Androgens and estrogens induce seasonal-like growth of song nuclei in the adult songbird brain

In seasonally breeding songbirds, the brain regions that control song behavior undergo dramatic structural changes at the onset of each annual breeding season. As spring approaches and days get longer, gonadal testosterone (T) secretion increases and triggers the growth of several song control nuclei. T can be converted to androgenic and estrogenic metabolites by enzymes expressed in the brain. This opens the possibility that the effects of T may be mediated via the androgen receptor, the estrogen receptor, or both. To test this hypothesis, we examined the effects of two bioactive T metabolites on song nucleus growth and song behavior in adult male white-crowned sparrows. Castrated sparrows with regressed song control nuclei were implanted with silastic capsules containing either crystalline T, 5alpha-dihydrotestosterone (DHT), estradiol (E(2)), or a combination of DHT+E(2). Control animals received empty implants. Song production was highly variable within treatment groups. Only one of seven birds treated with E(2) alone was observed singing, whereas a majority of birds with T or DHT sang. After 37 days of exposure to sex steroids, we measured the volumes of the forebrain song nucleus HVc, the robust nucleus of the archistriatum (RA), and a basal ganglia homolog (area X). All three steroid treatments increased the volumes of these three song nuclei when compared to blank-implanted controls. These data demonstrate that androgen and estrogen receptor binding are sufficient to trigger seasonal song nucleus growth. These data also suggest that T's effects on seasonal song nucleus growth may depend, in part, upon enzymatic conversion of T to bioactive metabolites.     

Lack of a synergistic effect between estradiol and dihydrotestosterone in the masculinization of the zebra finch song system

Previous studies have suggested that both major active metabolites of testosterone, estradiol (E₂) and dihydrotestosterone (DHT), are needed for complete masculinization of the brain regions that control song in passerine birds. However, DHT treatment of hatchling female zebra finches has only small masculinizing effects on the song system. To assess whether E₂ and DHT have a synergistic effect on the masculinization of the zebra finch song system, female zebra finches were given Silastic implants of E₂ on the day of hatching (day 1) either without any additional hormone treatment or in combination with DHT on days 1, 14, or 70. At 105 to 110 days of age, we measured the volumes of Area X, higher vocal center (HVC), robust nucleus of the archistriatum (RA), soma sizes in HVC, RA, and the lateral magnocellular nucleus of the neostriatum (lMAN), and neuron density and number in RA. E₂ masculinized all of the measures in the song system with the exception of the number of neurons in RA. DHT did not synergize with E₂ to produce any additional masculinization of the attributes measured. These data demonstrate that the combination of E₂ and DHT did not result in the complete masculinization of the song control nuclei and argue against the importance of androgen in sexual differentiation of the song system. © 1995 John Wiley & Sons, Inc.     

Seasonal changes in avian song control circuits do not cause seasonal changes in song discrimination in song sparrows

In seasonally breeding songbirds, brain nuclei of the song control system that act in song perception change in size between seasons. It has been hypothesized that seasonal regression of song nuclei may impair song discrimination. We tested this hypothesis in song sparrows (Melospiza melodia), a species in which males share song types with neighbors and must discriminate between similar songs in territorial interactions. We predicted that song sparrows with regressed song systems would have greater difficulty in discriminating between similar songs. Sparrows were housed either on short days (SD) and had regressed song circuits, or were exposed to long days and implanted with testosterone (LD+T) to induce full growth of the song circuits. We conducted two experiments using a GO/NO-GO operant conditioning paradigm to measure song discrimination ability of each group. Birds learned four (experiment 1) or three (experiment 2) pairs of song types sequentially, with each pair more similar in the number of shared song elements and thus more difficult to discriminate. Circulating T levels differed between the SD and LD+T groups. The telencephalic song nuclei HVc, RA, and area X were larger in the LD+T birds. The two groups of sparrows did not differ, however, in their ability to learn to discriminate between shared song types, regardless of the songs' similarity. These results suggest that seasonal changes in the song control system do not affect birds' ability to make difficult song discriminations.     

