Sexually dimorphic SCAMP1 expression in the forebrain motor pathway for song production of juvenile zebra finches

Mechanisms regulating sexual differentiation of the zebra finch song system are not well understood. The present study was designed to more fully characterize secretory carrier membrane protein 1 (SCAMP1), which was identified in a cDNA microarray screen as showing increased expression in the forebrains of developing male compared with female zebra finches. We completed the sequence of the open reading frame and used in situ hybridization to compare mRNA in song control regions of juvenile (25-day-old) individuals. Expression was significantly greater in the HVC (used as a proper name) and robust nucleus of the arcopallium (RA) in males than in females. Immunohistochemistry revealed that SCAMP1 protein is also expressed in these two brain regions, and qualitatively appears greater in males. Western analysis confirmed that the protein is increased in the telencephalon of males when compared with females at 25 days of age. These results are consistent with the idea that SCAMP1 is involved in masculinization of these brain areas, perhaps facilitating the survival of cells within them.     

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.     

Enhanced gene expression in the forebrain of hatchling and juvenile male zebra finches

The molecular mechanisms regulating sexual differentiation of the brain are largely unknown, although progress is being made, particularly in some mammalian systems. To uncover more of the key factors, a screen was conducted for genes involved in sexually dimorphic development of the neural song system in zebra finches. cDNA microarrays were initially used to compare gene expression in the telencephalons of hatchling and juvenile males and females. Then, real-time quantitative polymerase chain reaction (PCR) was employed to confirm sex differences, and the brain regions expressing the cDNAs of interest were localized using in situ hybridization. Several genes, including those likely to encode two ribosomal proteins (RPL17 and RPL37), SCAMP1, ZNF216, and a COBW-domain containing protein, showed enhanced expression in the telencephalon of males compared to females. In several cases, expression in the song control nuclei specifically was detected only in males. Interestingly, the sequences of some of these cDNAs shared substantial homology with regions of the chicken Z chromosome (male birds are ZZ, females ZW). Thus, we have identified genes likely to be involved in masculinization of the structure and/or function of the song circuit, some of which could be initial triggers for the sexual differentiation process.     

Song discrimination learning in zebra finches

Young male and female zebra finches, Taeniopygia guttata, were tested for their ability to discriminate between the song of their father or tutor and that of another male in two types of preference test, one with a male whose song was dissimilar to that of the father or tutor and one with a male who sang a similar song, on removal from the father, at 4 months and at 6 months of age. Birds that were isolated from their father at 25 days (group A) did not show a preference for their father's song whereas those isolated from the father at 35 days (groups B and C) learnt to prefer their father's song. In addition, group C, which were housed with tutor 1 from 35 to 70 days and with tutor 2 from 4 to 6 months of age, preferred their tutors' songs to those of other males. If one compares the strength of preference in the two types of test, group B showed a weaker preference for the father in tests with the father and a similar male. Further, group C showed a weaker preference in the test with tutor 1 versus a similar male. The possible influence of tutor, sibling and own song on the discrimination of the father's song is discussed, and the results are compared with those for song performance learning. Finally, the mother birds preferred their mate's song.     

Early exposure leads to repeatable preferences for male song in female zebra finches

This study addressed the question of how early learning processes in females influence later preferences for a male trait. I tested whether exposure to song alone (of a male other than the father) was sufficient for inducing a stable (repeatable) preference in female zebra finches (Taeniopygia guttata) by limiting early exposure to tape tutoring. A group of controls heard no songs before also being tested in adulthood. Repeated tests for preferences for tutor or unfamiliar song were made, interspersed with additional tests involving new songs. Preferences were tested in an operant task where pecking of response keys led to song playback. Most females significantly preferred one of the two songs in a given test. In the first test, the relative preference for the tutor song was significantly higher for the tutored than for the control females. Subsequently, tutored females' preferences for the tutor song remained higher on average, but the two groups did not differ significantly. However, tutored, but not untutored females' preferences were highly repeatable between tests, suggesting that early exposure to song might lead to a consolidation ol choice behaviour, a previously unknown effect of early exposure to song in female songbirds.     

Behavioral discrimination of sexually dimorphic calls by male zebra finches requires an intact vocal motor pathway

Vocal communication between zebra finches includes the exchange of long calls (LCs) as well as song. By using this natural call behavior and quantifying the LCs emitted in response to playbacks of LCs of other birds, we have previously shown that adult male zebra finches have a categorical preference for the LCs of females over those of males. Female LCs are acoustically simpler than male LCs, which include complex acoustic features that are learned during development. Production of these male-typical features requires an intact nucleus RA, the sexually dimorphic source of the main telencephalic projection to brainstem vocal effectors. We have now made bilateral lesions of RA in 17 adult males and tested their discrimination behavior in the call response situation. Lesioned birds continue to call, but lose the male-typical preference for female LCs. The degree of loss is correlated with the extent of RA damage. Further, the simplified LCs of males with RA lesions have a variable duration that is correlated with stimulus features. In effect, the call response behavior of lesioned males becomes like that of females. Apparently, in the absence of RA, the remaining intact structures receive different call information than RA normally does, and/or process it differently. This suggests that the vocal motor nucleus RA could play a role in the transformation of a signal encoding the salience of stimulus parameters into a control signal that modulates the probability and strength of responding.     

