In social interactions utilizing vocal communication signals, the acoustic features of the signals carry emotional information (Altenmüller et al., 2013; Darwin, 1872; Seyfarth and Cheney, 2003). Listeners receive and analyze the acoustic information, compare it with previous experiences, identify the salience and valence of such information, and respond with appropriate behaviors. These integrated functions depend on brain circuits that include the amygdala, a region located within the temporal lobe that is recognized to play a role in orchestrating emotional responses to salient sensory stimuli (LeDoux, 2000; McGaugh, 2002; Sah et al., 2003). The amygdalar target of auditory inputs from the thalamus and cortex is the basolateral amygdala (BLA) (LeDoux et al., 1984; Romanski and LeDoux, 1993; Shi and Cassell, 1997; Tsukano et al., 2019). By integrating this auditory input with other sensory inputs and inputs from other limbic areas, BLA neurons shape appropriate behavioral responses (Beyeler et al., 2016; Gründemann et al., 2019; Namburi et al., 2015; Namburi et al., 2016) via projections to downstream targets such as the nucleus accumbens (Ambroggi et al., 2008; Stuber et al., 2011) and central nucleus of the amygdala (Ciocchi et al., 2010).

The BLA processes vocal and other acoustic information in a context-dependent manner (Gadziola et al., 2016; Grimsley et al., 2013; Matsumoto et al., 2016; Wenstrup et al., 2020; Wiethoff et al., 2009). Contextual information may arise from inputs associated with other sensory modalities (e.g., somatic sensation or olfaction) (Grimsley et al., 2013; Lanuza et al., 2004; McDonald, 1998), but in other cases the contextual information is associated with an animal’s internal state. Sources of internal state cues to BLA include brain circuits involving modulatory neurochemicals (i.e., neuromodulators), known to affect the processing of sensory signals, thus shaping attention, emotion, and goal-directed behaviors (Bargmann, 2012; Likhtik and Johansen, 2019; Schofield and Hurley, 2018). While previous work has demonstrated a role for some neuromodulators—dopamine (DA) and acetylcholine (ACh)—in the production of social vocalizations (Inagaki et al., 2020, Rojas-Carvajal et al., 2022; Silkstone and Brudzynski, 2020), it remains unclear how these and other neuromodulators contribute to vocal processing in social interactions.

This study investigates contextual information provided by neuromodulatory inputs to the BLA in response to vocal communication signals. Our hypothesis is that these salient vocalizations elicit distinct patterns of neuromodulator release into the BLA, by which they shape the processing of subsequent meaningful sensory information. We further hypothesize that these neuromodulatory patterns may depend on longer-term processes that are critical to vocal communication: to experience with these behaviors and the accompanying vocalizations, to sex, and to estrous stage in females. To test these hypotheses, we conducted playback experiments in a mouse model to understand the behavioral and neuromodulator responses to salient vocalizations associated with very different behavioral states.

To study how vocalizations affect behaviors and release of neurochemicals within BLA, we first developed highly salient vocal stimuli associated with appetitive (mating) and aversive (restraint) behaviors of CBA/CaJ mice. From more intense, mating-related interactions between adult male and female mice that included female head-sniffing and attempted or actual mounting (Gaub et al., 2016; Ghasemahmad, 2020, see Materials and methods), we selected several sequences of vocalizations to form a 20-min mating vocal stimulus. These sequences included ultrasonic vocalizations (USVs) with harmonics, steps, and complex structure, mostly emitted by males, and low-frequency harmonic calls (LFHs) emitted by females (Figure 1A, C; Finton et al., 2017; Gaub et al., 2016; Ghasemahmad, 2020; Hanson and Hurley, 2012). During short periods of restraint, mice produce distinctive mid-frequency vocalizations (MFVs) that are associated with anxiety-related behaviors and increased release of the stress hormone corticosterone (Dornellas et al., 2021; Grimsley et al., 2016; Niemczura et al., 2020). From vocal sequences produced by restrained mice, we created a 20-min vocal stimulus, primarily containing MFVs and fewer USV and LFH syllables (Figure 1B, C).