Brain aromatase, 5α‐reductase,and 5β‐reductase change seasonally in wild male song sparrows: Relationship to aggressive and sexual behavior

In many species, territoriality is expressed only during the breeding season, when plasma testosterone (T) is elevated. In contrast, in song sparrows (Melospiza melodia morphna), males are highly territorial during the breeding (spring) and nonbreeding (autumn) seasons, but not during molt (late summer). In autumn, plasma sex steroids are basal, and castration has no effect on aggression. However, inhibition of aromatase reduces nonbreeding aggression, suggesting that neural steroid metabolism may regulate aggressive behavior. In wild male song sparrows, we examined the neural distribution of aromatase mRNA and seasonal changes in the activities of aromatase, 5α‐, and 5β‐reductase, enzymes that convert T to 17β‐estradiol, 5α‐dihydrotestosterone (5α‐DHT, a potent androgen), or 5β‐DHT (an inactive metabolite), respectively. Enzyme activities were measured in the diencephalon, ventromedial telencephalon (vmTEL, which includes avian amygdala), caudomedial neostriatum (NCM), and the hippocampus of birds captured during spring, molt, or autumn. Aromatase and 5β‐reductase changed seasonally in a region‐specific manner. Aromatase in the diencephalon was higher in spring than in molt and autumn, similar to seasonal changes in male sexual behavior. Aromatase activity in the vmTEL was high in both spring and autumn but significantly reduced at molt, similar to seasonal changes in aggression. 5β‐Reductase was not elevated during molt, suggesting that low aggression during molt is not a result of increased inactivation of androgens. These data highlight the relevance of neural steroid metabolism to the expression of natural behaviors by free‐living animals. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 209–221, 2003     

Androgens and the immunocompetence handicap hypothesis: unraveling direct and indirect pathways of immunosuppression in song sparrows

The immunocompetence handicap hypothesis proposes that testosterone (T)-dependent sexual signals are honest indicators of male health or genetic quality because only high-quality males are able to withstand the obligate effects of T-induced immunosuppression. In birds, the basic assumption that T suppresses immune function is equivocal, and the physiological mechanisms underlying T-induced immunosuppression remain to be investigated. We explored the proximate pathways of T-induced immunosuppression in song sparrows (Melospiza melodia) by treating captive nonbreeding males with different androgens and measuring several components of acquired immune function. Males implanted with T suppressed cell-mediated and humoral immune responses compared to males implanted with 5alpha-dihydrotestosterone (DHT), dehydroepiandrosterone, or control (empty) implants. Furthermore, T treatment increased plasma levels of corticosterone and decreased body mass and fat stores in relation to other treatments. The failure of DHT to depress immune function suggests that T-induced immunosuppression does not occur through a direct pathway because both T and DHT bind to androgen receptors on target cells. Instead, we outline indirect pathways that are likely responsible for suppression of the avian immune system that include stress-induced immunosuppression, aromatization to estrogen, and alterations in energy allocation that constrain expenditures toward immune system activation.     

Seasonal‐like plasticity of spontaneous firing rate in a songbird pre‐motor nucleus

Many animals exhibit seasonal changes in behavior and its underlying neural substrates. In seasonally breeding songbirds, the brain nuclei that control song learning and production undergo substantial structural changes at the onset of each breeding season, in association with changes in song behavior. These changes are largely mediated by photoperiod‐dependent changes in circulating concentrations of gonadal steroid hormones. Little is known, however, about whether changes in the electrophysiological activity of neurons accompany the dramatic morphological changes in the song nuclei. Here we induced seasonal‐like changes in the song systems of adult white‐crowned sparrows and used extracellular recording in acute brain slices from those individuals to study physiological properties of neurons in the robust nucleus of the arcopallium (RA), a pre‐motor nucleus necessary for song production. We report that: RA neurons from birds in breeding condition show a more than twofold increase in spontaneous firing rate compared to those from nonbreeding condition; this change appears to require both androgenic and estrogenic actions; and this change is intrinsic to the RA neurons. Thus, neurons in the song circuit exhibit both morphological and physiological adult seasonal plasticity. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Seasonal-like plasticity of spontaneous firing rate in a songbird pre-motor nucleus

Many animals exhibit seasonal changes in behavior and its underlying neural substrates. In seasonally breeding songbirds, the brain nuclei that control song learning and production undergo substantial structural changes at the onset of each breeding season, in association with changes in song behavior. These changes are largely mediated by photoperiod-dependent changes in circulating concentrations of gonadal steroid hormones. Little is known, however, about whether changes in the electrophysiological activity of neurons accompany the dramatic morphological changes in the song nuclei. Here we induced seasonal-like changes in the song systems of adult white-crowned sparrows and used extracellular recording in acute brain slices from those individuals to study physiological properties of neurons in the robust nucleus of the arcopallium (RA), a pre-motor nucleus necessary for song production. We report that: RA neurons from birds in breeding condition show a more than twofold increase in spontaneous firing rate compared to those from nonbreeding condition; this change appears to require both androgenic and estrogenic actions; and this change is intrinsic to the RA neurons. Thus, neurons in the song circuit exhibit both morphological and physiological adult seasonal plasticity.     

