Complex modulation of singing behavior by testosterone in an open-ended learner, the European Starling

In many temperate zone songbird species males only produce song during the breeding season, when plasma testosterone (T) levels are high. Males of some species sing throughout the year, even when T levels are low, indicating a dissociation between high T levels and song rate. Given that few studies have taken advantage of these species, we compare here song traits expressed under high versus low T concentrations and we study the role of testosterone in adult song learning in the European Starling, an open-ended learner in which repertoire size dramatically increases with age. We performed a detailed comparison of song complexity and song rate between fall and spring in 6-year-old intact male European starlings. In parallel, we investigated whether potential seasonal changes were regulated by the gonadally induced increase in plasma T, by comparing seasonal changes in intact and castrated males of the same age (castrated as juveniles during their first fall) and by subsequently experimentally elevating T in half of the castrated males. While song rate and stereotypy did not differ between intacts and castrates or between fall and spring, both groups increased their average song bout length from fall to spring, but only intact males increased their repertoire size, indicating that effects of seasonal T changes differ between song traits. Intact males overall displayed a larger song repertoire and a longer bout length than the castrates, and implantation with T caused a turnover in repertoire composition in castrates. However, as the castrates had never experienced high T levels and yet displayed a markedly higher repertoire size than that of typical yearling males, this suggests that the progressive increase of song repertoire with age in male starlings is not dependent on gonadal T, although it may be T-enhanced.     

Testosterone increases repertoire size in an open-ended learner: An experimental study using adult male European starlings (Sturnus vulgaris)

Song in songbirds is a learned secondary sexual behavior, first acquired during a sensitive phase of juvenile development, which is affected by hormones such as testosterone (T). While the latter has received much attention, the potential involvement of T in the adult repertoire changes observed in a number of species is much less understood. Yet, this may prove essential to understand the role of song as a sexually selected trait. We therefore performed a T-implantation experiment during the non-breeding season (when T is basal), using adult male European starlings (Sturnus vulgaris), a songbird species in which song repertoire size (and composition) changes seasonally and increases with age. Repertoire size increased rapidly in T-males, but not in control males, indicating a role for T in repertoire size changes. This increase resulted from a lower proportion of dropped song types in T-males than in control males, while the proportion of added song types did not differ between both groups. Interestingly, the observed repertoire turnover (adding and removing song types from the repertoire) in both groups, suggests that elevated plasma T levels were not essential for changes in repertoire composition (contrary to repertoire size). Finally, T-males (but not control males) significantly increased their song rate, while neither group showed a significant change in their song bout length and phrase repetition rate. Taken together, our results suggest a role for T in adult song learning and provide new insights into the information content of repertoire size and song bout length as sexually selected traits.     

Testosterone increases repertoire size in an open-ended learner: An experimental study using adult male European starlings (Sturnus vulgaris)

Song in songbirds is a learned secondary sexual behavior, first acquired during a sensitive phase of juvenile development, which is affected by hormones such as testosterone (T). While the latter has received much attention, the potential involvement of T in the adult repertoire changes observed in a number of species is much less understood. Yet, this may prove essential to understand the role of song as a sexually selected trait. We therefore performed a T-implantation experiment during the non-breeding season (when T is basal), using adult male European starlings (Sturnus vulgaris), a songbird species in which song repertoire size (and composition) changes seasonally and increases with age. Repertoire size increased rapidly in T-males, but not in control males, indicating a role for T in repertoire size changes. This increase resulted from a lower proportion of dropped song types in T-males than in control males, while the proportion of added song types did not differ between both groups. Interestingly, the observed repertoire turnover (adding and removing song types from the repertoire) in both groups, suggests that elevated plasma T levels were not essential for changes in repertoire composition (contrary to repertoire size). Finally, T-males (but not control males) significantly increased their song rate, while neither group showed a significant change in their song bout length and phrase repetition rate. Taken together, our results suggest a role for T in adult song learning and provide new insights into the information content of repertoire size and song bout length as sexually selected traits.     