We next asked whether these salient vocal stimuli, associated with very different behavioral states, could elicit distinct behaviors and patterns of neuromodulator release into the BLA. We focused on the neuromodulators ACh and DA, since previous work suggests that these neuromodulatory systems interact in the emission of positive and negative vocalizations (Rojas-Carvajal et al., 2022; Silkstone and Brudzynski, 2020). Our experiments combined playback of the vocal stimuli, behavioral tracking and observations (see Table 1 for descriptions), and microdialysis of BLA extracellular fluid in freely moving mice (Figure 1D).

Prior to the study, male and female mouse subjects had no experience with sexual or restraint behaviors. On the first 2 days of the experiment, mice in an experienced group (EXP, n = 31) were each exposed to 90-min sessions with mating and restraint behaviors in a counterbalanced design (Figure 1D). Mice were then implanted with a guide cannula for microdialysis. On the playback/sample collection day (Day 6), a microdialysis probe was inserted into the guide cannula. After a 4-hr period of mouse habituation and probe equilibration, we recorded behavioral reactions and sampled extracellular fluid from the BLA before (Pre-Stim) and during a 20-min playback period, divided into two 10-min stimulation/collection/observation periods that are designated Stim 1 and Stim 2 (Figure 1D, E; see also Materials and methods). Each mouse received playback of either the mating or restraint stimuli, but not both: same-day presentation of both stimuli would require excessively long playback sessions, the condition of the same probe would likely change on subsequent days, and quality of a second implanted probe on a subsequent day was uncertain. Fluids were analyzed using a liquid chromatography/mass spectrometry (LC/MS) technique that allowed simultaneous measurement of several neurochemicals and their metabolites in the same dialysate samples, including ACh, DA, and the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) (see Materials and methods). All neurochemical results during Stim 1 and Stim 2 periods are expressed as a percentage relative to the Pre-Stim control period. However, raw values of ACh, DA, and 5-HIAA are reported in source data files (Figure 3—source data 1, Figure 3—source data 2, Figure 3—source data 3). Data are reported only from mice with more than 75% of the microdialysis probe implanted within the BLA (Figure 2).

We first describe tests to examine whether playback of mating and restraint vocalizations results in different behavioral and neurochemical responses in male mice. We observed that two behaviors, still-and-alert and flinching, showed differential effects of playback type, increasing during restraint playback relative to mating playback during the Stim 1 period (Figure 3A, B). There were also distinct patterns of ACh and DA release into the BLA depending on the type of vocalization playback (Figure 3C, D). Thus, in response to restraint vocalizations, we observed an increase in ACh concentration during both Stim 1 (M ± SD: +24 ± 31% re Pre-Stim baseline) and Stim 2 (+25 ± 16%) playback windows. Mating vocalizations, however, resulted in a decrease in ACh release during both playback periods (Stim 1: −19 ± 21.0%; Stim 2: −16.4 ± 24.0%) (Figure 3C). DA release displayed opposite patterns to ACh, increasing during playback of mating vocalizations during Stim 1 (+12.0 ± 27.4%) and Stim 2 (+25 ± 39% re baseline), but decreasing during playback of restraint vocal sequences (Stim 1: −11.0 ± 11.2%; Stim 2: −22 ± 25.6%) (Figure 3D). For ACh, levels differed significantly between the mating and restraint groups during both Stim 1 and Stim 2, while DA levels differed significantly only during Stim 2 (Figure 3C, D). In contrast, the serotonin metabolite 5-HIAA showed no distinct pattern over time following playback of either vocal stimulus, nor significant differences between groups (Figure 3E). These findings suggest that both behavioral responses and ACh and DA release are modulated in listening male mice by the affective content of social vocalizations.

As male and female mice emit different vocalizations during courtship and mating (Finton et al., 2017; Grimsley et al., 2013; Neunuebel et al., 2015; Sales, 1972), we tested whether playback of vocal interactions associated with mating (Figure 1A) results in different behavioral and neurochemical responses in listening male and female mice. Since our testing included both estrus and non-estrus females, we further examined the estrous effect on neurochemical release and behavioral reactions.

