The effects of an aromatization inhibitor on the reproductive behavior of male zebra finches

Recent evidence indicates that aromatizable androgens are more effective than nonaromatizable androgens in restoring normal levels of sexual behavior in castrated male zebra finches (Poephila guttata). To determine whether the efficacy of treatment with aromatizable androgens, is in part due to their conversion to estrogens, castrated male finches were treated with androstenedione (AE), an aromatizable androgen, and their sexual and aggressive behavior was compared with that of castrates treated with AE plus 1,4,6-androstatriene-3,17-dione (ATD), an aromatization inhibitor. Males treated with AE + ATD showed less courtship activity and less copulatory behavior than AE-treated males, and were unlikely to have nests. Estradiol (E), when given concurrently with AE + ATD, reversed the inhibitory effects of ATD and restored levels of courtship and copulation to those observed in AE-treated males. Only AE- and AE + ATD + E-treated males displayed aggressive behaviors, but the frequency of such behaviors was so low that there were no significant differences across groups. These data affirm the importance of estrogen in the control of reproductive activities in male zebra finches and indicate that aromatization may be an obligatory step for maintaining normal levels of sexual and aggressive behavior.     

Undirected Song Encourages the Breeding Female Zebra Finch to Remain in the Nest

Wild zebra finches sing frequently during the breeding season, but the vast majority of song is of the undirected song type that is not directed at any individual, and the function of which is obscure — it appears to be ignored by all potential recipients. It is sung close to the nest-site, has a peak in production during the egg-laying period, and diminishes thereafter. The incidence of undirected song is positively correlated with extra-pair courtship, a finding consistent with the hypothesis that it is a means of advertising availability for extra-pair matings. Typically, undirected song occurred outside the nest when the female was inside, and a positive relationship was found between the amount of singing given by the male during the 5-min interval immediately after the female entered the nest and the time she subsequently spent inside the nest. Keeping the partner inside the nest during her fertile period is an advantage to the male: it serves as a form of paternity protection against other males and it allows him opportunities to pursue his own extra-pair matings. Occupancy of the nest during laying is also a means of guarding against intraspecific brood parasitism, which was high at this colony.     

The Onset of Song Learning and Song Tutor Selection in Fledgling Zebra Finches

We investigated song development in the pre‐independent zebra finch (aged 15–35 d), a period when neural pathways for song learning and production are forming and social influences outside the family are limited. Expt 1 investigated the onset and the minimum duration of tutoring needed for song learning in fledglings. We found most begin to learn song from 25 d of age and need about 10 d contact with the father tutor to make accurate copies. This onset corresponds with major developments in the formation of the neural circuitry implicated in song acquisition. Subsong also begins on day 25 suggesting that the sensory and motor phases of song learning fully overlap in the zebra finch. Our findings support the hypothesis that the song circuitry is fully functional by 35 d of age and the sensitive phase for zebra finches extends therefore from about days 25–65. However, only the first 10 d of this period are necessary to learn a tutor's song with fair accuracy. Expt 2 investigated the role of the paternal bond, spatial proximity and mating status in a fledgling's choice of song tutor where the father was the sole parent. Young chose the father over single unrelated males (expt 2a) or unrelated males in company with their female partners (expt 2b). Given the close spatial proximity of both potential tutors to the fledglings it is probably the filial bond, established via paternal care that is the cause of this preference. Zebra finches sing the same song phrase in two contrasting contexts: female‐directed song during pre‐copulatory courtship and undirected song where no female or display is involved. In expt 3 we tested the song learning preference of pre‐independent young for two categories of non‐paternal tutors: those singing predominantly female‐directed song and those singing exclusively undirected song. There was a small, but significant, preference for fledgling zebra finches to copy songs from males that sang female‐directed song. This preference is consistent with the hypothesis that young males not only learn the acoustic features of their tutor's song but also the visual and dynamic movements that constitute the courtship display.     

