Testosterone and the incidence of hormone target cells in song-control nuclei of adult canaries

Adult male canaries learn to produce high-amplitude complex courtship songs each breeding season, whereas females do not, and brain nuclei involved with the production of song behavior are much larger in breeding males than in nonbreeding males or females (Nottebohm, 1980, 1981). However, treatment of adult females with testosterone (T) causes them to produce male-like song and stimulates pronounced growth of some song-control brain nuclei such as the caudal nucleus of the ventral hyperstriatum (HVc). We reexamined the effects of T on song-control nuclei in deafened birds. In order to examine whether the pattern of hormone accumulation varies as a function of circulating testosterone levels we described the distribution of testosterone-concentrating cells in HVc and the magnocellular nucleus of the anterior neostriatum (MAN) in hearing adult male, female, and T-treated female canaries, as well as in deaf T-treated and untreated females. In contrast to our previous findings (Bottjer, Schoonmaker, and Arnold, 1986a), we observed no tendency in this study for testosterone-induced growth of HVc to be attenuated in deafened birds. There was no difference between deaf and hearing birds in the incidence of labeled cells within HVc. We also observed no sex or hormone-induced differences in the percentage of hormone-concentrating cells in HVc: normal females have approximately the same proportion of hormone target cells as do males and T-treated females. However, males normally have many more neurons in HVc than do control females, and systemic exposure to testosterone induces a pronounced increase in the number of HVc neurons of adult females. Therefore, the absolute number of hormone target cells in HVc is likely to be much greater in males and T-treated females than in normal females. As in HVc, there were no differences among groups in the proportion of labeled cells within lateral MAN (IMAN), a nucleus that has been implicated in song learning (Bottjer, Miesner and Arnold, 1984). In contrast, the incidence of hormone target cells in medial MAN (mMAN) did vary as a function of hormonal condition: although mMAN of normal females is rarely visible in Nissl-stained sections and cells in this region are not hormone labeled, mMAN is clearly visible in Nisslstained sections of males and T-treated females and contains many hormone-labeled cells. This testosterone-induced change in the phenotype of mMAN cells suggests a possible role for mMAN in learned song behavior.     

Testosterone and the incidence of hormone target cells in song-control nuclei of adult canaries.

Adult male canaries learn to produce high-amplitude complex courtship songs each breeding season, whereas females do not, and brain nuclei involved with the production of song behavior are much larger in breeding males than in nonbreeding males or females (Nottebohm, 1980, 1981). However, treatment of adult females with testosterone (T) causes them to produce male-like song and stimulates pronounced growth of some song-control brain nuclei such as the caudal nucleus of the ventral hyperstriatum (HVc). We reexamined the effects of T on song-control nuclei in deafened birds. In order to examine whether the pattern of hormone accumulation varies as a function of circulating testosterone levels we described the distribution of testosterone-concentrating cells in HVc and the magnocellular nucleus of the anterior neostriatum (MAN) in hearing adult male, female, and T-treated female canaries, as well as in deaf T-treated and untreated females. In contrast to our previous findings (Bottjer, Schoonmaker, and Arnold, 1986a), we observed no tendency in this study for testosterone-induced growth of HVc to be attenuated in deafened birds. There was no difference between deaf and hearing birds in the incidence of labeled cells within HVc. We also observed no sex or hormone-induced differences in the percentage of hormone-concentrating cells in HVc: normal females have approximately the same proportion of hormone target cells as do males and T-treated females. However, males normally have many more neurons in HVc than do control females, and systemic exposure to testosterone induces a pronounced increase in the number of HVc neurons of adult females. Therefore, the absolute number of hormone target cells in HVc is likely to be much greater in males and T-treated females than in normal females. As in HVc, there were no differences among groups in the proportion of labeled cells within lateral MAN (IMAN), a nucleus that has been implicated in song learning (Bottjer, Miesner and Arnold, 1984). In contrast, the incidence of hormone target cells in medial MAN (mMAN) did vary as a function of hormonal condition: although mMAN of normal females is rarely visible in Nissl-stained sections and cells in this region are not hormone labeled, mMAN is clearly visible in Nissl-stained sections of males and T-treated females and contains many hormone-labeled cells. This testosterone-induced change in the phenotype of mMAN cells suggests a possible role for mMAN in learned song behavior.     

