Social context-induced song variation affects female behavior and gene expression

Abstract

Social cues modulate the performance of communicative behaviors in a range of species, including humans, and such changes can make the communication signal more salient. In songbirds, males use song to attract females, and song organization can differ depending on the audience to which a male sings. For example, male zebra finches (Taeniopygia guttata) change their songs in subtle ways when singing to a female (directed song) compared with when they sing in isolation (undirected song), and some of these changes depend on altered neural activity from a specialized forebrain-basal ganglia circuit, the anterior forebrain pathway (AFP). In particular, variable activity in the AFP during undirected song is thought to actively enable syllable variability, whereas the lower and less-variable AFP firing during directed singing is associated with more stereotyped song. Consequently, directed song has been suggested to reflect a "performance" state, and undirected song a form of vocal motor "exploration." However, this hypothesis predicts that directed-undirected song differences, despite their subtlety, should matter to female zebra finches, which is a question that has not been investigated. We tested female preferences for this natural variation in song in a behavioral approach assay, and we found that both mated and socially naive females could discriminate between directed and undirected song-and strongly preferred directed song. These preferences, which appeared to reflect attention especially to aspects of song variability controlled by the AFP, were enhanced by experience, as they were strongest for mated females responding to their mate's directed songs. We then measured neural activity using expression of the immediate early gene product ZENK, and found that social context and song familiarity differentially modulated the number of ZENK-expressing cells in telencephalic auditory areas. Specifically, the number of ZENK-expressing cells in the caudomedial mesopallium (CMM) was most affected by whether a song was directed or undirected, whereas the caudomedial nidopallium (NCM) was most affected by whether a song was familiar or unfamiliar. Together these data demonstrate that females detect and prefer the features of directed song and suggest that high-level auditory areas including the CMM are involved in this social perception.

Figure 1. Diagram of Behavioral Assay and Sonograms Illustrating Song Differences between and within Males

(A) Diagram of the two-choice behavior assay. Tests began when females were in the center chamber containing food, water, and a perch. Stimuli were played alternately from speakers at each end of the cage, and females moved freely into the side chambers. Thick lines in the side and center chambers indicate perches. (B) Examples of a directed song bout from an unfamiliar male and directed and undirected song bouts from a mate. Introductory notes are indicated with open lines, and motifs are indicated with black lines. In these examples, there are more motifs in both of the directed songs (unfamiliar and mate's song) than the undirected song. (C) Sonograms of single motifs from each of the song examples. These examples highlight the much greater differences between the songs of different birds (e.g., unfamiliar male versus mate) than between songs produced in the two contexts by the same bird (mate's directed versus mate's undirected).

Figure 2. Females Prefer the Directed Songs of Their Mate

(A) Mean percent of time spent in the unfamiliar directed and mate's directed song chambers. Individuals are plotted as diamonds. Females spent significantly more time in the mate's directed song chamber (*p < 0.05). (B) Mean percent of time spent in the mate's undirected and directed song chambers. Individuals are plotted as diamonds. Females spent significantly more time in the directed song chamber (*p < 0.05).

Figure 3. Mated and Naive Females Prefer Unfamiliar Directed Songs to Unfamiliar Undirected Songs

(A) Mean percent of time that mated females spent in the unfamiliar undirected and directed song chambers for the four behavior tests on which females displayed a preference. Females significantly preferred unfamiliar directed to undirected songs (*p < 0.05). (B) Mean percent of time that naive females spent in the unfamiliar undirected and directed song chambers. Seven females showed significant preferences for at least one of the stimuli. Because some females responded to stimuli from more than one male, diamonds represent the average response for each female. Naive females significantly preferred directed song to undirected song (*p < 0.05). (C) Mated females tested on their mate's songs spent significantly more time in the directed song chamber (*p < 0.05) than did naive or mated females tested on unfamiliar songs. (D) Number of undirected and directed song bouts produced over a 6-h period by males housed with their mates. While directed song bouts tended to be longer and accompanied by additional courtship behaviors and copulation attempts, males sang significantly more undirected song bouts (*p < 0.05).

Figure 4. Song Variability and Singer Identity Influence Preferences for Directed Song

(A) Plot of the correlation between the percent of time females spent in the directed chamber and the percent difference between the directed and undirected stimuli in the CV of the fundamental frequency of harmonic stacks. When the CV was higher in directed songs than in undirected songs, the preference for directed song was weaker than when the CV was higher in undirected songs. (B) Whether females responded to unfamiliar songs depended on which male the songs came from. Graphed is the number of naive (gray bars) and mated (open bars) females that showed a preference for the directed songs of each of the six unfamiliar males. Overall, there was a significant effect of stimulus male on the responses of naive females (p < 0.05) but not a significant effect on the responses of mated females.

Figure 5. The Number of ZENK-Expressing Cells in the MLd Is Equally Increased by All Stimuli

(A) Diagram of the region sampled within the MLd. Dashed line indicates the area shown in the images in (B). DM, dorsomedial nucleus of the intercollicular complex; OT, optic tectum. (B) Representative images of ZENK staining in the MLd in each of the stimulus groups. (C) Mean number of cells per area in the MLd. There were no significant differences between any of the groups in the number of ZENK expressing cells.

Figure 6. The Number of ZENK-Expressing Cells in the NCM Is Preferentially Increased by Unfamiliar Stimuli

(A) Diagram of the regions sampled within NCM. HP, hippocampus; NCMc, caudal region of NCM; NCMv, ventral region of NCM. Both regions were similar to those used in ZENK cell counts in NCM in other studies (see Methods). (B) Representative images of ZENK staining in NCM (caudal region) in each of the stimulus groups. (C) Mean number of cells per area in the caudal and ventral regions of the NCM. Females that heard unfamiliar directed songs had significantly more ZENK-expressing cells than females that heard their mate's directed or undirected songs. (*p < 0.05).

Figure 7. The Number of ZENK-Expressing Cells in the CMM Is Preferentially Increased by Directed Stimuli

(A) Diagram of the region sampled within CMM. HP, hippocampus. (B) Representative images of ZENK staining in CMM in each of the stimulus groups. (C) Mean number of cells per area in the CMM. Females that heard their mate's undirected songs had significantly fewer ZENK-expressing cells than females that heard their mate's directed songs or unfamiliar directed songs (*p < 0.05).