Estrogen contributes to seasonal plasticity of the adult avian song control system

Songbirds show dramatic neural plasticity as adults, including large-scale anatomical changes in discrete brain regions ("song control nuclei") controlling the production of singing behavior. The volumes of several song control nuclei are much larger in the breeding season than in the nonbreeding season, and these seasonal neural changes are regulated by plasma testosterone (T) levels. In many cases, the effects of T on the central nervous system are mediated by neural conversion to estradiol (E(2)) by the enzyme aromatase. The forebrain of male songbirds expresses very high levels of aromatase, in some cases adjacent to song control nuclei. We examined the effects of aromatase inhibition and estrogen treatment on song nuclei size using wild male songbirds in both the breeding and nonbreeding seasons. In breeding males, aromatase inhibition caused the volume of a telencephalic song control nucleus (HVC) to decrease, and this effect was partially rescued by concurrent estrogen replacement. In nonbreeding males, estradiol treatment caused HVC to grow to maximal spring size within 2 weeks. Overall, these data suggest that aromatization of T is an important mediator of song control system plasticity, and that estradiol has neurotrophic effects in adult male songbirds. This study demonstrates that estrogen can affect adult neural plasticity on a gross anatomical scale and is the first examination of estrogen effects on the brain of a wild animal.     

Time course of changes in Gambel's white-crowned sparrow song behavior following transitions in breeding condition

Seasonal changes in behavior and in its underlying neural substrate are common across animal taxa. These changes are often triggered by steroid sex hormones. Song in seasonally breeding songbirds provides an excellent example of this phenomenon. In these species, dramatic seasonal changes mediated by testosterone and its metabolites occur in adult song behavior and in the neural circuitry controlling song. While song rate can quickly change in response to seasonal breeding cues, it is unknown how quickly other aspects of song change, particularly the stereotypy of song phonology and syntax. In this study we determined whether and how quickly song rate, phonology, and syntax change in response to breeding and non-breeding physiological cues. We asked these questions using Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), a closed-ended learner with well-characterized changes in the neural circuitry controlling song behavior. We exposed ten photosensitive sparrows to long-day photoperiod and implanted them with subcutaneous testosterone pellets (day 0) to simulate breeding conditions. We continuously recorded song and found that song rate increased quickly, reaching maximum around day 6. The stereotypy of song phonology changed more slowly, reaching maximum by day 10 or later. Song syntax changed minimally after day 6, the earliest time point examined. After 21 days, we transitioned five birds from breeding to non-breeding condition. Song rate declined precipitously. These results suggest that while song rate changes quickly, song phonology changes more slowly, generally following or in parallel with previously investigated changes in the neural substrate.     

Rapamycin blocks the neuroprotective effects of sex steroids in the adult birdsong system

In adult songbirds, the telencephalic song nucleus HVC and its efferent target RA undergo pronounced seasonal changes in morphology. In breeding birds, there are increases in HVC volume and total neuron number, and RA neuronal soma area compared to nonbreeding birds. At the end of breeding, HVC neurons die through caspase‐dependent apoptosis and thus, RA neuron size decreases. Changes in HVC and RA are driven by seasonal changes in circulating testosterone (T) levels. Infusing T, or its metabolites 5α‐dihydrotestosterone (DHT) and 17 β‐estradiol (E2), intracerebrally into HVC (but not RA) protects HVC neurons from death, and RA neuron size, in nonbreeding birds. The phosphoinositide 3‐kinase (PI3K)‐Akt (a serine/threonine kinase)‐mechanistic target of rapamycin (mTOR) signaling pathway is a point of convergence for neuroprotective effects of sex steroids and other trophic factors. We asked if mTOR activation is necessary for the protective effect of hormones in HVC and RA of adult male Gambel's white‐crowned sparrows (Zonotrichia leucophrys gambelii). We transferred sparrows from breeding to nonbreeding hormonal and photoperiod conditions to induce regression of HVC neurons by cell death and decrease of RA neuron size. We infused either DHT + E2, DHT + E2 plus the mTOR inhibitor rapamycin, or vehicle alone in HVC. Infusion of DHT + E2 protected both HVC and RA neurons. Coinfusion of rapamycin with DHT + E2, however, blocked the protective effect of hormones on HVC volume and neuron number, and RA neuron size. These results suggest that activation of mTOR is an essential downstream step in the neuroprotective cascade initiated by sex steroid hormones in the forebrain.     