Neuron loss and addition in developing zebra finch song nuclei are independent of auditory experience during song learning

In zebra finches early auditory experience is critical for normal song development. Young males first listen to and memorize a suitable song model and then use auditory feedback from their own vocalizations to mimic that model. During these two phases of vocal learning, song-related brain regions exhibit large, hormone-induced changes in volume and neuron number. Overlap between these neural changes and auditory-based vocal learning suggests that processing and acquiring auditory input may influence cellular processes that determine neuron number in the song system. We addressed this hypothesis by measuring neuron density, nuclear volume, and neuron number within the song system of normal male zebra finches and males deafened prior to song learning (10 days of age). Measures were obtained at 25, 50, 65, and 120 days of age, and included four song nuclei: the hyperstriatum ventralis pars caudalis or higher vocal center (HVc), Area X, the robust nucleus of the archistriatum (RA), and the lateral magnocellular nucleus of the anterior neostriatum (IMAN). In both HVc and Area X, nuclear volume and neuron number increased markedly with age in both normal and deafened birds. The volume of RA also increased with age and was not affected by early deafening. In IMAN, deafening also did not affect the overall age-related loss of neurons, although at 25 days neuron number was slightly less in deafened than in normal birds. We conclude that while the addition and loss of neurons in the developing song system may provide plasticity essential for song learning, these changes do not reflect learning.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Hormone accumulation in song regions of the canary brain.

[3H]Testosterone (T) was injected into male and female canaries (Serinus canarius), a species in which females are able to sing but do so more rarely and more simply than males. Autoradiographic analysis revealed that males and females have equal proportions of cells labeled by T or its metabolites in four song control nuclei: the high vocal center (HVC), the lateral portion of the magnocellular nucleus of the anterior neostriatum (IMAN), the robust nucleus of the archistriatum (RA), and the hypoglossal motor nucleus (nXII). Labeled cells were also observed in both sexes in the medial portion of MAN, and in hypothalamic nuclei. In both sexes, labeled cells in HVC, IMAN, RA, and nXII were larger than unlabeled cells. There were no sex differences in the size of either labeled or unlabeled cells in these song nuclei. The density of labeled cells per unit volume of tissue did not differ between the sexes in any song nucleus analyzed. However, because males have larger HVC and RA than females, males have a greater total number of hormone-sensitive cells in these regions than do females. Comparison of these results with measures of hormone accumulation in zebra finches and tropical duetting wrens suggests that the complexity of song that a bird can produce is correlated with the total number of hormone-sensitive cells in song nuclei.     

Tamoxifen fails to block estradiol accumulation, yet is weakly accumulated by the juvenile zebra finch anterior hypothalamus: an autoradiographic study.

In experiment 1, we used autoradiographic procedures to examine whether tamoxifen could displace 3H-estradiol labeling in the anterior hypothalamus and the caudal nucleus of the ventral hyperstriatum (HVc) of ovariectomized 20-day-old female zebra finches. There was no significant reduction in labeling of cells by 3H-estradiol in birds preinjected with unlabeled tamoxifen. In experiment 2, we found that injections of 3H-tamoxifen caused-weak labeling of cells in the anterior hypothalamus of 20-day-old male and female zebra finches. These results are compatible with the idea that tamoxifen does not block the action of estradiol in the brain of zebra finches, and suggest that the effects of early tamoxifen treatment on the morphology of the song system may reflect central actions of tamoxifen.     

