Recovery of song preferences after excitotoxic HVC lesion in female canaries

The courtship solicitation display (CSD) of the female canary is a model to study estrogen dependent auditory preferences for male songs. The forebrain auditory-vocal nucleus, HVC, is part of the circuit that determines such preferences. To further develop this model we show that bilateral excitotoxic lesions of the medial part of HVC involving between 18-60% of the bilateral nucleus are behaviorally effective while complete unilateral lesions are not. Further, we show that animals recover their song preferences over a period of several months after the lesion. This functional recovery does not involve anatomical recovery of the HVC. Even 9 months after the lesion, the HVC size of these females was similar to that of females sacrificed 2 days after the lesion and thus was 40 +/- 8% smaller compared to normal females. Further, ipsilaterally, the lesion procedure transiently disturbed the neurochemistry, such as GAD-mRNA expression, in the part of HVC that did not undergo cell death. These results suggest that the integrity of the lateral part of at least one HVC is required to perform CSD in response to relevant auditory stimuli.     

Yolk testosterone, postnatal growth and song in male canaries

Avian eggs contain substantial amounts of maternal yolk androgens, which have been shown to modulate offspring phenotype. The first studies on the functional consequences of maternal yolk androgens have focused on early life stages and their role in sibling competition. However, recent longitudinal studies reported long-lasting effects of maternal yolk androgens on offspring phenotype, mostly concerning traits that are sensitive to androgens. This suggests that maternal yolk androgens could play an important role in sexual selection, since the expression of many male sexual characters is testosterone-dependent. Using male canaries as a model, we examined the consequences of an experimental elevation of yolk testosterone concentrations on early development as well as long-lasting effects particularly on song, which is one of the most important sexual characters in male songbirds. Elevated yolk testosterone concentrations inhibited male growth, possibly in interaction with an existent ectoparasite exposure. Males hatched from testosterone-treated eggs (T-males) did not have enhanced competitive skills, in contrast to previous studies. The elevation of yolk testosterone concentrations delayed song development but did not affect adult song phenotype. This is intriguing, as yolk testosterone possibly induced developmental stress, which is known to reduce song quality. We hypothesize that yolk testosterone has either no direct effect on adult song phenotype, or that positive effects are merged by the negative effects of developmental stress. Finally, females mated with T-males invested more in their clutch indicating that females either assess T-males as more attractive (differential allocation hypothesis) or compensated for lower offspring viability (compensation hypothesis).     

Female canaries that respond and discriminate more between male songs of different quality have a larger song control nucleus (HVC) in the brain

In male songbirds the song control pathway in the forebrain is responsible for song production and learning. In most species, females do not sing and have smaller nuclei in the song control pathway. Although the function of the pathway in females is assumed to be associated with the perception of male song, there is little direct evidence to support this view. In this study on female canaries, we investigate the role of two key nuclei in the song control pathway (HVC and lMAN) in relation to playback of male song. Male canaries produce elaborate songs that function to attract and stimulate females. The songs are constructed from smaller units called syllables, and special syllables with a more complex structure (sexy syllables) are known to induce females to perform copulation solicitation displays (CSD) as an invitation to mate. By using computer-edited experimental songs, we first show that females discriminate between songs by producing significantly more CSD to those containing sexy syllables. We then sectioned the brains and used in situ hybridization to reveal song nuclei containing androgen receptors. We report positive correlations between the size of HVC and both total CSD response and the amount of discrimination between sexy and nonsexy songs. We found no such relationships between these measures and the size of lMAN. These results provide some evidence to support the view that, in female canaries HVC is involved in female perception and discrimination of male song. The results also have implications for the evolution of complex male songs by sexual selection and female choice.     

LMAN lesions prevent song degradation after deafening without reducing HVC neuron addition

In some songbirds perturbing auditory feedback can promote changes in song structure well beyond the end of song learning. One factor that may drive vocal change in such deafened birds is the ongoing addition of new vocal-motor neurons into the song system. Without auditory feedback to guide their incorporation, the addition of these new neurons could disrupt the established song pattern. To assess this hypothesis, the authors determined if neuronal recruitment into the vocal motor nucleus HVC is affected by neural signals that influence vocal change in adult deafened birds. Such signals appear to be conveyed via LMAN, a nucleus in the anterior forebrain that is necessary for vocal change after deafening. Here the authors tested whether LMAN lesions might restrict song degradation after deafening by reducing the addition or survival of new HVC neurons that would otherwise corrupt the ongoing song pattern. Using [3H]thymidine autoradiography to identify neurons generated in adult zebra finches, it was shown here that LMAN lesions do not reduce the number or percent of new HVC neurons surviving for either several weeks or months after [3H]thymidine labeling. However, the authors confirmed previous reports that LMAN lesions restrict vocal change after deafening. These data suggest that neurons incorporated into the adult HVC may form behaviorally adaptive connections without requiring auditory feedback, and that any role such neurons may play in promoting vocal change after adult deafening requires anterior forebrain pathway output.     