Playback of mating vocalizations resulted in some general and some sex-based differences in behavioral responses. For instance, all groups displayed increased attending behavior (Figure 4A). In females, regardless of estrous stage, rearing decreased and still-and-alert behavior increased significantly relative to male mice (Figure 4B, C). This change in motor activity was further supported by video tracking results showing a significant decrease in distance traveled by female mice in response to mating vocal playback (significant sex effect (F(1,15) = 9.3, p = 0.008, ŋ² = 0.4)). However, one behavioral change was estrous dependent: females in estrus displayed a strikingly higher number of flinching behaviors compared to males and non-estrus females during both Stim 1 and Stim 2 periods (Figure 4D). Our analysis of neuromodulator responses to mating vocalization playback revealed an estrous-dependent modulation of ACh levels during playback. ACh concentration in estrus females increased during both Stim 1 and Stim 2 periods, whereas ACh decreased in both males and non-estrus females (Figure 4E). Moreover, during playback, post hoc comparison with Bonferroni correction showed that the ACh in estrus females was significantly higher (Stim 1, +29.3 ± 27.0%; Stim 2, +25.5 ± 22%) than both males (Stim 1, −18 ± 21.0%; Stim 2, −16.0 ± 24.0%) and non-estrus females (Stim 1, −6.0 ± 14%; Stim 2, −35.0 ± 29.6%). DA release showed increases during mating playback within all three experimental groups (males: Stim 1, +12.0 ± 27.0%, Stim 2, +26.0 ± 39.1%; estrus females: Stim 1, +24.1 ± 37.0%, Stim 2, +23.0 ± 27.0%; non-estrus females: Stim 1, +27.0 ± 17.0%, Stim 2, +49.0 ± 48.0%), but no significant differences among groups (Figure 4F). Similar to male groups in restraint and mating playback, the 5-HIAA release patterns in females showed no clear modulation pattern during mating vocal playback or differences among groups (Figure 4G).

Like male mice exposed to restraint vocalizations, estrus females showed robust and significant increases in flinching behavior in response to mating vocalizations. Both groups displayed increased ACh release. This supports the possible involvement of ACh in shaping such behavior in both males listening to restraint calls and in estrus females listening to mating vocalizations. Note that neuromodulator release, including ACh, has been previously linked to motor behaviors (Wall and Woolley, 2020), but the changes in ACh that we observed showed no relationship with behaviors involving motor activity such as rearing (restraint males: Stim 1: n = 6, r = 0.1, p = 0.8; Est females: n = 6, r = 0.7, p = 0.08), locomotion (Stim 1: restraint males, n = 6, r = −0.2, p = 0.7; Est females: n = 6, r = 0.3, p = 0.6), or distance traveled (Stim 1: restraint males, n = 6, r = 0.16, p = 0.8; Est females: n = 6, r = −0.6, p = 0.2). This suggests that the observed changes in ACh reflect the valence of these vocalizations.

All EXP mice used in the above experiments had undergone a single session each to experience mating and restraint conditions prior to the playback session on Day 6 (Figure 1D). Does such experience shape the release patterns of these neuromodulators in response to vocal playback? We tested male and female mice under identical vocal playback conditions as previous groups, except that they did not receive the restraint and mating experiences (INEXP groups). Since only one INEXP female was in a non-estrus stage during the playback session, our analysis of the effect of experience included only estrus females and males.

Several behavioral responses to vocalization playback differed between EXP and INEXP mice in a sex- or context-dependent manner. For example, EXP estrus females showed significantly increased sound-evoked flinching behaviors compared to INEXP estrus females (Figure 5A) in response to mating vocal sequences. These experience effects were not observed in males in response to mating or restraint vocal playback. Furthermore, the differences in flinching behavior among EXP groups (male-mating vs male-restraint or estrus female-mating) were not evident among INEXP groups. For rearing behavior, EXP males responding to mating vocalizations showed an increase compared to INEXP males (Figure 5B). This pattern was not observed during restraint playback for males or mating playback in estrus females. These findings indicate that behavioral responses to salient vocalizations result from interactions between sex of the listener or context of vocal stimuli with the previous behavioral experience associated with these vocalizations.