Cell death during development of a forebrain nucleus involved with vocal learning in zebra finches

Lateral MAN (magnocellular nucleus of the anterior neostriatum) is a forebrain nucleus that is known to be importantly involved with vocal learning in juvenile male zebra finches only during a restricted period of the learning process: lesions of lMAN completely disrupt song behavior in zebra finches prior to 50 days of age but have little or no effect in older juvenile or adult birds. The development of lMAN, as of other song-control regions, is delayed until the time that song behavior is being learned. Lateral MAN undergoes a substantial loss of neurons between 25 and 55 days of age, a time that encompasses initial stages of vocal production as well as the interval during which lMAN lesions become ineffective. In this study, we measured both the time course of neuronal loss and the incidence of pyknotic cells within lMAN during the period of cell loss. There is a pronounced loss of neurons from lMAN between 20 and 35 days, after which the adult number of neurons is established. The incidence of pyknosis is greatest at 20 days, around the time when the loss of live cells is also most pronounced, suggesting that the loss of neurons from lMAN is attributable to cell death. The loss of neurons occurs well before lesions of lMAN become ineffective in disrupting vocal behavior. Thus the neurons remaining in lMAN after the period of cell loss apparently undergo a substantial change in function at the time lesions lose effectiveness (about 55-60 days).     

Hormone accumulation in song regions of the canary brain

[³H]Testosterone (T) was injected into male and female canaries (Serinus canarius), a species in which females are able to sing but do so more rarely and more simply than males. Autoradiographic analysis revealed that males and females have equal proportions of cells labeled by T or its metabolites in four song control nuclei: the high vocal center (HVC), the lateral portion of the magnocellular nucleus of the anterior neostriatum (IMAN), the robust nucleus of the archistriatum (RA), and the hypoglossal motor nucleus (nXII). Labeled cells were also observed in both sexes in the medial portion of MAN, and in hypothalamic nuclei. In both sexes, labeled cells in HVC, IMAN, RA, and nXII were larger than unlabeled cells. There were no sex differences in the size of either labeled or unlabeled cells in these song nuclei. The density of labeled cells per unit volume of tissue did not differ between the sexes in any song nucleus analyzed. However, because males have larger HVC and RA than females, males have a greater total number of hormone-sensitive cells in these regions than do females. Comparison of these results with measures of hormone accumulation in zebra finches and tropical duetting wrens suggests that the complexity of song that a bird can produce is correlated with the total number of hormone-sensitive cells in song nuclei. © 1992 John Wiley & Sons, Inc.     

Tamoxifen fails to block estradiol accumulation,yet is weakly accumulated by the juvenile zebra finch anterior hypothalamus: An autoradiographic study

In experiment 1, we used autoradiographic procedures to examine whether tamoxifen could displace ³H-estradiol labeling in the anterior hypothalamus and the caudal nucleus of the ventral hyperstriatum (HVc) of ovariectomized 20-day-old female zebra finches. There was no significant reduction in labeling of cells by ³H-estradiol in birds preinjected with unlabeled tamoxifen. In experiment 2, we found that injections of ³H-tamoxifen causedweak labeling of cells in the anterior hypothalamus of 20-day-old male and female zebra finches. These results are compatible with the idea that tamoxifen does not block the action of estradiol in the brain of zebra finches, and suggest that the effects of early tamoxifen treatment on the morphology of the song system may reflect central action of tamoxifen.     

The relationship between rates of HVc neuron addition and vocal plasticity in adult songbirds

In adulthood, songbird species vary considerably in the extent to which they rely on auditory feedback to maintain a stable song structure. The continued recruitment of new neurons into vocal motor circuitry may contribute to this lack of resiliency in song behavior insofar as new neurons that are not privy to auditory instruction could eventually corrupt established neural function. In a first step to explore this possibility, we used a comparative approach to determine if species differences in the rate of vocal change after deafening in adulthood correlate positively with the extent of HVc neuron addition. We confirmed previous reports that deafening in adulthood changes syllable phonology much more rapidly in bengalese finches than in zebra finches. Using [(3)H]thymidine autoradiography to identify neurons generated in adulthood, we found that the proportion of new neurons in the HVc one month after labeling was nearly twice as great in bengalese than in zebra finches. Moreover, among the subset of HVc vocal motor neurons that project to the robust nucleus of the archistriatum, the incidence of [(3)H]thymidine-labeled neurons was nearly three times as great in bengalese than in zebra finches. This correlation between the proportion of newly added neurons and the rate of song deterioration supports the hypothesis that HVc neuron addition may disrupt stable adult song production if new neurons cannot be "trained" via auditory feedback.     