Paradoxical hypermasculinization of the zebra finch song system by an antiestrogen

Exogenous estrogens, when administered to hatchling female zebra finches, masculinize the morphology and function of their neural vocal control system. The first of two experiments evaluated whether tamoxifen citrate is an antiestrogen in zebra finches, and the second determined whether it would block the masculinization hypothesized to be caused in hatchling males by the males' endogenous estradiol. In the first experiment adult female zebra finches were ovariectomized and injected for 10 days with estradiol benzoate (EB), tamoxifen, EB and tamoxifen combined, or vehicle (control). The dependent variable was oviduct weight. The EB-stimulated growth of the oviduct was blocked by tamoxifen, which had no effects when administered alone. Thus, tamoxifen acts as an antiestrogen in the zebra finch oviduct. In Experiment 2, male and female zebra finches were treated with tamoxifen or vehicle for the first 20 days after hatching. The males were castrated at 20 days. At 60 days we compared the song control regions of experimental and control males and females. In both sexes tamoxifen increased the somatic areas of neurons in RA (robust nucleus of the archistriatum), HVc (caudal nucleus of the ventral hyperstriatum), and MAN (magnocellular nucleus of the anterior neostriatum). Tamoxifen also increased the volumes of HVc, RA, MAN, and Area X in males. Thus, tamoxifen failed to block masculinization of males, but masculinized females and hypermasculinized males. Tamoxifen's hypermasculinization of the male and masculinization of the female song system is paradoxical given that (1) estradiol does not have similar effects on the male song system, and (2) tamoxifen antagonizes the effects of EB in the oviduct.     

Is female preference for male repertoires due to sensory bias?

There are several possible explanations for the female preference for male repertoires in birds. These males are older, and have better territories; thus there are functional reasons for females to prefer these males. However, there is an alternative explanation; females may habituate less quickly to song repertoires than single songs. I tested whether females have a non-functional, sensory bias for male song repertoires, by testing female preference for a repertoire in zebra finches (Taenopygia guttata), a species in which males possess a single stereotyped song. Females chose between a male repertoire of four different phrases created from the song phrase of one individual and that of one of those phrases repeated four times (natural zebra finch song). Females were also given a choice between the above repertoire and a song made from the phrases of four related males (''family'' stimulus). I tested female preference by training females to press a button for presentation of a song stimulus, and counting the number of button presses. Females preferred the song repertoire to a single phrase song, and did not differentiate between the repertoire and song phrases from four males. Evidence from the Estrildidae indicates that having a single song is the ancestral state for zebra finches, so the preference is not ancestral.     

Social context-induced song variation affects female behavior and gene expression

Social cues modulate the performance of communicative behaviors in a range of species, including humans, and such changes can make the communication signal more salient. In songbirds, males use song to attract females, and song organization can differ depending on the audience to which a male sings. For example, male zebra finches (Taeniopygia guttata) change their songs in subtle ways when singing to a female (directed song) compared with when they sing in isolation (undirected song), and some of these changes depend on altered neural activity from a specialized forebrain-basal ganglia circuit, the anterior forebrain pathway (AFP). In particular, variable activity in the AFP during undirected song is thought to actively enable syllable variability, whereas the lower and less-variable AFP firing during directed singing is associated with more stereotyped song. Consequently, directed song has been suggested to reflect a “performance” state, and undirected song a form of vocal motor “exploration.” However, this hypothesis predicts that directed–undirected song differences, despite their subtlety, should matter to female zebra finches, which is a question that has not been investigated. We tested female preferences for this natural variation in song in a behavioral approach assay, and we found that both mated and socially naive females could discriminate between directed and undirected song—and strongly preferred directed song. These preferences, which appeared to reflect attention especially to aspects of song variability controlled by the AFP, were enhanced by experience, as they were strongest for mated females responding to their mate's directed songs. We then measured neural activity using expression of the immediate early gene product ZENK, and found that social context and song familiarity differentially modulated the number of ZENK-expressing cells in telencephalic auditory areas. Specifically, the number of ZENK-expressing cells in the caudomedial mesopallium (CMM) was most affected by whether a song was directed or undirected, whereas the caudomedial nidopallium (NCM) was most affected by whether a song was familiar or unfamiliar. Together these data demonstrate that females detect and prefer the features of directed song and suggest that high-level auditory areas including the CMM are involved in this social perception.     

