Research report
Differential expression of the immediate early genes FOS and ZENK following auditory stimulation in the juvenile male and female zebra finch

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Abstract

The brains of adult zebra finches (Taeniopygia guttata) are tuned to the songs of conspecifics. In adult males, the caudomedial neostriatum (NCM) responds to zebra finch song, and in adult females the NCM and hippocampus (HP) are active following exposure to zebra finch song more than other auditory stimuli. The caudal hyperstriatum ventrale (cHV) in both sexes also responds to song, but in females not as selectively as the NCM and HP. While much is known about the adult perceptual circuit, less is known about its development. The present study exposed d30 male and female zebra finches to conspecific or heterospecific song, tones or silence, and examined the densities of FOS- and ZENK-immunoreactive nuclei in the NCM, cHV and HP. Significant interactions existed between sex and auditory stimulus condition for both immediate early genes, but they were in opposite directions. That is, across the three regions, FOS-immunoreactive neurons were increased in females following presentation of conspecific songs; males did not show an effect of stimulus exposure. In contrast, the density of ZENK-positive neurons was increased in males, but not females, following zebra finch song exposure. The FOS results demonstrate that some neural responses required for song perception may develop earlier in females than males; data on ZENK induction suggest the opposite. Overall, differences in juvenile immediate early gene activation suggest either that males and females employ divergent neural mechanisms for song perception or that the developmental trajectories leading to common neural responses differ.

Introduction

In adulthood, regions of the brains of zebra finches (Taeniopygia guttata) are fine-tuned to respond to the songs of their own species. Adult males that perceive their own song or other males’ songs show upregulation of the immediate early genes FOS and/or ZENK in the caudomedial neostriatum (NCM) and caudal hyperstriatum ventrale (cHV) [4], [8], [13], [16], [17], [18], [19], [24]. In NCM, heterospecific song presentations do not provoke such strong ZENK responses as conspecific song [16]; this induction by zebra finch song is qualitatively similar in males and females [16]. Similarly, a higher density of FOS-immunoreactive nuclei is seen in the NCM of females following stimulation with conspecific song compared to heterospecific song [2]. Interestingly, this pattern is also detected in the hippocampus (HP). FOS activation is equivalent in cHV following zebra finch and non-zebra finch song, yet both are increased compared to presentations of tone and silence [2].

Although much is known about the neural substrates of auditory perception in adult songbirds, less is known about its development. The expression of one immediate early gene, ZENK, has been quantified in juvenile zebra finches, but in NCM only [9], [24]. While an inducible ZENK response is not detected at post-hatching day 20 (d20), expression at d30 is significantly increased relative to a silence control following both conspecific and heterospecific song but not tones [9], [24]. In fact, the heterospecific response to song at this age can be considerably higher than in adults [24]. Juvenile males raised with their siblings and mother but no exposure to adult song show a similar ZENK induction to animals reared normally; birds reared with their mother only show ZENK induction to song similar to basal levels [9]. Electrophysiological specificity for conspecific song does exist in NCM at d30 and does not differ between the sexes. However, it appears that males and females were not considered separately in the parallel analysis of ZENK induction [24]. These results suggest that song-selective genomic responses in NCM, at least, become fine-tuned with developmental experience. In order to obtain a more complete understanding of the neural responses involved in male and female zebra finch perceptual development, it is important to investigate immediate early gene responses elicited by auditory stimulation in more detail.

The following experiment was conducted to determine whether in response to song exposure: (1) sex differences in juvenile immediate early gene expression exist; (2) FOS and ZENK are differentially expressed; and (3) the pattern of immediate early gene immunoreactivity in developing male and female zebra finches is similar to that observed in adults. Neuronal activation was assessed at d30 because, in addition to allowing comparisons to prior work (see above), the age is critical for song learning in both sexes. That is, it is during the period in which males form templates of their fathers’ songs, but before they produce their own [22], and the experience of female zebra finches at this developmental stage is critical for later responses to song. They do not show the normal choice for their father’s song over that of other males if isolated from song at d25 [5], whereas isolation at d35 does not impact their preference [5], [20].

Section snippets

Animals

Juvenile male and female zebra finches (d30; day of hatch equals d1; see below for sample sizes) were obtained from the breeding colony at Michigan State University. Animals were housed in communal aviaries consisting of approximately seven breeding pairs and their young. Free access to seed and water was provided along with once-weekly supplements of spinach or oranges and a mixture of hard-boiled eggs and bread. All stimulus exposures were done during the light portion of the light/dark cycle

Results

The most striking results were interactions between sex and stimulus type that were in opposite directions for the two immediate early genes: the induction of FOS and ZENK differed in males and females (Fig. 1). For FOS (interaction: F=4.47, p=0.008), females (F=3.37, p=0.035), but not males (F=2.01, p=0.138), showed a significant effect of stimulus exposure. For ZENK (F=2.85, p=0.049), the effect of stimulus exposure was marginally significant in males (F=3.05, p=0.050) but not in females (F

Summary

The brains of juvenile (d30) male and female zebra finches respond differently to auditory stimuli. Juvenile females show specificity for zebra finch song across the NCM, cHV and HP via FOS expression but not ZENK, whereas the same areas in males show specificity for zebra finch song as measured by ZENK but not FOS expression. Although direct comparisons to adult data cannot be made because tissue was not processed concurrently, the patterns observed in the present and previous studies suggest

Acknowledgements

The authors thank David McFarlane for technical assistance, Stephanie Fuerhing and Katie Grausam for help with tissue collection and processing, and Tomoko Hattori for bird care. The FOS antibody was generously supplied by Dr Els D’Hondt from the laboratory of Dr Frans Vandesande, Catholic University of Leuven, Belgium. This work was supported by NIH grant MH55488 (JW).

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