Behavioral / microdialysis experiments test how playback of affective vocal signals alters behaviors and neuromodulator release into the BLA.

A. Short sample of mating vocal sequence used in playback experiments. Recording was obtained during high-intensity mating interactions and included ultrasonic vocalizations (USVs), likely emitted by the male, as well as low frequency harmonic (LFH) calls likely emitted by the female. B. Short sample of restraint vocal sequence used in playback experiments. Recording was obtained from an isolated, restrained mouse (see Material and Methods) and consisted mostly of mid-frequency vocalization (MFV) syllables. C. Syllable types in mating playback sequences differ substantially from those in restraint playback sequences. Percentages indicate frequency-of-occurrence of syllable types across all examplars used in mating or restraint vocal stimuli (nMating = 545, nRestraint = 622 syllables). D. Experimental design in playback experiment. Days 1 and 2: each animal experienced restraint and mating behaviors (counterbalanced order across subjects). Day 2: a microdialysis guide tube was implanted in the brain above the BLA. Day 6: the microdialysis probe was inserted through guide tube into the BLA. Playback experiments began after several hours of habituation/equilibration. Behavioral observations and microdialysis sampling were obtained before, during, and after playback of one vocalization type. Microdialysis samples were analyzed using LC/MS method described in Materials and Methods. E. Detailed sequencing of vocal stimuli, shown here for mating playback. A 20-min period of vocal playback was formed by seven repeated stimulus blocks of 170 s. The stimulus blocks were composed of five vocal exemplars of variable length, with each exemplar followed by an equal duration of silence. Stimuli during the Stim1 and Stim 2 playback windows thus included identical blocks but in slightly altered patterns.

Behavioral and neuromodulator responses to vocal playback in male mice differ by behavioral context of vocalizations.

A, B. Boxplots show number of occurrences of specified behavior in 10-min observation periods before (Pre-Stim) and during (Stim 1, Stim 2) playback of mating or restraint vocal sequences (nMating = 7, nRestraint = 6). Note that playback sequences during Stim 1 and Stim 2 periods were identical within each group. During playback, the restraint group increased still-and-alert behavior compared to the mating group (A) (context: F (1,11) = 9.6, p=0.01, ŋ2=0.5), and increased flinching behavior (B) (time*context: F (1.1,12.2) =6.3, p=0.02, ŋ2=0.4) compared to the Pre-Stim baseline and to the mating group. C, D. Boxplots show differential release of acetylcholine (ACh) or dopamine (DA), relative to the Pre-Stim period, during mating and restraint vocal playback (nMating = 9, nRestraint = 7). C. Significant differences in ACh for restraint (increase) playback in Stim 1 and Stim 2 vs Pre-Stim baseline and vs males in mating playback (time*context: F(2,28)=6.6,p=0.004, ŋ2=0.32). D. Significant differences for DA for mating (increase) over time and vs restraint playback (time*context: F(2,28)=6.9,p=0.004, ŋ2=0.33). E. Across EXP male subjects in vocal playback, the number of flinching behaviors during Stim 1 was positively correlated with changes in ACh concentration relative to the Pre-Stim period. (n=13, Pearson r=0.6, p=0.03). A-D. Statistical testing examined all time windows within groups and all intergroup comparisons within time windows. Only significant tests are shown. Repeated measures GLM: *p<0.05, **p<0.01, ***p<0.001 (Bonferroni post hoc test). Time windows comparison: 95% confidence intervals. See Data Analysis section in Materials and Methods for description of box plots.

Behavioral and neuromodulator responses to playback of mating vocal sequences differ by sex and female estrous stage.

A-D. Occurrences of specified behaviors in 10-min periods before (Pre-Stim) and during (Stim 1, Stim 2) mating vocal playback (nMale = 7, nEstrous Fem = 6, nNon-estrous Fem = 5). A. Attending behavior increased during Stim 1 in response to mating vocal playback, regardless of sex or estrous stage (time*sex: F (2,30) = 0.12; p=0.9, ŋ2=0.008; time*estrous: F (2,30) = 1.1; p=0.4, ŋ2=0.07). B-C. Females regardless of estrous stage reared less (sex: F (1,15) =10.22, p=0.006; ŋ2=0.4; estrous: F (1,15) =0.2, p=0.7, ŋ2=0.01) and displayed more Still-and-Alert behaviors (Sex: F (1,15) =5.17, p=0.04, partial ŋ2= 0.3, estrous: (1,15) =0.07, p=0.8, partial ŋ2= 0.005) than males during mating vocal playback D. Estrus females, but not non-estrus females or males, showed a significant increase in flinching behavior during Stim 1 and Stim 2 periods (time*estrous: F (2,30) =9.0, p=0.001, ŋ2=0.4). E, F. Changes in concentration of acetylcholine (ACh) or dopamine (DA) relative to the Pre-Stim period, evoked during Stim 1 and Stim 2 periods of vocal playback (nMale = 9, nEstrus Fem = 8, nNon-estrus Fem = 7). E. Release of ACh during mating playback increased in estrus females (Stim1, Stim 2) but decreased in males and non-estrus females (Stim 2). Among groups, there was a significant estrous effect (time*estrous: F (2,42) =10.0, p<0.001, ŋ2=0.32), increasing above Pre-Stim period in estrus females. F. DA release during mating playback increased in all groups relative to Pre-Stim period (time: F (2,42) = 12.4, p<0.001, ŋ2=0.4), with no significant sex (F (1,21) =0.2, p=0.6, ŋ2=0.01) or estrous effect (F (1,21) =0.8, p=0.4, ŋ2=0.04). G. Across EXP subjects responding to mating playback, number of flinching behaviors during Stim 1 was positively correlated with change in ACh concentration, relative to Pre-Stim period (n=18, Pearson r=0.56, p=0.02). A-F: Repeated measures GLM: *p<0.05, **p<0.01, ***p<0.001 (Bonferroni post hoc test). Time windows comparison: 95% confidence intervals.