Testosterone treatment increases the metabolic capacity of adult avian song control nuclei

In songbirds, the size of brain nuclei that control song learning and production change seasonally. These changes are mainly controlled by seasonal changes in plasma testosterone (T) concentration. One hypothesis to explain why it may be adaptive for these areas to regress in the fall is that this would decrease the metabolic demand of maintaining a large song system when singing is reduced or absent. We used a marker for cellular metabolism to examine birds with regressed song nuclei and compared them to birds whose song nuclei were induced to grow by administration of exogenous T. Photorefractory male Gambel's white-crowned sparrows were captured during their autumnal migration and kept in outdoor aviaries on a natural photoperiod. We implanted birds with Silastic capsules containing T or with empty implants. Three weeks later the birds were sacrificed. We assayed the brains for cytochrome oxidase (CO) activity and measured the volume of four song nuclei: HVc, RA, 1MAN, and area X. All four nuclei increased in volume in response to T treatment. T treatment increased the metabolic capacity of area X, HVc, and RA relative to surrounding tissue but had no effect on the metabolic capacity of 1MAN. These results support the hypothesis that song nuclei are more metabolically active under the influence of T than they are when plasma T levels are low.     

Seasonal changes in intrinsic electrophysiological activity of song control neurons in wild song sparrows

Song behavior and its underlying neural substrate can change seasonally in adult songbirds. To test whether environmental cues induce seasonal changes in electrophysiological characteristics of song control neurons, we measured in vitro intrinsic neuronal activity in the song control nucleus RA of adult male song sparrows (Melospiza melodia) in both the fall non-breeding and spring breeding seasons. We found that RA neurons in spring-captured birds show a more than threefold increase in spontaneous firing rate compared to those from fall-captured birds. We conclude that environmental cues are sufficient to induce seasonal changes in electrophysiological properties of song control neurons, and that changes in these properties may underlie seasonal changes in song behavior.     

Seasonal changes in song nuclei and song behavior in Gambel's white-crowned sparrows

In males of several songbird species, the morphology of forebrain nuclei that control song changes seasonally. The only seasonally breeding songbird in which seasonal changes in the structure of song control nuclei have been reported not to occur is the nonmigratory Nuttall's subspecies of white-crowned sparrow. In the present study, we manipulated photoperiod and plasma testosterone concentrations in captive male white-crowned sparrows of the migratory Gambel's subspeices. Males exposed to photoperiods and plasma testosterone concentrations typical of those experienced by wild breeding males had larger song control nuclei than males held on a winter photoperiod. We also found seasonal change in stereotypy of spectral and temporal parameters of song in wild Gambel's white-crowned sparrows. We hypothesize that seasonal changes in song control nuclei may correlate with seasonal changes in song stereotypy. © 1995 John Wiley & Sons, Inc.     

The role of androgen receptors in regulating territorial aggression in male song sparrows

This paper examines the role that androgen receptors (ARs) play in modulating aggressive behavior in male song sparrows, Melospiza melodia morphna. Song sparrows are seasonally breeding, territorial birds that maintain year-round territories with male-female pair bonds formed during the spring breeding season. Plasma testosterone levels peak as territories are established and mates acquired. In late summer, testosterone levels fall and remain basal during the non-breeding season. We examined the role of ARs in regulating territorial aggression in captive song sparrows under short- and long-day conditions as well as just prior to, and at the start of the breading season in freely living birds using the nonsteroidal antiandrogen flutamide to block AR function. Birds were implanted with either empty or drug filled silastic implants for 18 to 42 days and then challenged with a novel male decoy to assess the individual birds level of male-male aggression. Freely living birds remained on their home territory and underwent a simulated territorial intrusion, whereas laboratory-held birds were assessed using a laboratory simulated territorial intrusion and remained in their home cage. Experimental treatment of male song sparrows decreased aggressive behavior during the pre-breeding life history substage (March-April) in freely living birds as well as in laboratory-held birds under long-day (16L:8D) conditions. During the early breeding substage (April-May) there was no measurable effect of flutamide treatment on aggressive behavior, nor was there a difference in behavior in the (8L:16D) laboratory birds. This demonstrates that ARs are an important component of the neuroendocrine control of aggressive behavior. Given that flutamide only affected aggression during the pre-breeding substage and in LD birds, the results suggest that AR dependent control of aggressive behavior changes as song sparrow life history states change.     