Comparative study of long-term reduction of high-frequency song syllables in deafened zebra and Bengalese finches: implications for the role of auditory feedback in song maintenance

Auditory feedback is necessary for adult song maintenance in both oscines and psittacines. Although belonging to phylogenically separated orders, deafened adult oscine Bengalese finches and psittacine budgerigars exhibit similarities in certain aspects of song changes. An interesting question is whether these birds share common mechanisms for song maintenance. Therefore, it is important to compare the effects of deafening on adult song patterns among and within orders. Although zebra and Bengalese finches are closely related oscine species, few studies have performed direct, long-term, quantitative comparisons of their songs after deafening because suitable song characteristics have not been identified. Based on our previous findings for Bengalese finch songs, we analyzed zebra finch songs over 9 months after deafening, focusing on changes in the number of syllables categorized according to fundamental frequencies. Deafened zebra finches demonstrated a gradual but significant decrease in high-frequency syllables and a tendency to increase low-frequency syllables, similar to deafened Bengalese finches. Although this change took longer in zebra finches, the altered proportion of syllables eventually stabilized. Results indicated that adult songs show similar aspects after auditory deprivation, and that neural mechanisms involved in the maintenance of high-frequency song syllables, using auditory feedback, may be present in both finches despite species differences.     

Lesions of a telencephalic nucleus in male zebra finches: Influences on vocal behavior in juveniles and adults

Male zebra finches learn to sing during a restricted phase of juvenile development. Song learning is characterized by the progressive modification of unstable song vocalizations by juvenile birds during development, a process that leads to the production of stereotyped vocal patterns as birds reach adulthood. The medial magnocellular nucleus of the anterior neostriatum (mMAN) is a small cortical region that has been implicated in song behavior based on its neuronal projection to the High Vocal Center (HVC), a nucleus that is critical for adult vocal production and presumably also plays a role in song learning. To assess the function of mMAN in song, ibotenic acid lesions of this brain region were made in juvenile male zebra finches during the period of vocal learning (40-50 days of age) and in adult males that were producing stable song (>90 days of age). Birds lesioned as juveniles produced highly abnormal, poor quality song as adults. Although the overall song quality of birds lesioned as adults was not highly disrupted or abnormal, the postoperative song behavior of these birds was discernibly different due to slight increases in variability of vocal production, particularly at the onset of singing. These results demonstrate that mMAN plays some important role in vocal production during the sensitive period for song learning, and is also important for consistent initiation and stereotyped production of adult song behavior.     

Manipulations of sensory experiences during development reveal mechanisms underlying vocal learning biases in zebra finches

Biological predispositions in learning can bias and constrain the cultural evolution of social and communicative behaviors (e.g., speech and birdsong), and lead to the emergence of behavioral and cultural “universals.” For example, surveys of laboratory and wild populations of zebra finches (Taeniopygia guttata) document consistent patterning of vocal elements (“syllables”) with respect to their acoustic properties (e.g., duration, mean frequency). Furthermore, such universal patterns are also produced by birds that are experimentally tutored with songs containing randomly sequenced syllables (“tutored birds”). Despite extensive demonstrations of learning biases, much remains to be uncovered about the nature of biological predispositions that bias song learning and production in songbirds. Here, we examined the degree to which “innate” auditory templates and/or biases in vocal motor production contribute to vocal learning biases and production in zebra finches. Such contributions can be revealed by examining acoustic patterns in the songs of birds raised without sensory exposure to song (“untutored birds”) or of birds that are unable to hear from early in development (“early‐deafened birds”). We observed that untutored zebra finches and early‐deafened zebra finches produce songs with positional variation in some acoustic features (e.g., mean frequency) that resemble universal patterns observed in tutored birds. Similar to tutored birds, early‐deafened birds also produced song motifs with alternation in acoustic features across adjacent syllables. That universal acoustic patterns are observed in the songs of both untutored and early‐deafened birds highlights the contribution motor production biases to the emergence of universals in culturally transmitted behaviors.     

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.     