The effect of auditory distractors on song discrimination in male canaries (Serinus canaria)

Male songbirds such as canaries produce complex learned vocalizations that are used in the context of mate attraction and territory defense. Successful mate attraction or territorial defense requires that a bird be able to recognize individuals based on their vocal performance and identify these songs in a noisy background. In order to learn more about how birds are able to solve this problem, we investigated, with a two-alternative choice procedure, the ability of adult male canaries to discriminate between conspecific song segments from two different birds and to maintain this discrimination when conspecific songs are superimposed with a variety of distractors. The results indicate that male canaries have the ability to discriminate, with a high level of accuracy song segments produced by two different conspecific birds. Song discrimination was partially maintained when the stimuli were masked by auditory distractors, but the accuracy of the discrimination progressively declined as a function of the number of masking distractors. The type of distractor used in the experiments (other conspecific songs or different types of artificial white noise) did not markedly affect the rate of deterioration of the song discrimination. These data indicate that adult male canaries have the perceptual abilities to discriminate and selectively attend to one ongoing sound that occurs simultaneously with one or more other sounds. The administration of a noradrenergic neurotoxin did not impair markedly the discrimination learning abilities although the number of subjects tested was too small to allow any firm conclusion. In these conditions, however, the noradrenergic lesion significantly increased the number failures to respond in the discrimination learning task suggesting a role, in canaries, of the noradrenergic system in some attentional processes underlying song learning and processing.     

Female canaries produce eggs with greater amounts of testosterone when exposed to preferred male song

Male birdsong has a great influence in the stimulation of female reproduction. However, female physiological responsiveness to song may depend on the degree of complexity of male song. This is expected because females of iteroparous organisms may increase their fitness by matching their reproductive investment to the predicted value of each reproductive attempt. To the extent that the expression of male ornaments is a signal of male quality, we expect females to increase their investment when paired to highly ornamented males. However, female investment may be cryptic and difficult to detect, such as androgen content in the eggs. In this study, we exposed female canaries (Serinus canaria) to attractive and unattractive song repertoires using a crossover design. As predicted, females invested greater concentrations of testosterone in their eggs when exposed to attractive repertoires than when exposed to unattractive repertoires. This implies that song repertoires convey important information about the reproductive value of a given male and suggests that testosterone deposition in egg yolk may be costly.     

Effect of artificially increased day length in winter on female domesticated canaries

Three groups of female canaries were given different schedules of increasing day-length between October and December: all groups responded in the same way. Three groups of female canaries were given a 20 hour light/four hour dark schedule at different periods outof the breeding season (October-December, October-January, and January-February).All experimental birds became defeathered and vascular. There was an increase in skin tactile sensitivity in October-December but not in January-February. Nest-building was induced in all months. Only six out of 41 experimental birds laid eggs. Eleven birds which did not lay showed marked incubation behaviour. Discussion centres around the hormonal control of brood patch formation. The possibility that the absence of an effect of photostimulation on sensitivity in January-February is due to shortage of progesterone is considered. These light schedules induced more nest-building than injected oestrogen: no conclusions are yet possible on the hormonal control of next-building.     

Bilateral LMAN lesions cancel differences in HVC neuronal recruitment induced by unilateral syringeal denervation

Twenty-six-day-old male zebra finches received (1) unilateral section of their tracheosyringeal nerve, (2) bilateral lesions of the lateral magnocellular nucleus of the anterior neostriatum (LMAN), and (3) both operations. All birds were kept with an adult, singing male as a tutor until day 65. Tracheo-syringeal nerve-cut birds were able to imitate this model, but LMAN-lesioned birds were not. Bromodeoxyuridine, a marker of cell division, was injected intramuscularly during post-hatching days 61-65 and all birds were killed at 91 days of age. The number of bromodeoxyuridine+ neurons in the high vocal center of the tracheosyringeal-cut birds was twice as high in the intact as in the nerve cut side. This asymmetry disappeared when nerve section was combined with bilateral LMAN lesions. The latter operation, by itself, had no effect on new neuron counts. We suggest that the single nerve cut produced a hemispheric asymmetry in learning, reflected in new neuron recruitment, which disappeared when LMAN lesions blocked learning.     

