Lastly, stimulating Esr1⁺ Amg_C/M neurons was similarly efficient for suppressing USVs in both males and females. Taken together, this evidence suggests that Esr1⁺ Amg_C/M-PAG neurons mediate vocal suppression in both male and female mice. Though GABAergic Esr1⁺ neurons in the hypothalamus and glutamatergic Esr1 +neurons in the posterior amygdala have been implicated in mediating sexual behaviors and aggression, to our knowledge this is a novel role for GABAergic Esr1⁺ neurons in the Amg_C/M-PAG.

The experiments described in the prior sections establish that activating Amg_PAG neurons in male and female mice suppresses spontaneous USV production during social encounters with novel females, that these neurons respond similarly to an aversive predator odorant that also suppresses USV production, and that they display similar molecular phenotypes. We next sought to understand how these vocal-suppressing Amg_C/M-PAG neurons interact with vocal promoting signals to influence USV production in the service of social affiliation. Although the drive and affiliative function are well understood for male courtship USVs, the drivers and functions for USVs produced by previously isolated female mice during encounters with novel females are not known. During courtship, male mice produce vocalizations robustly (Chabout et al., 2015; Neunuebel et al., 2015; Portfors, 2007; Tschida et al., 2019; Warren et al., 2018). As females prefer more vocal males (Tschida et al., 2019), vocalizing in the presence of a female facilitates a male’s mating success. In contrast, it is unclear whether the USVs that a previously isolated female produces in response to a novel female function in an affiliative or aggressive manner. For these reasons, we next sought to characterize the interaction of vocal promoting and vocal suppressing signals more fully in the amygdala of male mice.

The decision to suppress ongoing vocalizations depends on the integration of external cues with interoceptive information. Given that Amg_C/M-PAG neurons suppress vocalizations by directly inhibiting vocal gating neurons in the PAG, they are likely to integrate a wide range of inputs that help regulate this decision. We conducted a series of additional anatomical and functional experiments in male mice to better understand the nature of this integrative process. We first conducted monosynaptically restricted transsynaptic rabies tracing from Amg_C/M-PAG neurons to map their inputs (Figure 4A). To specifically label inputs to Amg_C/M neurons that project to PAG we injected the caudolateral PAG with AAV-retro-Cre and the Amg_C/M with Cre-dependent helper viruses that express rabies glycoprotein (G), a protein required for transsynaptic spread (Callaway and Luo, 2015). Two weeks later, we injected the same amygdalar regions with a pseudotyped G-deleted rabies virus, which when complemented by the wild-type rabies G glycoprotein, resulted in expression of GFP in neurons that make synapses onto Amg_C/M-PAG neurons (Figure 4A; N=4 males).

This approach revealed that Amg_C/M-PAG neurons in male mice receive monosynaptic input from numerous cortical regions that process a wide variety of sensory and interoceptive information, including the piriform, motor, somatosensory, insular and auditory cortices (Figure 4B; Bhattacharjee et al., 2021; Gogolla, 2017; Shipley and Ennis, 1996; Zatorre et al., 2002). We also observed that Amg_C/M-PAG neurons receive input from many other forebrain and midbrain regions, including the medial and lateral POA, the bed nucleus of the stria terminalis (BNST), the ventral tegmental area, and the nucleus accumbens shell (Figure 4C–D), which have been variously implicated in defensive behaviors, sexual-social interactions, reward and satisfaction (Breitfeld et al., 2015; Michael et al., 2020; Paredes, 2003; Salgado and Kaplitt, 2015).

Of particular interest to us was the labeling of cell bodies in the medial POA (Figure 4E), a region that contains neurons whose activation elicits USVs via a disinhibitory circuit motif within the PAG (Chen et al., 2021; Michael et al., 2020). This anatomical organization raises the possibility that Amg_C/M-PAG cells are a cellular locus where USV-suppressing information can be weighed against USV-promoting signals from the POA. Therefore, we focused on characterizing the social and USV-related information that POA axons convey to the Amg_C/M, and the function of the synapses that POA neurons make on Amg_C/M-PAG neurons in male mice.