Aromatase inhibition affects testosterone‐induced masculinization of song and the neural song system in female canaries

Songbirds have a specialized steroid‐sensitive network of brain nuclei, the song system, for controlling song. Most nuclei of the song system express androgen receptors, and the sensory‐motor integration nucleus High Vocal Center (HVC) alone also expresses estrogen receptors. Apart from expressing estrogen receptors in the vocal control system, songbirds are unique among birds because they have high concentrations of the estrogen‐synthesizing enzyme aromatase in the neostriatum surrounding HVC. However, the role of estrogen in controlling the development of the song structure has been scarcely investigated. In this work, we show that blocking the production of estrogen during testosterone‐induced song motor development in adult female canaries alters the song pattern compared to control females treated with testosterone only. These effects were correlated with inhibition of the expression of estrogen‐sensitive genes, such as brain‐derived nerve growth factor, in HVC. The expression of the ATP‐synthase gene, an indicator of cell activity, in HVC, and the size of HVC, were not affected by the treatment. Our results provide the first example of estrogen‐sensitive mechanisms controlling the structural features of adult birdsong. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 370–379, 2003     

Coordinated and dissociated effects of testosterone on singing behavior and song control nuclei in canaries (Serinus canaria)

Temperate zone songbirds that breed seasonally exhibit pronounced differences in reproductive behaviors including song inside and outside the breeding season. Springlike long daylengths are associated with increases in plasma testosterone (T) concentrations, as well as with increases in singing and in the volume of several brain nuclei known to control this behavior. The mechanisms whereby T can induce changes in behavior and brain, and whether or not these effects are differentially regulated, have recently begun to be examined, as has the question of the relative contributions of T and its androgenic and estrogenic metabolites to the regulation of this seasonal behavioral and neural plasticity. In this experiment, we examined the effects of T, 5alpha-dihydrotestosterone, or 17beta-estradiol treatment on castrated male canaries housed on short days and compared neural and behavioral effects in these males to similarly-housed males given only blank implants. We observed that only T treatment was effective in eliciting significant increases in singing behavior after 11 days of hormone exposure. In addition, T alone was effective in increasing the volume of a key song production nucleus, HVC. However, at this time, none of the steroids had any effects on the volumes of two other song control nuclei, Area X of the medial striatum and the robust nucleus of the arcopallium (RA), that are efferent targets of HVC, known to be regulated by androgen in canaries and also to play a role in the control of adult song. T can thus enhance singing well before concomitant androgen-induced changes in the song control system are complete.     

Action of androgen and estrone implants on sexual behavior and reproductive organs of castrated male rabbits

Sexually mature but inexperienced male rabbits were castrated, immediately implanted with either testosterone (T), estrone (E₁), dihydrotestosterone (DHT), T + E₁, or DHT + E₁, and tested for male sexual behavior. Other castrates were not implanted, and testing was either begun immediately (Ca-I) or delayed for 4 weeks (Ca-D). Intact males served as controls (C). Latency to mount a teaser female and to ejaculate into an artificial vagina was tested twice in a morning three times per week for 8 weeks. Then, animals were sacrificed, and reproductive organs were measured. The Ca-I group responded slowly to sexual training and ceased nearly all sexual activity by 8 weeks, whereas the Ca-D males seldom displayed interest in the teaser female. Intact controls and the T and T + E₁, groups all responded to the teaser and mounted and ejaculated within a few seconds. DHT and E₁, individually maintained the sexual activity of castrates equivalent to that of C for 4–5 weeks, but the time required to mount and, particularly, to ejaculate increased thereafter. The results with DHT + E₁ were equivocal in that castrates with this hormone combination sustained sexual activity equivalent to that of the controls for 7 weeks, but one animal in particular became sexually inactive the last week of the experiment. Penis weight was at least partially maintained in all implanted castrates. Accessory sex gland weight was smallest in the DHT group and was significantly increased in the T + E₁ and DHT + E₁ groups. The largest ejaculates of fluid were obtained in the group receiving E₁ alone. These results may be interpreted to indicate a role of both androgen and estrogen centrally and peripherally in the rabbit.     