Social Context–Induced Song Variation Affects Female Behavior and Gene Expression

Social cues modulate the performance of communicative behaviors in a range of species, including humans, and such changes can make the communication signal more salient. In songbirds, males use song to attract females, and song organization can differ depending on the audience to which a male sings. For example, male zebra finches (Taeniopygia guttata) change their songs in subtle ways when singing to a female (directed song) compared with when they sing in isolation (undirected song), and some of these changes depend on altered neural activity from a specialized forebrain-basal ganglia circuit, the anterior forebrain pathway (AFP). In particular, variable activity in the AFP during undirected song is thought to actively enable syllable variability, whereas the lower and less-variable AFP firing during directed singing is associated with more stereotyped song. Consequently, directed song has been suggested to reflect a “performance” state, and undirected song a form of vocal motor “exploration.” However, this hypothesis predicts that directed–undirected song differences, despite their subtlety, should matter to female zebra finches, which is a question that has not been investigated. We tested female preferences for this natural variation in song in a behavioral approach assay, and we found that both mated and socially naive females could discriminate between directed and undirected song—and strongly preferred directed song. These preferences, which appeared to reflect attention especially to aspects of song variability controlled by the AFP, were enhanced by experience, as they were strongest for mated females responding to their mate's directed songs. We then measured neural activity using expression of the immediate early gene product ZENK, and found that social context and song familiarity differentially modulated the number of ZENK-expressing cells in telencephalic auditory areas. Specifically, the number of ZENK-expressing cells in the caudomedial mesopallium (CMM) was most affected by whether a song was directed or undirected, whereas the caudomedial nidopallium (NCM) was most affected by whether a song was familiar or unfamiliar. Together these data demonstrate that females detect and prefer the features of directed song and suggest that high-level auditory areas including the CMM are involved in this social perception.     

Acoustic characteristics, early experience, and endocrine status interact to modulate female zebra finches' behavioral responses to songs

Female songbirds use male songs as an important criterion for mate selection. Properties of male songs are thought to indicate the male's quality as a potential mate. Song preferences in female zebra finches are known to be influenced by two factors--early auditory experience and the acoustic characteristics of males' songs. Studies often investigate song preferences by priming females with estrogen. However, estrogenic influences on song preferences have not been studied. We investigated the relative influence of early auditory experience, acoustic features of songs, and estrogen availability on song responsiveness in female zebra finches. Juvenile female zebra finches were tutored for 10 days with 40 songs per day with one of three acoustically different song types--simple songs, long-bout songs or complex songs. A fourth group of females was untutored. Aside from this brief song exposure, females were raised and maintained without exposure to male songs. During adulthood, females' behavioral responses to the three song types were tested under three hormone conditions--untreated, estradiol-treated and 1,4,6-androstatriene-3,17-dione (ATD)-treated (to lower endogenous estrogen). Based on the results of our study, four conclusions can be drawn. First, song responsiveness in female zebra finches is strongly affected by minimal early acoustic experience. Second, inexperienced female zebra finches are inherently biased to respond more to complex songs over other song types Third, although female zebra finches are inherently biased to respond more to complex songs, early acoustic experience may either reinforce or weaken this inherent responsiveness to complex songs. Fourth, estrogen selectively accentuates song responsiveness in acoustically-experienced female zebra finches.     

Hormonal specificity and activation of sexual behavior in male zebra finches

Castrated zebra finches receiving one of six hormone treatments were given three weekly tests with different females and their sexual behavior was contrasted with that of two control groups consisting of intact or castrated males given implants of cholesterol. The six hormone treatments were: two aromatizable androgens, testosterone (T) and androstenedione (AE); two nonaromatizable androgens, androsterone (AN) and dihydrotestosterone (DHT); an estrogen, estradiol (E); or a combination of E + DHT. Half the males receiving DHT received the 5α-isomer, half received the 5β-isomer. Castration significantly reduced the proportion of males which courted females, total courtship displays, high-intensity courtship displays, beak wiping activity, and significantly increased the latencies to show these behaviors compared to intact males. Castrated males never attempted to mount a female. All of these measures of courtship and copulatory behavior were restored to normal levels only by treatments providing both estrogenic and α-androgenic metabolites (i.e., T, AE, E + αDHT). AE was clearly the most effective of these, raising behavior significantly above normal on several measures. AN treatment was more effective than αDHT on all measures and not significantly different from intact birds on some. Treatment with E, αDHT, βDHT, or E + βDHT was totally ineffective. Surprisingly, females only solicited males whose hormone treatments provided estrogenic metabolites. Not only did they solicit males given aromatizable androgens, which showed high rates of courtship activity, they also solicited males given E or E + βDHT, some of which never even courted. Castration and hormone treatment also affected body and syringeal weight, but in opposite directions. Castration increased body weight while decreasing syringeal weight. Hormone treatments providing α-androgenic metabolites decreased body weight and increased syrinx weight. Treatments supplying estrogen as well were slightly more effective.     