To measure the activity of POA inputs to the Amg_C/M, we used intersectional methods to express GCaMP8s in Esr1⁺ POA neurons and recorded bulk fluorescence from their axon terminals in the Amg_C/M (Figure 5A; N=5 male Esr1-Cre mice). We then measured the activity of POA axons in the Amg_C/M during the male’s social interactions with novel female and male mice, 2MT, or novel plastic toys. In contrast to the activity of Amg_C/M-PAG neurons measured in these various conditions (Figure 1G), POA_Esr1-Amg terminal activity showed a large and sustained increase at the onset of social interactions with female mice, a moderate sustained increase at the onset of non-aggressive social interactions with other male mice, and no response at the onset of or during trials where the mouse encountered a dish containing 2MT or a novel plastic toy (Figure 5B). In addition to the large and sustained increase in activity upon the introduction of a social partner, POA_Esr1-Amg terminal activity further increased at the onset of subsequent social interactions and at the onset of individual USV bouts (Figure 5C–D). However, POA_Esr1-Amg terminal activity remained low when aligned to the onsets of the mouse’s investigation of a dish containing 2MT or the onsets of attacks by male conspecifics (Figure 5E and F). Taken together, these findings support the idea that POA_Esr1-Amg axons in the Amg_C/M-PAG of male mice are highly active during social encounters in which USVs are produced, but not in situations in which Amg_C/M-PAG activity is high and USV production is suppressed. Therefore, in addition to being strongly activated by stimuli or aggressive social interactions that suppress USV production (Figure 1D, F and J), Amg_C/M-PAG neurons receive inputs from the POA that convey information about pro-vocal social contexts and USV production.

These various observations strongly suggest that the POA neurons that provide input to the amygdala are actually USV-promoting neurons. Because activating POA_PAG cells that express Esr1 is sufficient to promote USV production (Chen et al., 2021; Karigo et al., 2021; Michael et al., 2020), we first investigated whether POA cells that project to the Amg_C/M (POA_AMG cells, labeled by intersectional methods; Figure 6A) are part of this Esr1-expressing subpopulation of POA cells. We found that the majority (~82%) of GFP-labeled POA_AMG cells express Esr1 (Figure 6B, N=298/362 cells from 2 male mice). We further noted that axon terminals from GFP-labeled POA_AMG cells were present in the caudolateral PAG, the same region where PAG-USV neurons are located (Figure 6C, see also Tschida et al., 2019). To directly test whether POA_AMG cells are USV-promoting, we used intersectional viral methods to express ChR2 in POA_AMG cells (Figure 6D). We found that exciting the cell bodies of these ChR2-expressing POA_AMG cells with blue light was sufficient to trigger USVs in male mice in the absence of social cues (Figure 6E–G; Figure 6—video 1; N=4 male mice showed increased USV rates in response to this optogenetic stimulation). The effects on mean USV rate, success rate of eliciting USVs, and the mean latency to elicited USVs using the POA_AMG optogenetic stimulation approach were not significantly different from the USV-promoting effects achieved by stimulating POA_PAG neurons or Esr1⁺ POA terminals in PAG (Michael et al., 2020; Figure 6H). Therefore, the POA cells that convey pro-vocal social signals and USV-related signals to the Amg_C/M are Esr1⁺ also project to the caudolateral PAG and directly promote USV production.

Prior studies show that the vast majority of POA_PAG neurons, including those that promote USV production, release the inhibitory neurotransmitter GABA (Chen et al., 2021; Michael et al., 2020). Therefore, our current observations raise the interesting possibility that the Amg_C/M-PAG cells that suppress USVs are directly inhibited by the POA_PAG neurons that promote USV production. To test this hypothesis, we first injected the PAG with AAV-retro-Cre, the POA with AAV-flex-ChR2, and the amygdala with AAV-flex-tdTomato (Figure 6I). Several weeks later, we performed in vitro whole-cell voltage clamp recordings from visually identified Amg_C/M-PAG neurons while optogenetically activating POA_PAG axon terminals in brain slices containing the amygdala. We visually targeted our recordings to tdTomato⁺ Amg_C/M-PAG neurons in the presence of TTX and 4AP to isolate monosynaptic currents. Optogenetically activating POA_PAG axon terminals in the amygdala evoked inhibitory postsynaptic currents (IPSCs) in a majority (9/13) of Amg_C/M-PAG (td-Tomato+) neurons (Figure 6J; mean current = 141.9 pA at 0 mV in TTX/4AP; currents with reversal potentials at –70 mV were identified as IPSCs, and no EPSCs were observed at –70 mV in the presence of TTX and 4AP). Bath application of the GABA_A receptor antagonist gabazine SR-95531 abolished these optogenetically evoked IPSCs in all four cases in which it was applied (Figure 6J, 4/9 recordings). Therefore, POA_PAG cells provide monosynaptic inhibitory input onto Amg_C/M-PAG neurons. These various findings support the idea that USV-suppressing Amg_C/M-PAG neurons, which are strongly activated by predator cues and social threats, are also directly inhibited by USV-promoting POA neurons.

Further information and requests for resources and reagents should be directed to the corresponding authors, Richard Mooney (mooney@neuro.duke.edu).