Reproductive state modulates testosterone-induced singing in adult female European starlings (Sturnus vulgaris)

Abstract

European starlings (Sturnus vulgaris) exhibit seasonal changes in singing and in the volumes of the neural substrate. Increases in song nuclei volume are mediated at least in part by increases in day length, which is also associated with increases in plasma testosterone (T), reproductive activity, and singing behavior in males. The correlations between photoperiod (i.e. daylength), T, reproductive state and singing hamper our ability to disentangle causal relationships. We investigated how photoperiodic-induced variation in reproductive state modulates the effects of T on singing behavior and song nuclei volumes in adult female starlings. Female starlings do not naturally produce measureable levels of circulating T but nevertheless respond to exogenous T, which induces male-like singing. We manipulated photoperiod by placing birds in a photosensitive or photorefractory state and then treated them with T-filled or empty silastic implants. We recorded morning singing behavior for 3 weeks, after which we assessed reproductive condition and measured song nuclei volumes. We found that T-treated photosensitive birds sang significantly more than all other groups including T-treated photorefractory birds. All T-treated birds had larger song nuclei volumes than with blank-treated birds (despite photorefractory T-treated birds not increasing song-rate). There was no effect of photoperiod on the song nuclei volumes of T-treated birds. These data show that the behavioral effects of exogenous T can be modulated by reproductive state in adult female songbirds. Furthermore, these data are consistent with other observations that increases in singing rate in response to T are not necessarily due to the direct effects of T on song nuclei volume.

Keywords: Birdsong; Enkephalin; Female songbird; HVC; Neuroplasticity; Photoperiodism; Song; Testosterone.

Figure 1

Sound spectrographs of T-treated adult female starling songs. The x-axis represents time and the y-axis frequency. Amplitude is represented by the brightness of the vocalization (dark = low amplitude, bright = high amplitude). Displayed are songs from two different T-treated females in two different photoperiodic conditions taken from the same day of study, A) a photosensitive T-treated female starling and B) a T-treated photorefractory female starling. As illustrated, starling song is a complex arrangement of harmonically variable vocalizations and though rich in acoustic diversity the various syllable phrases that make up starling song can be characterized into one of four motif or phrase types. The labeled boxes highlight exemplars of the four phrase types; whistle, warble/variable, rattle, and high frequency phrases.

Figure 2

Bar graph illustrating the mean number of songs produced per 2 hour recording session. Black bars represent T-treated photosensitive birds. Dark gray bars represent photosensitive control birds. Light gray bars represent T-treated photorefractory birds. White bars represent photorefractory controls. In general T was much more effective in inducing high rates of singing in photosensitive than in photorefractory female starlings. See results section for more detail.

Figure 3

Bar graphs illustrating the analysis of song repertoire in the photosensitive and photorefractory T-treated female starlings. A) Differences in the relative repertoire size in the T-treated photosensitive (black bars) vs the photorefractory females (gray bars). Too few blank-treated females sang to perform this analysis in the control birds. B) Mean number of phrases of each of the four types defined in figure 1 in the T-treated photosensitive (black bars) vs the photorefractory females (gray bars).

Figure 4

Bar graphs illustrating different measures of the effectiveness of T-treatment. A) Concentrations of T in the blood based on measurements via RIA on samples collected 3 weeks after implantation just prior to brain collection. T-treated birds had significantly higher T than blank-treated birds. T was not different between T-treated photosensitive (black bars) and T-treated photorefractory (gray bars) females. B) Bar graphs illustrating the mean beak score for females in all the groups. The higher the score the higher the percentage of the beak that is yellow in color rather than black. The beak becomes fully yellow eventually if T is present in the blood. Photosensitive (black bars) T-treated birds had beaks that had a much higher percentage covered in yellow than photorefractory T-treated birds or the blank-treated control groups. See text for more details.

Figure 5

Bar graph illustrating the mean ovary and oviduct mass for all the experimental groups. Photosensitive females not treated with T (dark gray bars) had the largest ovary mass.

Figure 6

Photomicrographs of the regions of interest and bar graphs illustrating the mean volume of 4 key forebrain song nuclei in the females in all 4 experimental groups. Representative photomicrographs of the staining quality show the regions of interest for volume reconstruction, namely, song nuclei A) Area X, B) LMAN, C) HVC, and D) RA. E) Bar graphs show that T-treatment resulted in a marked increase in the volume of all four nuclei in both the photosensitive and photorefractory birds compared with blank-treated birds. There were no significant differences in the mean volume of the song nuclei between the T-treated photorefractory and the T-treated photosensitive birds. In the case of HVC and RA nucleus volume was larger in the photosensitive blank-treated control birds than in the photorefractory blank-treated birds. Black bars represent T-treated photosensitive birds. Dark gray bars represent photosensitive control birds. Light gray bars represent T-treated photorefractory birds. White bars represent photorefractory control birds. Abbreviations: HVC used as its proper name; LMAN, lateral magnocellular nucleus of the anterior nidopallium; RA, robust nucleus of the arcopallium.