Photoperiodicity and circannual levels of LH, FSH, and testosterone in normal and castrated male, white-tailed deer

To establish the relation between photoperiodicity and the levels of LH, FSH, and testosterone (T) in plasma, three intact and three castrated adult male white-tailed deer were sampled once a month for 2-3 years. The rang of average LH levels in controls varied between 0.8 and 2.0 ng/mL; the levels in castrates were considerably higher, 3.4 to 8.9 ng/mL. Average levels of FSH varied in controls between 25 and 112 ng/mL and in castrates between 141 and 240 ng/mL. A significant correlation between the seasonal time course of LH and FSH was found in castrated, but not in intact bucks. In castrates both gonadotropins exhibit two major elevations coinciding with spring and fall equinoxes in March and September. The seasonal time course of FSH in castrates correlates highly with seasonal levels of FSH in controls. However, the time course of the LH curve in controls is substantially different from the curve in castrates, presumably owing to feedback mechanisms. A possible role of testicular estradiol in this feedback is discussed. In controls, peak T levels are reached in December, i.e., 3 months after maximum levels of FSH and 5 months after peak levels of LH were detected. It appears that male deer undergo two periods of reproductive stimulation (one in the spring, the other in the fall). However, the organism responds with the full range of gonadal and behavioral mechanisms leading to the initiation of the rut only during the fall.     

Context-dependent effects of castration and testosterone treatment on song in male European starlings

Most seasonally breeding songbirds display dramatic seasonal fluctuations in plasma testosterone (T) levels and mate attraction behaviors, including song. However, males of some songbird species, such as the European starling (Sturnus vulgaris), continue to sing at high levels after the breeding season, when T levels are basal. In male starlings song during the breeding season functions mainly to attract mates, whereas song during the nonbreeding season appears unrelated to reproduction. This suggests that song produced in a context unrelated to female courtship, unlike song directed toward females, is not regulated by plasma T. In captive males housed in large outdoor aviaries we explored the relationship between plasma T and song produced during the breeding season within and outside a courtship context. This was achieved by determining the effects of castration and subsequent T treatment on song and mate attraction behaviors in both the presence and the absence of a female. Compared to intact males, castrated males did not show reduced song activity in the absence of a female for at least 6 months after the operation, strongly suggesting that the expression of noncourtship song is not regulated by plasma T. Likewise, we found that experimentally elevating T levels in castrated males did not affect noncourtship song rates. However, control castrated males receiving empty implants tended to show reduced noncourtship song rates after implantation. This may have been due to a suppressive effect caused by the presence of the T-implanted castrated males in the same aviary. In contrast, courtship singing was clearly controlled by plasma T: it was abolished by castration and restored by subsequent T replacement when males were housed both individually and in a group situation. High plasma levels of T also appeared necessary for the activation of three other behavioral traits critical for mate attraction, namely, nesthole occupancy, spending time (singing) in a nesthole, and carrying green nesting material into a nesthole.     

Seasonal Changes in Courtship Song and the Medial Preoptic Area in Male European Starlings (Sturnus vulgaris)

In male starlings (Sturnus vulgaris) courtship song plays a critical role in mate attraction. During the breeding season courtship song occurs prior to copulation and appears to reflect male sexual arousal. Outside the breeding season starlings sing, but song appears unrelated to reproduction. The aromatization of testosterone (T), likely within the medial preoptic nucleus (POM), is critical for the expression of male sexual arousal. The present study was performed to determine whether seasonal changes in the POM might relate to seasonal changes in courtship singing behavior in male starlings. T concentrations, the volume of the POM, and aromatase within the POM were examined both during and outside of the breeding season in male starlings. Song was also recorded at these times both with and without a female present. The POM was largest and contained dense aromatase immunostaining only during the spring breeding season, when T concentrations were highest and males responded to a female with an increase in courtship song. Outside the breeding season the volume of the POM was small, T concentrations were low, and males displayed no changes in song expression in response to female conspecifics. Song bout length was positively related to POM volume, and males sang longer songs in spring. Only males with nestboxes in spring responded to a female, and the POM tended to be larger in these males, suggesting that nestbox possession might influence neuroplasticity within the POM. Overall, the findings suggest that T-dependent plasticity and aromatase activity within the POM might regulate courtship singing in a wild songbird.     