Song learning with audiovisual compound stimuli in zebra finches

We investigated the effects of audiovisual compound training on song learning in zebra finches, Taeniopygia guttata. In the first experiment, presentation of a stuffed adult zebra finch male was found to be reinforcing to zebra finch males in an operant task. In a separate experiment, zebra finch males were reared without their father from day 7 after hatching onwards. Between 35 and 76 days, they were placed in isolation and exposed to taped songs of a zebra finch male, according to a random schedule (20 presentations/h). For half of the birds, presentation of the song coincided with presentation of a stuffed zebra finch male. For the remaining birds, each presentation of the song was followed by presentation of a stuffed male. The birds were subsequently isolated until day 142, when their own songs were recorded and analysed. Birds in both groups shared significantly more song elements with their tutor songs than with an unfamiliar song. There was no significant difference in song learning between the groups. These results confirm that zebra finches can learn part of their songs from taped tutor songs. Furthermore, simultaneous presentation of the tutor song and a relevant, salient visual stimulus is not superior to sequential presentation. Copyright 1999 The Association for the Study of Animal Behaviour.     

Courtship song, male agonistic encounters, and female mate choice in the house cricket,Acheta domesticus (Orthoptera: Gryllidae)

Whether female crickets choose among males based on characteristics of the courtship song is uncertain, but in many species, males not producing courtship song do not mate. In the house cricket,Acheta domesticus, we examined whether a female chose or rejected a male based on his size, latency to chirp, latency to produce courtship song, or rate of the high-frequency pulse of courtship song (“court rate”). We confirmed that females mated only with males that produced courtship song, but we found no evidence that the other factors we measured affected a female’s decision to mate. In addition, we investigated whether the outcome of male agonistic encounters affected the subsequent production of courtship song. In one experiment, we observed courtship and mating behavior when a single female was placed with a pair of males following a 10-min interaction period between the two males. Winners of male agonistic encounters had higher mating success. However, winners and losers of agonistic encounters were not different in their likelihood or latency to produce courtship song or in the number of times they were disrupted by the other male in the pair. In a second experiment, we allowed two males to interact for a 10-min period, but following this interaction period, we placed a female with each male separately and observed courtship and mating behavior. The mating success of winners and losers was not different under these circumstances, and we found no differences between winners and losers in any subsequent courtship or mating behavior examined. We conclude that winning agonistic encounters influences a male’s mating success in ways other than his production of courtship song and this effect is lost when winning and losing males are separated and each is given an opportunity to mate.     

Forebrain circuits underlying the social modulation of vocal communication signals

Across vertebrate species, signalers alter the structure of their communication signals based on the social context. For example, male Bengalese finches produce faster and more stereotyped songs when directing song to females (female‐directed [FD] song) than when singing in isolation (undirected [UD] song), and such changes have been found to increase the attractiveness of a male's song. Despite the importance of such social influences, little is known about the mechanisms underlying the social modulation of communication signals. To this end, we analyzed differences in immediate early gene (EGR‐1) expression when Bengalese finches produced FD or UD song. Relative to silent birds, EGR‐1 expression was elevated in birds producing either FD or UD song throughout vocal control circuitry, including the interface nucleus of the nidopallium (NIf), HVC, the robust nucleus of the arcopallium (RA), Area X, and the lateral magnocellular nucleus of the anterior nidopallium (LMAN). Moreover, EGR‐1 expression was higher in HVC, RA, Area X, and LMAN in males producing UD song than in males producing FD song, indicating that social context modulated EGR‐1 expression in these areas. However, EGR‐1 expression was not significantly different between males producing FD or UD song in NIf, the primary vocal motor input into HVC, suggesting that context‐dependent changes could arise de novo in HVC. The pattern of context‐dependent differences in EGR‐1 expression in the Bengalese finch was highly similar to that in the zebra finch and suggests that social context affects song structure by modulating activity throughout vocal control nuclei. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 47–63, 2016     