Estrogen accumulation in zebra finch song control nuclei: implications for sexual differentiation and adult activation of song behavior

In zebra finches the gonadal steroid estradiol (E2) directs the sexual differentiation of neural regions controlling song and synergizes with androgens to stimulate song in adulthood. To identify regions where E2 may act to exert these effects, steroid autoradiographic techniques were used to assess cellular accumulation of 3[H]-E2 or its metabolites within various nuclei of the zebra finch brain. In Experiment 1 we examined brains from juvenile females, still within the critical period for E2's effect on sexual differentiation. In Experiment 2 the pattern and extent of labeling in adult male brains was determined following injection of 3[H]-E2, 3[H]-testosterone, or 3[H]-dihydrotestosterone. The results suggest that, both during development and in adulthood, most song-control nuclei contain few E2-accumulating cells. In contrast, many cells densely labeled by 3[H]-E2 or its metabolites are present in the hypothalamus and in close proximity to one song-control region, the hyperstriatum ventralis pars caudalis (HVc). The distribution of these latter cells overlaps with cells that project to another song-related nucleus, Area X. Thus, in Experiment 3 fluorescent retrograde tracing and steroid autoradiographic techniques were combined to determine if E2-accumulating cells project to Area X in adult males. Although a few retrogradely labeled cells were lightly labeled by 3[H]-E2 or its metabolites, for the most part these appear to be two distinct populations of cells. The sparse accumulation of E2 in the zebra finch song system contrasts with that described in other song birds and has important implications as to the mechanism of E2 action on the developing and mature song system.     

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.     

Testosterone decreases the potential for song plasticity in adult male zebra finches

Zebra finches are age-limited learners; males crystallize their songs at 90 days and do not subsequently alter those songs. However, a variety of interventions, including deafening and syringeal denervation, result in long-term changes to the crystallized song. These changes can be prevented by lesioning nucleus LMAN. As different social contexts for song production result in differential activation of LMAN, we asked whether the social context experienced by adult males would affect their ability to alter their songs in response to syringeal denervation. Males able to see and direct their songs to females made fewer changes to their songs than did males that could hear but not see females, but this trend was not significant. The volume of a male's HVc, a forebrain song control nucleus, also failed to predict the degree to which a male would change his song. However, testis mass was significantly correlated with the number of changes made to the song, indicating that variations in testosterone modulate adult song plasticity. We directly tested the effect of circulating testosterone on adult song plasticity by implanting adult males with either testosterone or flutamide, a testosterone receptor blocker, and tracking song changes triggered by ts nerve injury. As predicted, males implanted with testosterone changed their songs less than did males that received flutamide implants. These results suggest that the high testosterone concentrations associated with sexual maturity and song crystallization in zebra finches continue to act in adult males to reduce the potential for vocal plasticity.     

Changes in adult zebra finch song require a forebrain nucleus that is not necessary for song production

Male zebra finches normally crystallize song at approximately 90 days and do not show vocal plasticity as adults. However, changes to adult song do occur after unilateral tracheosyringeal (ts) nerve injury, which denervates one side of the vocal organ. We examined the effect of placing bilateral lesions in LMAN (a nucleus required for song development but not for song maintenance in adults) upon the song plasticity that is induced by ts nerve injury in adults. The songs of birds that received bilateral lesions within LMAN followed by right ts nerve injury silenced, on average, 0.25 syllables, and added 0.125 syllables (for an average turnover of 0.375 syllables), and changed neither the frequency with which individual syllables occurred within songs nor the motif types they used most often. In contrast, the songs of birds that received sham lesions followed by ts nerve injury lost, on average, 1.625 syllables, silenced 0.125 syllables, and added 0.75 syllables, turning over an average of 2.5 syllables. They also significantly changed both the frequency with which individual syllables were included in songs and the motif variants used. Thus, song plasticity induced in adult zebra finches with crystallized songs requires the presence of LMAN, a nucleus which had been thought to play a role in vocal production only during song learning. Although the changes to adult songs induced by nerve transection are more limited than those that arise during song development, the same circuitry appears to underlie both types of plasticity.