Bi-directional sexual dimorphisms of the song control nucleus HVC in a songbird with unison song

Sexually dimorphic anatomy of brain areas is thought to be causally linked to sex differences in behaviour and cognitive functions. The sex with the regional size advantage (male or female) differs between brain areas and species. Among adult songbirds, males have larger brain areas such as the HVC (proper name) and RA (robust nucleus of the arcopallium) that control the production of learned songs. Forest weavers (Ploceus bicolor) mated pairs sing a unison duet in which male and female mates learn to produce identical songs. We show with histological techniques that the volume and neuron numbers of HVC and RA were > or =1.5 times larger in males than in females despite their identical songs. In contrast, using in-situ hybridizations, females have much higher (30-70%) expression levels of mRNA of a number of synapse-related proteins in HVC and/or RA than their male counterparts. Male-typical and female-typical sexual differentiation appears to act on different aspects of the phenotypes within the same brain areas, leading females and males to produce the same behaviour using different cellular mechanisms.     

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.     

Social context affects testosterone-induced singing and the volume of song control nuclei in male canaries (Serinus canaria)

The contribution of social factors to seasonal plasticity in singing behavior and forebrain nuclei controlling song, and their interplay with gonadal steroid hormones are still poorly understood. In many songbird species, testosterone (T) enhances singing behavior but elevated plasma T concentrations are not absolutely required for singing to occur. Singing is generally produced either to defend a territory or to attract a mate and it is therefore not surprising that singing rate can be influenced by the sex and behavior of the social partner. We investigated, based on two independent experiments, the effect of the presence of a male or female partner on the rate of song produced by male canaries. In the first experiment, song rate was measured in dyads composed of one male and one female (M-F) or two males (M-M). Birds were implanted with T-filled Silastic capsules or with empty capsules as control. The number of complete song bouts produced by all males was recorded during 240 min on week 1, 2, 4, and 8 after implantation. On the day following each recording session, brains from approximately one-fourth of the birds were collected and the volumes of the song control nuclei HVC and RA were measured. T increased the singing rate and volume of HVC and RA but these effects were affected by the social context. Singing rates were higher in the M-M than in the M-F dyads. Also, in the M-M dyads a dominance-subordination relationship soon became established and dominant males sang at higher rates than subordinates in T-treated but not in control pairs. The differences in song production were not reflected in the size of the song control nuclei: HVC was larger in M-F than in M-M males and within the M-M dyads, no difference in HVC or RA size could be detected between dominant and subordinate males. At the individual level, the song rate with was positively correlated with RA and to a lower degree HVC volume, but this relationship was observed only in M-M dyads, specifically in dominant males. A second experiment, carried out with castrated males that were all treated with T and exposed either to another T-treated castrate or to an estradiol-implanted female, confirmed that song rate was higher in the M-M than in the M-F condition and that HVC volume was larger in heterosexual than in same-sex dyads. The effects of T on singing rate and on the volume of the song control nuclei are thus modulated by the social environment, including the presence/absence of a potential mate and dominance status among males.     

Hormonal acceleration of song development illuminates motor control mechanism in canaries

In songbirds, the ontogeny of singing behavior shows strong parallels with human speech learning. As in humans, development of learned vocal behavior requires exposure to an acoustic model of species‐typical vocalizations, and, subsequently, a sensorimotor practice period after which the vocalization is produced in a stereotyped manner. This requires mastering motor instructions driving the vocal organ and the respiratory system. Recently, it was shown that, in the case of canaries (Serinus canaria), the diverse syllables, constituting the song, are generated with air sac pressure patterns with characteristic shapes, remarkably, those belonging to a very specific mathematical family. Here, we treated juvenile canaries with testosterone at the onset of the sensorimotor practice period. This hormone exposure accelerated the development of song into stereotyped adultlike song. After 20 days of testosterone treatment, subsyringeal air sac pressure patterns of song resembled those produced by adults, while those of untreated control birds of the same age did not. Detailed temporal structure and modulation patterns emerged rapidly with testosterone treatment, and all previously identified categories of adult song were observed. This research shows that the known effect of testosterone on the neural circuits gives rise to the stereotyped categories of respiratory motor gestures. Extensive practice of these motor patterns during the sensorimotor phase is not required for their expression. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 943–960, 2010     

Oviduct development and its relation to other aspects of reproduction in domesticated canaries

The histology and development of the oviduct of unpaired female canaries during the natural breeding season were examined. The histology resembles that of the fowl. The tubular glands develop by invagination of the epithelial cells, and albumen granules subsequently form in their cytoplasm. The epithelium differentiates into ciliated columnar cells and goblet cells. The histology of the magnum is uniform along its length at all stages of development.
Oviduct development is closely correlated with that of the ovary. In its early stages there appears to be little correlation between oviduct development and that of the brood patch or of nest-building. The formation of albumen coincides with the enlargement of one ovarian follicle more than the rest, the completion of defeathering, and intensive nest-building behaviour.     