Social facilitation of male song by male and female conspecifics in the zebra finch, Taeniopygia guttata

Zebra finches are a ubiquitous model system for the study of vocal learning in animal communication. Their song has been well described, but its possible function(s) in social communication are only partly understood. The so-called ‘directed song’ is a high-intensity, high-performance song given during courtship in close proximity to the female, which is known to mediate mate choice and mating. However, this singing mode constitutes only a fraction of zebra finch males’ prolific song output. Potential communicative functions of their second, ‘undirected’ singing mode remain unresolved in the face of contradicting reports of both facilitating and inhibiting effects of social company on singing. We addressed this issue by experimentally manipulating social contexts in a within-subject design, comparing a solo versus male or female only company condition, each lasting for 24 h. Males’ total song output was significantly higher when a conspecific was in audible and visible distance than when they were alone. Male and female company had an equally facilitating effect on song output. Our findings thus indicate that singing motivation is facilitated rather than inhibited by social company, suggesting that singing in zebra finches might function both in inter- and intrasexual communication.     

Food, stress, and reproduction: Short-term fasting alters endocrine physiology and reproductive behavior in the zebra finch

Stress is thought to be a potent suppressor of reproduction. However, the vast majority of studies focus on the relationship between chronic stress and reproductive suppression, despite the fact that chronic stress is rare in the wild. We investigated the role of fasting in altering acute stress physiology, reproductive physiology, and reproductive behavior of male zebra finches (Taeniopygia guttata) with several goals in mind. First, we wanted to determine if acute fasting could stimulate an increase in plasma corticosterone and a decrease in corticosteroid binding globulin (CBG) and testosterone. We then investigated whether fasting could alter expression of undirected song and courtship behavior. After subjecting males to fasting periods ranging from 1 to 10 h, we collected plasma to measure corticosterone, CBG, and testosterone. We found that plasma corticosterone was elevated, and testosterone was decreased after 4, 6, and 10 h of fasting periods compared with samples collected from the same males during nonfasted (control) periods. CBG was lower than control levels only after 10 h of fasting. We also found that, coincident with these endocrine changes, males sang less and courted females less vigorously following short-term fasting relative to control conditions. Our data demonstrate that acute fasting resulted in rapid changes in endocrine physiology consistent with hypothalamo-pituitary-adrenal axis activation and hypothalamo-pituitary-gonadal axis deactivation. Fasting also inhibited reproductive behavior. We suggest that zebra finches exhibit physiological and behavioral flexibility that makes them an excellent model system for studying interactions of acute stress and reproduction.     

Zebra Finch Mates Use Their Forebrain Song System in Unlearned Call Communication

Unlearned calls are produced by all birds whereas learned songs are only found in three avian taxa, most notably in songbirds. The neural basis for song learning and production is formed by interconnected song nuclei: the song control system. In addition to song, zebra finches produce large numbers of soft, unlearned calls, among which “stack” calls are uttered frequently. To determine unequivocally the calls produced by each member of a group, we mounted miniature wireless microphones on each zebra finch. We find that group living paired males and females communicate using bilateral stack calling. To investigate the role of the song control system in call-based male female communication, we recorded the electrical activity in a premotor nucleus of the song control system in freely behaving male birds. The unique combination of acoustic monitoring together with wireless brain recording of individual zebra finches in groups shows that the neuronal activity of the song system correlates with the production of unlearned stack calls. The results suggest that the song system evolved from a brain circuit controlling simple unlearned calls to a system capable of producing acoustically rich, learned vocalizations.     