A major finding is that prior experience with mating and restraint behaviors shaped patterns of ACh release in response to vocal playback; there was a significant interaction of context and experience for ACh (F(1,39) = 12.7, p < 0.001; Figures 5C and 6A). Thus, ACh release was significantly different between INEXP and EXP males in restraint playback groups. A similar pattern was observed in INEXP estrus females in response to mating vocal playback, that is, the concentration of ACh re baseline failed to show such pronounced increases as observed in EXP females and showed a significant experience effect (sex*experience interaction: F(1,39) = 7.8, p = 0.008; Figures 5C and 6A). Furthermore, INEXP males in mating playback group failed to show the reduced ACh concentration observed in EXP males (INEXP Stim 1: +9.6 ± 15.0%; INEXP Stim 2: +6.1 ± 30.0; EXP Stim 1: −19.0 ± 21.0%; EXP Stim 2: −16.0 ± 24.0%), even though these differences were not significant (Figures 5C and 6A).

DA release patterns, which were weaker in EXP groups compared to ACh patterns (Figure 3C vs D ; Figure 4E vs F) showed no significant experience-dependent changes in any group (Figure 5D). There was no evidence that vocal playback altered DA release in any of the three groups of INEXP mice (Figure 6B), unlike what was observed in EXP groups (Figures 3D and 4F). 5-HIAA concentrations, which were unaffected by sex, estrous stage, or playback type (Figures 3E and 4G), were also unaffected by experience (Figure 6C).

Collectively, these data suggest that the playback vocalization type and estrous effects observed in ACh release patterns and behavioral reactions depend on previous experience with the corresponding behaviors.

Functional imaging studies in humans and mechanistic studies in other species provide substantial evidence that the amygdala participates in circuits that process vocalizations (Frühholz et al., 2016; Liebenthal et al., 2016; Sander et al., 2003; Wenstrup et al., 2020; Voytenko et al., 2023), assess the valence of appetitive and aversive cues (Kyriazi et al., 2018; O’Neill et al., 2018; Pignatelli and Beyeler, 2019; Smith and Torregrossa, 2021), and shape appropriate behavioral responses to these cues (Gründemann et al., 2019; Lim et al., 2009; Schönfeld et al., 2020; Zhang and Li, 2018). Nonetheless, how contextual information is delivered to the amygdala and contributes to vocal processing is not well understood. Since the amygdala receives strong projections from neuromodulatory brain centers (Aitta-Aho et al., 2018; Asan, 1997; Asan, 1998; Bigos et al., 2008; Bocchio et al., 2016; Carlsen et al., 1985), and since the role of these neurochemicals in providing internal state and contextual information is well documented (Bocchio et al., 2016; Jiang et al., 2016; Likhtik and Johansen, 2019), we hypothesized that release patterns of neuromodulators into the BLA provide contextual information during processing of affective vocalizations. Our results show that these emotionally charged vocalizations result in distinct release patterns of ACh and DA into the BLA of male and female mice. Furthermore, female hormonal state appears to influence ACh but not DA release into the BLA when processing mating vocalizations. Such context- or state-dependent changes were not observed in patterns of other neurochemicals (e.g., 5-HIAA), nor in the absence of experience with behaviors associated with the vocalizations. In particular, we showed that a single 90-min experience with intense behaviors is sufficient to establish strong, consistent patterns of ACh release into the amygdala. These data indicate that during analysis of affective vocalizations in the BLA, ACh, and DA provide experience-dependent, state- and context-related information that can potentially modulate sensory processing within the BLA and thus shape an individual’s response to these vocalizations.

The linkage between neuromodulator release and behavioral responses to vocalizations varies. The strongest case is for a link between increased ACh release and flinching behavior. Males in the restraint playback group and estrus females in the mating playback group both displayed significantly increased ACh release and flinching behavior, occurring in both playback periods. This suggests a mechanistic relationship. Other significant behavioral responses to vocal stimuli did not closely match the timing of neurochemical changes, suggesting a weak mechanistic link. Overall, it is not surprising that most behaviors and neurochemicals do not match well, given the poor temporal resolution of the neurochemical sampling. We believe that our results show the need for a finer analysis of DA and ACh release based on techniques for high temporal resolution of neurochemical measurements (e.g., genetically encoded ACh indicator; Jing et al., 2018), coupled with short interval behavioral observations. Only then can the role of the BLA and neuromodulator inputs in behavioral vocal responses be established more strongly. Furthermore, we note that no conclusion can be made regarding relationships between noradrenalin and serotonin concentrations and vocal playback, since these were not detected by the neurochemical analysis.