Seasonal changes in courtship song and the medial preoptic area in male European starlings (Sturnus vulgaris)

In male starlings (Sturnus vulgaris) courtship song plays a critical role in mate attraction. During the breeding season courtship song occurs prior to copulation and appears to reflect male sexual arousal. Outside the breeding season starlings sing, but song appears unrelated to reproduction. The aromatization of testosterone (T), likely within the medial preoptic nucleus (POM), is critical for the expression of male sexual arousal. The present study was performed to determine whether seasonal changes in the POM might relate to seasonal changes in courtship singing behavior in male starlings. T concentrations, the volume of the POM, and aromatase within the POM were examined both during and outside of the breeding season in male starlings. Song was also recorded at these times both with and without a female present. The POM was largest and contained dense aromatase immunostaining only during the spring breeding season, when T concentrations were highest and males responded to a female with an increase in courtship song. Outside the breeding season the volume of the POM was small, T concentrations were low, and males displayed no changes in song expression in response to female conspecifics. Song bout length was positively related to POM volume, and males sang longer songs in spring. Only males with nestboxes in spring responded to a female, and the POM tended to be larger in these males, suggesting that nestbox possession might influence neuroplasticity within the POM. Overall, the findings suggest that T-dependent plasticity and aromatase activity within the POM might regulate courtship singing in a wild songbird.     

Effects of testosterone and photoperiodic condition on song production and vocal control region volumes in adult male dark-eyed juncos (Junco hyemalis)

In seasonally breeding male oscines, song learning and expression are controlled by brain regions (vocal control regions, VCRs) that exhibit seasonal neural plasticity in adulthood. Several VCRs contain androgen receptors, and gonadal androgens play important roles in the control of seasonal structural and functional changes of VCRs. Recent studies also found that adult VCRs are influenced by factors other than gonadal hormones, including photoperiod, but the relative importance of these factors and their mechanisms of action are poorly understood. To address this issue, we investigated the contributions of photoperiod and testicular androgens to the regulation of VCR volumes and to the control of song expression in adult dark-eyed juncos, Junco hyemalis. Exposing castrated (CX) photosensitive males to long days (LD) enhanced their high vocal center (HVc) volumes compared to those of males held on short days (SD). These volumes were not further increased by concurrent testosterone (T) treatment, revealing a marked and gonadal androgen-independent stimulatory influence of photoperiod on the size of this brain region. HVc sizes were smaller in LD-exposed photorefractory than photosensitive males irrespective of whether birds were intact or had been castrated before photoperiodic manipulations, but HVc sizes increased in response to T treatment in intact photorefractory males. Thus, LD exposure can increase HVc volumes in the absence of gonadal T, but large volume induction in photorefractory males requires elevated plasma T levels. Testosterone treatment of SD-exposed photosensitive males increased HVc, but not Area X, MAN, or RA volumes. Only T-treated males sang and this treatment given to castrates was equally effective behaviorally when administered to photosensitive, photostimulated, or photorefractory juncos. This result indicates that the stimulating influence of LD exposure on HVc volumes is insufficient to induce song in the absence of elevated plasma T levels.     

Age- and behavior-related variation in volumes of song control nuclei in male European starlings

There is considerable interindividual variation in the volumes of song control nuclei. Sex and physiological condition appear to contribute to these differences; however, these factors alone do not account for all of the variation. Studies have attempted to relate differences in song behavior (i.e., song repertoire size) to variation in song nucleus volume, but have met with mixed success. In this article, two studies are presented that used male European starlings (Sturnus vulgaris) to explore the relationship between song nuclei volumes and age-related differences in song behavior and interindividual variation in song behavior in adults. The results of the first study showed that song repertoire size and song bout length were significantly greater in older adult than in yearling males. In addition, the volumes of the high vocal center (HVC) and nucleus robustus archistriatalis (RA) were significantly larger in older adults than yearlings. Area X of the parolfactory lobe did not differ significantly in volume between the two age classes. In the second study, both HVC and RA volume correlated positively with song bout length but not repertoire size among adult birds. Based on these results a new hypothesis is presented that states that variation in song nuclei volumes in starlings relates more to the amount of song produced than to the number of song types stored in memory. © 1996 John Wiley & Sons, Inc.     