Photoperiodic differences in a forebrain nucleus involved in vocal plasticity: Enkephalin immunoreactivity reveals volumetric variation in song nucleus lMAN but not NIf in male European starlings (Sturnus vulgaris)

Seasonal variation in the volume of various song control nuclei in many passerine species remains one of the best examples of naturally occurring adult neuroplasticity among vertebrates. The lateral portion of the magnocellular nucleus of the anterior nidopallium (lMAN) is a song nucleus that is important for song learning and seems to be critical for inducing variability in the song structure that is later pruned via a feedback process to produce adult crystallized song. To date, lMAN has not been shown to exhibit seasonal changes in volume, probably because it is difficult to resolve the boundaries of lMAN when employing histological methods based on Nissl staining. Here, lMAN_core volumes were examined in intact photostimulated (i.e., breeding), castrated photostimulated and photorefractory (i.e., nonbreeding) male starlings (Sturnus vulgaris) to investigate the degree of seasonal variation in brain morphology. We present data demonstrating that the volumes of the total MAN and lMAN_core delineated by enkephalin immunoreactivity are greater in photostimulated male starlings as compared to photorefractory males. Moreover, two other regions associated with the song system that have not been investigated previously in the context of seasonal plasticity namely (i) the medial portion of MAN (mMAN), and (ii) the nucleus interfacialis (NIf) did not display significant volumetric variation. We propose that greater lMAN_core volumes are associated with the increase in vocal plasticity that is generally observed prior to production of stereotyped song. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 751–763, 2010     

Androgen‐metabolizing enzymes show region‐specific changes across the breeding season in the brain of a wild songbird

The Lapland longspur (Calcarius lapponicus) is an arctic‐breeding songbird that shows rapid behavioral changes during a short breeding season. Changes in plasma testosterone (T) in the spring are correlated with singing but not territorial aggression in males. Also, T treatment increases song but not aggression in this species. In contrast, in temperate‐zone breeders, song and aggression are highly correlated, and both increase after T treatment. We asked whether regional or temporal differences in androgen‐metabolizing enzymes in the longspur brain explain hormone‐behavior patterns in this species. We measured the activities of aromatase, 5α‐reductase and 5β‐reductase in free‐living longspur males. Aromatase and 5α‐reductase convert T into the active steroids 17β‐estradiol (E₂) and 5α‐dihydrotestosterone (5α‐DHT), respectively. 5β‐Reductase deactivates T via conversion to 5β‐DHT, an inactive steroid. We examined seven brain regions at three stages in the breeding season. Overall, aromatase activity was high in the hypothalamus, hippocampus, and ventromedial telencephalon (containing nucleus taeniae, the avian homologue to the amygdala). 5β‐Reductase activity was high throughout the telencephalon. Activities of all three enzymes changed over time in a region‐specific manner. In particular, aromatase activity in the rostral hypothalamus was decreased late in the breeding season, which may explain why T treatment at this time does not increase aggression. Changes in 5β‐reductase do not explain the effects of plasma T on aggressive behavior. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 176–188, 1999     

Aromatase inhibition affects testosterone-induced masculinization of song and the neural song system in female canaries

Songbirds have a specialized steroid-sensitive network of brain nuclei, the song system, for controlling song. Most nuclei of the song system express androgen receptors, and the sensory-motor integration nucleus High Vocal Center (HVC) alone also expresses estrogen receptors. Apart from expressing estrogen receptors in the vocal control system, songbirds are unique among birds because they have high concentrations of the estrogen-synthesizing enzyme aromatase in the neostriatum surrounding HVC. However, the role of estrogen in controlling the development of the song structure has been scarcely investigated. In this work, we show that blocking the production of estrogen during testosterone-induced song motor development in adult female canaries alters the song pattern compared to control females treated with testosterone only. These effects were correlated with inhibition of the expression of estrogen-sensitive genes, such as brain-derived nerve growth factor, in HVC. The expression of the ATP-synthase gene, an indicator of cell activity, in HVC, and the size of HVC, were not affected by the treatment. Our results provide the first example of estrogen-sensitive mechanisms controlling the structural features of adult birdsong.