Spatial and behavioural measures of social discrimination by captive male zebra finches: Implications of sexual and species differences for recognition research

All bird species reproduce sexually and individuals need to correctly identify conspecifics for successful breeding. Captive zebra finches are a model system for studying the factors involved in species recognition and mate choice. However, male zebra finches’ behavioural responses in a spatial preference paradigm to a range of estrildid finch species, other than domesticated Bengalese finches, remain unknown. We investigated spatial and display responses of male zebra finch subjects to stimulus females between conspecific and four phylogeographically relevant finch species, in addition to female Bengalese finches. Surprisingly, male subjects did not show consistent spatial association with conspecific over heterospecific females. Overall, as predicted by sexual selection theory, the spatial proximity responses of males were less discriminatory compared to female zebra finches’ responses tested previously using the same paradigm. However, male subjects showed consistently more behavioural displays towards female conspecifics than heterospecifics which were positively related to the behavioural display rates of the respective female stimuli. Some male behavioural responses, other than song, also showed significant differences between the different stimulus species and consistently differed across individual test subjects, with the most individual subject variation seen in choice trials between female conspecific and Bengalese finch stimuli. The results are important for the design and interpretation of future behavioural and neurobiological experiments on species recognition systems using the zebra finch as a model species.     

Castration and antisteroid treatment impair vocal learning in male zebra finches.

Both song behavior and its neural substrate are hormone sensitive: castrated adult male zebra finches need replacement of gonadal steroids in order to restore normal levels of song production, and sex steroids are necessary to establish male-typical neural song-control circuits during early development. This pattern of results suggests that hormones may be required for normal development of learned song behavior, but evidence that steroids are necessary for normal neural and behavioral development during song learning has been lacking. We addressed this question by attempting to eliminate the effects of gonadal steroids in juvenile male zebra finches between the time of initial song production and adulthood. Males were castrated at 20 days of age and received systemic implants of either an antiandrogen (flutamide), an antiestrogen (tamoxifen), or both drugs. The songs of both flutamide- and tamoxifen-treated birds were extremely disrupted relative to normal controls in terms of the stereotypy and acoustic quality of individual note production, as well as stereotypy of the temporal structure of the song phrase. We did not discern any differences in the pattern of behavioral disruption between birds that were treated with either flutamide, tamoxifen, or a combination of both drugs. Flutamide treatment resulted in a reduced size of two forebrain nuclei that are known to play some role unique to early phases of song learning [lateral magnocellular nucleus of the anterior neostriatum (IMAN) and area X (X)], but did not affect the size of two song-control nuclei that are necessary for normal song production in adult birds [caudal nucleus of the ventral hyperstriatum (HVc) and robust nucleus of the archistriatum (RA)]. In contrast, treatment with tamoxifen did not result in any changes in the size of song-control nuclei relative to normal controls, and it blocked the effects of flutamide on the neural song-control system in birds that were treated with both drugs. Castration and antisteroid treatment exerted no deleterious effects on the quality of song behavior in adult birds, indicating that gonadal hormones are necessary for the development of normal song behavior during a sensitive period.     

Early life conditions that impact song learning in male zebra finches also impact neural and behavioral responses to song in females

Early life stressors can impair song in songbirds by negatively impacting brain development and subsequent learning. Even in species in which only males sing, early life stressors might also impact female behavior and its underlying neural mechanisms, but fewer studies have examined this possibility. We manipulated brood size in zebra finches to simultaneously examine the effects of developmental stress on male song learning and female behavioral and neural response to song. Although adult male HVC volume was unaffected, we found that males from larger broods imitated tutor song less accurately. In females, early condition did not affect the direction of song preference: all females preferred tutor song over unfamiliar song in an operant test. However, treatment did affect the magnitude of behavioral response to song: females from larger broods responded less during song preference trials. This difference in activity level did not reflect boldness per se, as a separate measure of this trait did not differ with brood size. Additionally, in females we found a treatment effect on expression of the immediate early gene ZENK in response to tutor song in brain regions involved in song perception (dNCM) and social motivation (LSc.vl, BSTm, TnA), but not in a region implicated in song memory (CMM). These results are consistent with the hypothesis that developmental stressors that impair song learning in male zebra finches also influence perceptual and/or motivational processes in females. However, our results suggest that the learning of tutor song by females is robust to disturbance by developmental stress. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018