Song acquisition in photosensitive and photorefractory male European starlings

We tested the ability of 1-year-old European starlings (Sturnus vulgaris) to acquire songs while in different physiological states. Photorefractory males, with low testosterone levels, learned songs as completely and as accurately as photosensitive males in full reproductive condition. This indicates that song acquisition in 1-year-old males does not depend on high levels of androgens. The ability to learn songs during the nonbreeding season may reflect the high song output of potential tutors through most of the year, including the photorefractory period, and may facilitate increases in song repertoire size in adulthood.     

Testosterone-induced aggression in adult female mice

Silastic implants of testosterone (T) and injections of testosterone propionate (TP) were used to study the effects of ovariectomy and androgen administration on the fighting behavior of adult female mice. A dose of T previously shown to hyperstimulate accessory organ growth in adult male castrates was sufficient to induce the complete behavioral repertoire of male-like aggression in females never before treated with exogenous androgen. As determined by radioimmunoassay, blood levels of T produced by implants containing an aggression-inducing dose of T (10-mg implant) were within the range of T concentrations observed in intact males. Following treatment with a 10-mg T implant, the aggressive behavior of ovariectomized females could be fully maintained with a dose of T (0.3-mg implant) that failed to maintain weight of the accessory organs in adult male castrates. In fact, females “androgenized” were subsequently more responsive to the aggression-activating properties of T than were males castrated after prenatal and perinatal androgen exposure.     

Differential effects of testosterone plus dihydrotestosterone on male courtship of castrated newts, Taricha granulosa

Sexually responsive Taricha granulosa males were castrated and implanted with testosterone (T), dihydrotestosterone (DHT), T plus DHT, or no steroid. The incidence of sexual behavior was highest in castrates implanted with T plus DHT. Newts implanted with T or DHT exhibited sexual behavior more frequently than did the untreated castrates, which exhibited none during the last 5 days of testing. A second experiment was conducted using sexually unresponsive males. The occurrence of sexual behavior remained low in castrates implanted with T plus DHT, untreated castrates, and intact control males. These results support the hypothesis that for this species of amphibian the presence of adequate levels of testicular androgen is a necessary, but not sufficient, condition for the manifestation of sexual behavior; the appearance of these behaviors requires, in addition, the presence of some nontesticular hormone.     

Influences of Sex,Castration, and Androgens on the Eumelanin and Pheomelanin Contents of Different Feathers in Wild Mallards

In mallards the bright nuptial plumage of the drake represents the neutral, sex hormone-independent coloration of the species that both sexes eventually exhibit after castration. We compared the pheo- and eumelanin contents of feathers from the head, breast, flank, and under-tail coverts in five groups of mallards after the post-nuptial molt in summer: intact hens, intact drakes, castrated drakes, castrated drakes injected with testosterone during the spring, and castrated drakes injected with 5α-dihydrotestosterone during the spring. In the head feathers and under-tail coverts, the gonadal hormones of the intact birds and the testosterone injections into castrates significantly reduced the eumelanin content, tended to increase the pheomelanin content, and, thereby, changed the melanin type from eumelanic in the untreated castrates to mixed melanic in the other three groups. The eumelanin contents of the flank feathers did not differ among the groups, but the pheomelanin contents at this site was significantly elevated in the two intact groups and the testosterone-treated compared to the uninjected castrates. Again, the melanin type changed from eumelanic in the castrates to mixed melanic in the other three groups. The high pheomelanin content of the breast feathers in the castrated birds was significantly reduced in the hens, intact drakes, and testosterone-injected castrates with a concomitant tendency for elevated eumelanin contents. At this site, a change occurred from pheomelanic to mixed melanic. 5α-dihydrotestosterone was clearly less effective than testosterone in affecting the melanin contents in castrates and resulted in an intermediate coloration. The differing effects of the two androgens might be a result of differences in their conversion to estrogens.     

