FOS and ZENK responses in 45-day-old zebra finches vary with auditory stimulus and brain region, but not sex

Abstract

Male zebra finches (Taeniopygia guttata) begin to sing around 45 days posthatch (d45) and tune their songs to match learned templates. Females never develop song, but they use male conspecific vocalizations for mate choice. While auditory perception is critical for both sexes, the responses of the immediate early genes (IEGs) ZENK and FOS differ in auditory brain areas of d30 males and females. The present study examined expression of these IEGs in the caudomedial nidopallium (NCM), caudomedial mesopallium (CMM; formerly cHV), and the hippocampus (HP) in both sexes at d45 in response to conspecific, heterospecific, or no songs. Overall, zebra finch song presentations resulted in the highest density of ZENK and FOS cells in each region analyzed, but expression varied across brain areas. Contrary to d30 birds, the IEG response patterns did not differ between the sexes. ZENK-immunoreactivity was significantly increased following exposure to conspecific songs compared to no songs, and zebra finch song presentations produced more FOS-immunoreactive nuclei than both heterospecific and no songs. While the pattern was consistent, significant effects of stimulus type were seen only in the NCM when the brain regions were analyzed separately. Furthermore, levels of FOS- and ZENK-immunoreactive neurons were higher in the lateral than medial NCM in both sexes. Along with previous work from our lab and others, these data suggest that at d45 neuronal responses within perceptual regions are still maturing on some levels, but IEG expression has acquired a number of adult characteristics.

Introduction

Communication in zebra finches (Taeniopygia guttata) involves the production and perception of a variety of vocal signals by both sexes. The most widely studied of these vocalizations, male song, is learned during a sensitive period in development when, beginning at approximately post-hatch day 25 (d25), sons memorize characteristics of their fathers songs. Although vocalizations are made by male and female zebra finches shortly after hatching, particularly in the form of begging calls [49], males do not begin to sing their own song until around d45, at which point they actively rehearse it [21] to closely match that of their tutor (“sensorimotor integration”) [5], [13], [17], [32]. Sensorimotor integration can be delayed or modified by preventing males from making or hearing their own vocalizations [35], [39], [40]. Furthermore, isolation of zebra finch males prior to the sensitive period for song learning until adulthood results in the production of an abnormal song [13], [17]. Females may undergo a similar period for learning as well: acoustic isolation at d25 [10] eliminates their preference for a father's song over that of another conspecific male, whereas this preference is maintained if isolation begins at d35 [30].

These results point to the importance, in both sexes, of hearing song during development. Electrophysiological recordings and the measurement of immediate early gene (IEG) responses, like those of c-FOS and ZENK (an acronym for Zif268, Egr-1, NGF1-A, Krox-24), have revealed regions of the brain important for the perception of song. A variety of auditory stimuli elicit activity in brain regions of adult male and female songbirds outside of the regions that control song learning and production. One of these regions, the caudomedial nidopallium (formerly caudomedial neostriatum; NCM; see [36] for recent avian brain nomenclature changes), responds primarily to conspecific songs [1], [3], [6], [8], [9], [12], [15], [16], [18], [19], [20], [23], [26], [27], [28], [29], [31], [33], [34], [37], [42], [45], [47], [48]. Responses within another auditory perceptual region, the caudomedial mesopallium (CMM; formerly “cHV”), are more variable, but activation similar to that observed in NCM has been shown [1], [2], [3], [4], [6], [12], [14], [15], [16], [19], [22], [23], [24], [25], [26], [28], [29], [33], [34], [38], [45]. FOS and ZENK expression in the zebra finch hippocampus (HP) following conspecific song presentations has also been detected in adult and juvenile males and females [1], [2], [3], [4], [6], [22], [24]. However, other studies examining ZENK have reported little, if any, IEG activity in response to song stimulation in the HP [14], [20], [26], and electrophysiological responses to auditory stimuli have not been observed in the structure [7].

Although a large volume of data from many oscine species details neural responses to auditory stimuli in adulthood, few studies have examined responses to auditory stimuli in the developing zebra finch. In d20 males and females, no ZENK response in NCM is detected following conspecific song presentations relative to a silence control, whereas both conspecific and heterospecific songs (but not tones) induce ZENK expression at d30 [20], [41]. Interestingly, electrophysiological specificity for conspecific song does exist in NCM at d20 and d30 and does not differ between males and females. We have shown that the brains of d30 male and female zebra finches respond differently to auditory stimuli. Overall, across the NCM, CMM and HP, juvenile females show specificity for zebra finch song as measured by levels of FOS- but not ZENK-immunoreactive cells, whereas the same areas in males show specificity for zebra finch song with ZENK but not FOS expression [2].

Whether ZENK or FOS continue to be expressed in males and females, respectively, in response to conspecific songs, or whether both IEG responses become equivalent in the two sexes as the birds become adults was unknown. The one study to simultaneously examine IEG activity in adult male and female zebra finches documented only that no qualitative difference existed in ZENK expression in the NCM following conspecific song presentations [29]; the lack of a difference in the IEG response mirrors the equivalence in electrophysiological recordings from the NCM of adult males and females in response to novel conspecific songs [9]. The specificity of NCM neurons to conspecific songs in adult female zebra finches, measured by FOS-immunoreactivity [1], parallels the distribution of FOS expression in adult males following tutor song exposure [3], but novel conspecific songs were not tested in these males. Although direct comparisons of both IEGs in the two sexes are needed, these results suggest that neurons within auditory perceptual regions in adult male and female zebra finches respond to specific auditory stimuli with similar patterns. To further gauge whether song-selective genomic responses in these regions become fine-tuned with developmental experience, the present experiment examined IEG responses at d45 in male and female zebra finches. This point in development was chosen because it is a time when a clear functional difference emerges, in that males but not females are beginning to produce song.