The BLA receives strong cholinergic projections from the basal forebrain (Aitta-Aho et al., 2018; Carlsen et al., 1985) that contribute to ACh-dependent processing of aversive cues and fear learning in the amygdala (Baysinger et al., 2012; Gorka et al., 2015; Jiang et al., 2016; Tingley et al., 2014). Our findings support these studies by demonstrating increased ACh release in BLA in EXP animals in response to playback of aversive vocalizations. Although the exact mechanism by which ACh affects vocal information processing in BLA is not clear yet, the result of ACh release onto BLA neurons seems to enhance arousal during emotional processing (Likhtik and Johansen, 2019). Our results, in conjunction with previous work, suggest mechanisms by which vocalizations affect ACh release and in turn drive behavioral responses (Figure 7A). Cholinergic modulation in the BLA is mediated via muscarinic and nicotinic ACh receptors on BLA pyramidal neurons and inhibitory interneurons (Aitta-Aho et al., 2018; Mesulam et al., 1983; Pidoplichko et al., 2013; Unal et al., 2015). During the processing of sensory information in the BLA, partially non-overlapping populations of neurons respond to cues related to positive or negative experiences (Namburi et al., 2015; Paton et al., 2006; Smith and Torregrossa, 2021). These neurons then project to different target areas involved in appetitive or aversive behaviors—the nucleus accumbens or central nucleus of the amygdala, respectively (Namburi et al., 2015).

In response to an aversive cue or experience (Figure 7A), released ACh affects BLA neurons according to their activity. If projection neurons are at rest, ACh may exert an inhibitory effect by activating nicotinic ACh receptors on local GABAergic interneurons, which in turn synapse onto the quiescent pyramidal neurons. This results in GABA-A-mediated inhibitory postsynaptic potentials in the pyramidal neurons. Alternately, direct activation of M1 ACh receptors on pyramidal neurons, activating inward rectifying K⁺ currents, may result in additional ACh-mediated inhibition (Figure 7A; Aitta-Aho et al., 2018; Pidoplichko et al., 2013; Unal et al., 2015). When BLA pyramidal neurons are already active due to strong excitatory input associated with aversive cues, M1 receptor activation can result in long afterdepolarizations that produce persistent firing lasting as long as ACh is present (Jiang et al., 2016; Unal et al., 2015). Such a process may explain persistent firing observed in single neuron responses to aversive social vocalizations in bats (Gadziola et al., 2012; Peterson and Wenstrup, 2012). Through this process of inhibiting quiescent neurons and enhancing activation and persistent firing in active neurons, ACh sharpens the population signal-to-noise ratio (SNR) during the processing of salient, aversive signals in the BLA. These neurons, processing negative cues, likely project to the central nucleus of the amygdala (CeA) to regulate defensive behaviors such as escape and avoidance (Figure 7A; Beyeler et al., 2016; Davis et al., 2010; Namburi et al., 2015). In agreement, our behavioral findings show increased behaviors such as flinching along with increased release of ACh during processing of aversive vocalizations. Such prolonged afterdepolarizations provide the appropriate condition for associative synaptic plasticity (Likhtik and Johansen, 2019) and underlie an increase in the ratio of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor current to N-methyl-D-aspartate (NMDA) receptor current among CeA-projecting neurons during processing of aversive cues.