Hormonal regulation of male reproductive behavior in the lizard Anolis sagrei: a test of the aromatization hypothesis

This study examined the hypothesis that aromatization of testosterone (T) to estradiol (E) is required to activate reproductive behavior in castrated male lizards (Anolis sagrei). Adult, reproductively active males were assigned to an intact control group or to one of four treatment groups. Treatment males were castrated and 1 week later three of the four castrated groups were implanted with subcutaneous pellets containing either 0.05 mg of E, 0.5 mg of T, or 0.5 mg of dihydrotestosterone (DHT). Two weeks after pellet implantation, males were tested with stimulus males, and 2 days later were tested with stimulus females. Behavioral tests were of 15-min duration and were videotaped. Significantly fewer E-treated castrates erected a crest in tests with stimulus males than did intact males. In tests with stimulus females, significantly fewer E-treated castrates displayed, neck-gripped, and intromitted than did intact males. Estradiol-treated castrates also showed significantly less display behavior than did intact males. However, aggressive and sexual behavior of DHT-treated castrates was not significantly different from that of intact males. The same was true for T-treated castrates with the exception that display behavior in tests with stimulus females was reduced compared to that of intact males. The results suggest that aromatization of T to E is not required for induction of androgen-dependent reproductive behavior in this lizard.     

Hormone-induced changes in identified cell populations of the higher vocal center in male canaries

Male canaries revise their vocal repertoire every year. Early work indicated that the volume and neuron number of the song-control nucleus HVC (Higher Vocal Center) declined in late-summer/fall as birds added and deleted syllables from their repertoire, and increased in spring as the set of song syllables stabilized to a fixed number. Seasonal variation in serum testosterone levels suggested that these changes in brain and behavior were regulated by testosterone (T). However, although initial studies describing growth and regression of HVC used Nissl-staining to define its borders, recent experiments that have measured the distribution of identified populations of HVC cells (projection neurons, hormone target cells) suggest that there are no seasonal changes in HVC volume or neuron number. In order to clarify the role of T in the regulation of HVC morphology, we castrated male canaries, maintained them on short (fall-like) days, and treated them with either T, antisteroid drugs, or nothing. After 1 month of treatment, we used a double-labeling technique to characterize HVC projection neurons and androgen target cells. The results showed that hormonal manipulation influenced HVC volume, the density and size of HVC cells, and the absolute number and percentage of androgen target cells in HVC. Hormonal manipulation did not influence the absolute number of cells in HVC. Moreover, the distribution of projection neurons, androgen target cells, and the Nissl-defined borders of HVC were closely aligned in all experimental groups, indicating that exposure to T and/or its metabolites (estradiol and dihydrotestosterone) regulates the overall size of HVC by affecting the distributions of both projection neurons and androgen target cells. Analysis of double-labeling results suggests that T specifically influences both cell size and the ability to accumulate androgen among HVC neurons that project to the robust nucleus of the archistriatum (RA). The results of this study show that steroid hormones exert potent effects on HVC morphology in male canaries, but differences between our results and studies of seasonal males suggest there may be additional factors that can regulate HVC morphology. © 1993 John Wiley & Sons, Inc.     

No overlap in song repertoire size between yearling and older Starlings Sturnus vulgaris

We recorded the song of 25 male Starlings Sturnus vulgaris to investigate differences in song characteristics between yearling and older males. Older males had significantly larger repertoire sizes and sang significantly longer average song bouts than yearling males. Since older males had almost twice as large a repertoire size as yearling males and since there was no overlap in repertoire size between yearling and older males, our data indicate that the repertoire size of Starlings increases after the first year of life. The largest difference between yearling and older males occurred in the 'variable song types' which are the song types containing most of the heterospecific imitations sung in a song bout.     

Testosterone-induced Singing in Female European Starlings (Sturnus vulgaris)

Adult female starlings (Sturnus vulgaris) were injected with testosterone in order to determine the song structures they had in memory. Those females had been caught in the wild in different geographical areas and had different ages. This hormone treatment clearly stimulated singing behaviour of females in isolation and revealed their ability to sing quite complex songs. Both categories of songs observed in males (warbling and whistles) were demonstrated and large repertoires were observed. Although the testosterone treatment may have altered the female song system and induced male-like vocalizations, it is interesting that none of the species-typic elements of male starlings was produced by the experimental females. Large changes were found in both repertoire size and composition over a year, revealing a high plasticity, even in older birds. We also found evidence that even under testosterone treatment, females do not necessarily sing all the songs they may have in memory.     