A single bout each of mating and restraint experience altered behaviors in response to vocal playback.

In all graphs, dots represent measures from individual animals obtained during the Stim 1 playback period; connected thick lines represent mean values across subjects. A. Experience increased flinching responses in estrus female mice but not in males in mating or restraint vocal playback (Females: nEXP = 6, nINEXP = 8, t=5.0, p<0.001; Male-mating: nEXP = 8, nINEXP = 7, t=0.4, p=0.7; Male-restraint: nEXP = 7, nINEXP = 7, t=0.6, p=0.6); INEXP male mating vs restraint: t=2.08, p=0.06; mating female vs male: t=0.8, p=0.4; EXP male mating vs restraint: t= 2.5, p=0.03; mating female vs male: t=4.3, p<0.001. B. Experience increased rearing responses in males exposed to mating playback (Male-mating: nEXP = 8, nINEXP = 7, t=3.2, p=0.007), but not in females (t=1.4, p=0.2) or males in restraint playback (t=0.4, p=0.7); INEXP male mating vs restraint: t=1.07, p=0.3, mating female vs male: t=1.7, p=0.1; EXP male mating vs restraint: t=2.0, p=0.08; mating female vs male: t=3.4, p=0.004. GLM repeated measures with Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001.

In INEXP mice, vocal playback failed to evoke significant changes in neuromodulator release.

Boxplots show change in concentration of the specified neuromodulator in 10-min playback periods (Stim 1, Stim 2) compared to the baseline level (Pre-Stim). A. ACh release shown for both playback periods (mating vs restraint: time*context: F (2,24) =0.56; p=0.6; male vs female: time*sex: F (2,24) =0.01; p=0.9). B. DA release shown for both playback periods (mating vs restraint: time*context: F (2,24) = 0.2; p=0.8; male vs female: time*sex: F (2,24) = 0.4; p=0.7). A, B. Statistical testing examined all time windows within groups and all intergroup comparisons within time windows. No tests were significant. Repeated measures GLM (Bonferroni post hoc test). Time windows comparison: 95% confidence intervals.

Proposed model for neuromodulation of salient vocalization processing via acetylcholine (ACh) and dopamine (DA) in the basolateral amygdala (BLA).

A. Cholinergic modulation of CeA-projecting neurons during aversive vocalization cue processing in the BLA. In the presence of aversive cues, ACh released from the basal forebrain acts on m1 ACh receptors (m1AChRs) to enhance the cue-induced excitatory responses of CeA-projecting neurons. In contrast, NAc-projecting neurons are quiescent, because they do not respond to aversive cues and are inhibited by interneurons that are activated through nicotinic ACh receptors (nAChRs). B. Dopaminergic modulation and enhancement of signal-to-noise ratio in response to reward-associated cues (appetitive vocalizations). When vocalizations or other rewarding cues are present, release of DA from VTA enhances D2R-mediated excitation in NAc-projecting neurons that are responsive to positive cues. In contrast, CeA-projecting neurons are not responsive to rewarding vocalizations and are inhibited by local interneurons. DA is thought to act on D1 DA receptors (D1Rs) in these local interneurons to shape a direct inhibition onto CeA-projecting neurons. Abbreviations: ACx: auditory cortex; BF, basal forebrain; CeA, central nucleus of amygdala; MGB, medial geniculate body; NAc, nucleus accumbens; VTA, ventral tegmental area.

Classification of behaviors during playback of vocalizations

Microdialysis probe locations for EXP and INEXP Groups.

Colored lines indicate recovered probe tracks that resulted from infusion of fluorescent tracers. Black solid lines indicate external capsule; black dashed lines indicate major amygdalar subdivisions. Inset: photomicrograph shows dextran-fluorescein labeling that marks the placement of the microdialysis probe. Abbreviations: B, basal nucleus of amygdala; CeA, central nucleus of amygdala; La, lateral nucleus of amygdala.

Concentration of serotonin metabolite 5-HIAA in EXP animals showed no significant context-, sex-, estrous-, or experience-dependent changes with vocal stimulus playback.

Boxplots show change in concentration of 5-HIAA in 10-min periods from the baseline level (Pre-Stim) during (Stim 1, Stim 2) vocal sequence playback. A. Comparison of mating and restraint groups of male mice (n Mating=9, n Restraint=7; time*context: F (2,28) = 0.97, p=0.4, ŋ2 =0.06). B. Comparison of male and female mice during mating sequence playback (nMale = 9, nEstrous Fem = 8, nNon-estrous Fem = 7, time*sex: F (2,42) = 0.2, p=0.8, ŋ2 =0.01; time*estrus: F (2,42) =1.2, p=0.3, ŋ2 =0.05). C. 5-HIAA concentration shown for both playback periods (mating vs restraint: time: F (1.3,15.6) =0.005, p=0.9; time*context: F (1.3,15.6) = 2.1; p=0.17; male vs female: time: F (1.3,15.7) =0.9; p=0.4; time* sex: F (1.3,15.7) = 0.3; p=0.7). nINEXP = 21. A-C. Statistical testing examined all time windows within groups and all intergroup comparisons within time windows. No tests were significant. Repeated measures GLM: ns: non-significant (Bonferroni post hoc test). Time windows comparison: 95% confidence intervals.