Dopaminergic innervation from the ventral tegmental area (Asan, 1998) acts on BLA neurons via D1 and D2 receptors, both G-protein-coupled receptors. DA is important in reward processing, fear extinction, decision making, and motor control (Ambroggi et al., 2008; Di Ciano and Everitt, 2004; Lutas et al., 2019). We observed increased DA release in the BLA in EXP groups in response to mating vocalizations both for males and for females across estrous stages. Electrophysiological studies show that DA enhances sensory processing in BLA neurons by increasing the population response SNR in a process like ACh (Kröner et al., 2005; Vander Weele et al., 2018). Thus, during processing of mating vocalizations or those related to other rewarding experiences, DA presence in the vicinity of BLA pyramidal neurons and interneurons is enhanced (Figure 7B). For neurons with elevated spiking activity during processing of appetitive vocalizations or other sensory stimuli, DA acts on D2 receptors of pyramidal cells to further enhance neuronal firing and result in persistent firing of these projection neurons. Conversely, in BLA projection neurons that do not respond to such positive cues, such as CeA-projecting neurons, DA exerts a suppressive effect directly via D1 receptors and indirectly by activating inhibitory interneuron feedforward inhibition (Kröner et al., 2005). The net result of this process in response to appetitive vocalizations is an enhancement of activity in the reward-responding neurons and suppression of activity in aversive-responding neurons. This process likely depends on the increase in synaptic plasticity via an enhanced ratio of AMPA receptor current to NMDA receptor current during processing such cues in NAc-projecting neurons in the BLA (Namburi et al., 2015; Otani et al., 2003; van Vugt et al., 2020). Our findings suggest that this may occur in the BLA in response to appetitive vocalizations.

As the results with males listening to restraint vocalizations demonstrate, increased ACh release in BLA is associated with processing aversive cues. How, then, should the increased ACh release in estrus females during mating vocal playback be interpreted? Previous work shows that neuromodulation of amygdalar and other forebrain activity is altered by sex hormone/receptor changes in males and females (Egozi et al., 1986; Kalinowski et al., 2023; Kirry et al., 2019; Matsuda et al., 2002; Mizuno et al., 2022; van Huizen et al., 1994). For instance, the cholinergic neurons that project to the BLA, originating in the basal forebrain, exhibit high expression of estrogen receptors that is influenced by a female’s hormonal state (Shughrue et al., 2000). During estrus, the enhanced circulating estrogen affects release of ACh and may influence neuronal networks and behavioral phenotypes in a distinct manner (Gibbs, 1996; McEwen, 1998). Thus, increased ACh release in estrus females may underlie increased attentional and risk-assessment behaviors in response to vocalization playback, as we observed with flinching behavior in this experimental group. Combined with DA increase, it may trigger both NAc and CeA circuit activation, resulting in both reward-seeking and cautionary behaviors in estrus females.

Our results demonstrate the strong impact of even limited experience in shaping behavioral and neuromodulatory responses associated with salient social vocalizations. In the adult mice, a single 90-min session of mating and of restraint, occurring 4–5 days prior to the playback experiment, resulted in consistent behavioral responses and consistent and enhanced ACh release into BLA for both vocalization types. As previous work shows (Huang et al., 2012; Nadim and Bucher, 2014; Pawlak et al., 2010), neuromodulatory inputs play crucial roles in regulating experience-dependent changes in the brain. However, it remains unclear whether the experience shapes neuromodulator release, or whether neuromodulators deliver the experience-related effect into the BLA. It is also unknown whether these experiences were specific to the type of vocalization playback associated with the experience, or whether either experience had a more generalized effect on responses to all vocalization playback, as suggested by the broad physiological effects of mating or restraint (Leuner et al., 2010; Arnold et al., 2019).

The interaction between ACh and DA is thought to shape motor responses to external stimuli (Lester et al., 2010). Our results support the view that a balance of DA and ACh may regulate the proper behavioral response to appetitive and aversive auditory cues. For instance, increased reward-seeking behavior (rearing and locomotion) in EXP males during mating vocalizations playback may result from the differential release of the two neuromodulators—decreased ACh and increased DA. Furthermore, the lack of this differential release may be the underlying cause for the lack of such responses in INEXP male mice. This supports the role of experience in tuning interactions of these two neuromodulators throughout the BLA, for shaping appropriate behaviors. Overall, the behavioral changes orchestrated by the BLA in response to emotionally salient stimuli are most likely the result of the interaction between previous emotional experiences, hormonal state, content of sensory stimuli, and sex of the listening animals.