Temporal relations of brood patch development, nest-building and egg-laying in domesticated canaries

Relationships were assessed between brood patch development, nest-building and egg-laying in 20 female canaries in the natural breeding season. Twelve of these were unpaired; eight were provided with males after two months of being kept singly. The later laying of some females was due to their more protracted development rather than to their later start. The completion of brood patch defeathering, a certain stage of its vascularity, and the start of intensive nest-building occurred within a few days of each other whatever the date of egg-laying. Differences from an earlier study of paired females which had only limited access to nest material can be understood in terms of the known effects of the male and the nest-cup on reproductive development.
All birds showed a period of intensive nest-building beginning about eight days before egg-laying, with a peak about three days before. In those females in which the period of intensive building lasted for more than eight days, there was an earlier peak about seven days previously. This, and also the building behaviour of many females even earlier in the season, are in harmony with the view that reproductive development is not progressive, but intermittent.     

Song repertoire size varies with HVC volume and is indicative of male quality in song sparrows (Melospiza melodia)

Complex birdsong is a classic example of a sexually selected ornamental trait. In many species, females prefer males with large song repertoires, possibly because repertoire size is limited by the size of song control nuclei which reflect developmental success. We investigated whether song repertoire size was indicative of brain area and male quality in song sparrows (Melospiza melodia) by determining if repertoire size was related to the volume of song control nucleus HVC, as well as several morphological, immunological and genetic indices of quality. We found that males with large repertoires had larger HVCs and were in better body condition. They also had lower heterophil to lymphocyte ratios, indicating less physiological stress and a robust immune system as measured by the number of lymphocytes per red blood cell. Song repertoire size also tended to increase with neutral-locus genetic diversity, as assessed by mean d2, but was not related to internal relatedness. Our results suggest several mechanisms that might explain the finding of a recent study that song sparrows with large song repertoires have higher lifetime fitness.     

Lesions of the anterior forebrain song control pathway in female canaries affect song perception in an operant task

Sexual behavior in female rats, typified by the lordosis reflex, is dependent upon estrogen action in the ventromedial nucleus of the hypothalamus (VMH) and its surrounding neuropil. However, the synaptic organization of this brain region remains unclear. Pseudorabies virus (PRV) was used to transneuronally label the neural network that innervates the lumbar epaxial muscles that execute the lordosis response. PRV-labeled neurons were identified within and subjacent to the VMH four days after injection of PRV into the back muscles. The pattern of labeling was defined in relation to three landmarks: the VMH core, as defined by Crystal Violet staining; the shell, as defined by the oxytocin fiber tract; and the cluster of estrogen receptor-containing cell nuclei. The pattern of PRV labeling in the VMH displayed a striking rostral-caudal gradient. In general, many of the PRV-labeled neurons were found in the oxytocin fiber tract, with far fewer in the core of the VMH. Furthermore, PRV-labeled neurons were rarely found in the cluster of estrogen receptor-containing neurons, and less than 3% of the PRV-labeled neurons were double labeled for estrogen receptor. The results suggest that oxytocin may directly influence these lordosis-relevant VMH projection neurons, whereas estrogen may have transsynaptic effects.     

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     

Differential geographic patterns in song components of male Albert’s lyrebirds

Geographic variation in bird song has received much attention in evolutionary studies, yet few consider components within songs that may be subject to different constraints and follow different evolutionary trajectories. Here, we quantify patterns of geographic variation in the socially transmitted “whistle” song of Albert''s lyrebirds (Menura alberti), an oscine passerine renowned for its remarkable vocal abilities. Albert''s lyrebirds are confined to narrow stretches of suitable habitat in Australia, allowing us to map likely paths of cultural transmission using a species distribution model and least cost paths. We use quantitative methods to divide the songs into three components present in all study populations: the introductory elements, the song body, and the final element. We compare geographic separation between populations with variation in these components as well as the full song. All populations were distinguishable by song, and songs varied according to the geographic distance between populations. However, within songs, only the introductory elements and song body could be used to distinguish among populations. The song body and final element changed with distance, but the introductory elements varied independently of geographic separation. These differing geographic patterns of within‐song variation are unexpected, given that the whistle song components are always produced in the same sequence and may be perceived as a temporally discrete unit. Knowledge of such spatial patterns of within‐song variation enables further work to determine possible selective pressures and constraints acting on each song component and provides spatially explicit targets for preserving cultural diversity. As such, our study highlights the importance for science and conservation of investigating spatial patterns within seemingly discrete behavioral traits at multiple levels of organization.