Status-appropriate singing behavior,testosterone and androgen receptor immunolabeling in male European starlings (Sturnus vulgaris)

Vocalizations convey information about an individual's motivational, internal, and social status. As circumstances change, individuals respond by adjusting vocal behavior accordingly. In European starlings, a male that acquires a nest site socially dominates other males and dramatically increases courtship song. Although circulating testosterone is associated with social status and vocal production it is possible that steroid receptors fine-tune status-appropriate changes in behavior. Here we explored a possible role for androgen receptors. Male starlings that acquired nest sites produced high rates of courtship song. For a subset of males this occurred even in the absence of elevated circulating testosterone. Immunolabeling for androgen receptors (ARir) was highest in the medial preoptic nucleus (POM) in males with both a nest site and elevated testosterone. For HVC, ARir was higher in dominant males with high testosterone (males that sang longer songs) than dominant males with low testosterone (males that sang shorter songs). ARir in the dorsal medial portion of the nucleus intercollicularis (DM) was elevated in males with high testosterone irrespective of dominance status. Song bout length related positively to ARir in POM, HVC and DM, and testosterone concentrations related positively to ARir in POM and DM. Results suggest that the role of testosterone in vocal behavior differs across brain regions and support the hypothesis that testosterone in POM underlies motivation, testosterone in HVC relates to song quality, and testosterone in DM stimulates vocalizations. Our data also suggest that singing may influence AR independent of testosterone and that alternative androgen-independent pathways regulate status-appropriate singing behavior.     

Immediate and long-term effects of testosterone on song plasticity and learning in juvenile song sparrows

Steroid sex hormones play critical roles in the development of brain regions used for vocal learning. It has been suggested that puberty-induced increases in circulating testosterone (T) levels crystallize a bird's repertoire and inhibit future song learning. Previous studies show that early administration of T crystallizes song repertoires but have not addressed whether new songs can be learned after this premature crystallization. We brought 8 juvenile song sparrows (Melospiza melodia) into the laboratory in the late summer and implanted half of them with subcutaneous T pellets for a two week period in October. Birds treated with T tripled their singing rates and crystallized normal songs in 2 weeks. After T removal, subjects were tutored by 4 new adults. Birds previously treated with T tended toward learning fewer new songs post T, consistent with the hypothesis that T helps to close the song learning phase. However, one T-treated bird proceeded to learn several new songs in the spring, despite singing perfectly crystallized songs in the fall. His small crystallized fall repertoire and initial lag behind other subjects in song development suggest that this individual may have had limited early song learning experience. We conclude that an exposure to testosterone sufficient for crystallization of a normal song repertoire does not necessarily prevent future song learning and suggest that early social experiences might override the effects of hormones in closing song learning.     

Copulatory behavior of adult tamarins (Saguinus fuscicollis) castrated as neonates or juveniles: Effect of testosterone treatment

The sexual interactions of Saguinus fuscicollis males castrated as neonates, at 37 days of age, or prepubertally with adult intact females were studied. Prepubertally castrated males were observed while receiving testosterone, and while being treated with saline. Males castrated neonatally or at 37 days of age were observed while receiving testosterone. Neonatal castrates had previously been studied without hormone treatment and therefore no control condition was included for these animals. Prepubertally castrated males showed Mounts, Mounts with Thrusts, and Sexual Tongue Flicking when treated with saline only. In three of the four males, all measures of sexual behavior increased with testosterone treatment. Neonatally castrated males had failed to display any mounting or thrusting without testosterone treatment during a previous study. During the present study, three of the four males did not respond to testosterone treatment with sexual behavior. The fourth male and one male castrated at 37 days of age displayed some sexual behavior. These results suggest that most neonatally castrated males are not able to respond to testosterone with the activation of copulatory behavior. The findings are consistent with the hypothesis that in callitrichids the sensitive period for behavioral differentiation is shifted into neonatal life. However, some neonatally castrated males show a weak response to testosterone. This may reflect an extended and perhaps partially prenatal period of sensitivity.