Factors limiting song acquisition in adult zebra finches

Song learning takes place in two separate or partially overlapping periods, a sensory phase in which a tutor song is memorized and a sensorimotor phase in which a copy of the model is produced. The stage of song development where song becomes stable and stereotyped is called crystallization. Adult birds usually do not learn new song in many species including the zebra finch. However, it is not known whether song crystallization as such or aging impedes adult learning. Exposure to loud noises prevents birds from developing and crystallizing their song, because they cannot control their voice by auditory feedback. Zebra finches even without previous experience of hearing or singing a song failed to learn a song model provided in adulthood. Thus, neither the absence of a tutor song nor the lack of song crystallization enables new song learning in adulthood, but age per se limits the ability or motivation to learn song. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

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     

Dynamic gene expression in the song system of zebra finches during the song learning period

The brain circuitry that controls song learning and production undergoes marked changes in morphology and connectivity during the song learning period in juvenile zebra finches, in parallel to the acquisition, practice and refinement of song. Yet, the genetic programs and timing of regulatory change that establish the neuronal connectivity and plasticity during this critical learning period remain largely undetermined. To address this question, we used in situ hybridization to compare the expression patterns of a set of 30 known robust molecular markers of HVC and/or area X, major telencephalic song nuclei, between adult and juvenile male zebra finches at different ages during development (20, 35, 50 days post‐hatch, dph). We found that several of the genes examined undergo substantial changes in expression within HVC or its surrounds, and/or in other song nuclei. They fit into broad patterns of regulation, including those whose expression within HVC during this period increases (COL12A1, COL 21A1, MPZL1, PVALB, and CXCR7) or decreases (e.g., KCNT2, SAP30L), as well as some that show decreased expression in the surrounding tissue with little change within song nuclei (e.g. SV2B, TAC1). These results reveal a broad range of molecular changes that occur in the song system in concert with the song learning period. Some of the genes and pathways identified are potential modulators of the developmental changes associated with the emergence of the adult properties of the song control system, and/or the acquisition of learned vocalizations in songbirds. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1315–1338, 2015     

Metabolic and respiratory costs of increasing song amplitude in zebra finches

Bird song is a widely used model in the study of animal communication and sexual selection, and several song features have been shown to reflect the quality of the singer. Recent studies have demonstrated that song amplitude may be an honest signal of current condition in males and that females prefer high amplitude songs. In addition, birds raise the amplitude of their songs to communicate in noisy environments. Although it is generally assumed that louder song should be more costly to produce, there has been little empirical evidence to support this assumption. We tested the assumption by measuring oxygen consumption and respiratory patterns in adult male zebra finches (Taeniopygia guttata) singing at different amplitudes in different background noise conditions. As background noise levels increased, birds significantly increased the sound pressure level of their songs. We found that louder songs required significantly greater subsyringeal air sac pressure than quieter songs. Though increased pressure is probably achieved by increasing respiratory muscle activity, these increases did not correlate with measurable increases in oxygen consumption. In addition, we found that oxygen consumption increased in higher background noise, independent of singing behaviour. This observation supports previous research in mammals showing that high levels of environmental noise can induce physiological stress responses. While our study did not find that increasing vocal amplitude increased metabolic costs, further research is needed to determine whether there are other non-metabolic costs of singing louder or costs associated with chronic noise exposure.     

Conspecific and heterospecific behavioural discrimination of individual odours by mound-building mice

We have tested the ability of male mound-building mice, Mus spicilegus, to discriminate on the basis of their social odours a) two males of their own species; and b) two males of the house mouse, Mus musculus domesticus. An habituation-dishabituation procedure was used. An experimental animal was presented with the scent from the same stimulus animal for four trials; on the fifth trial, scent from a second stimulus animal was presented. Male Mus spicilegus were able to discriminate the olfactory signatures of two mound-building mouse males but did not discriminate between the olfactory signatures of two house mouse males. The lack of inter-specific individual recognition is discussed in terms of specificity and attractive value of odour cues.     

Androstenedione therapy reinstates normal, not supernormal, song structure in castrated adult male zebra finches.

In previous studies, androstenedione (AE) replacement therapy restored the highest levels and intensities of courtship song displays in castrated male zebra finches of any hormone tested. Furthermore, female zebra finches responded strongly to AE-treated males and preferred intact males given small AE implants to unsupplemented males. In this study, we asked whether AE treatment might alter song structure, since male song is an important cue in mate choice by female zebra finches. Songs of adult males were recorded. The males were then castrated and given AE therapy and recorded again. No differences were found between the courtship or undirected songs males sang before castration and after AE treatment. As in previous studies, the structure of a male's courtship song differed significantly from his undirected song, and the structural differences between these two song types were not altered by AE treatment.     

Effects of housing condition and early corticosterone treatment on learned features of song in adult male zebra finches

Early developmental stress can have long-term physiological and behavioral effects on an animal. Developmental stress and early corticosterone (Cort) exposure affect song quality in many songbirds. Early housing condition can act as a stressor and affect the growth of nestlings and adult song, and improvements in housing condition can reverse adverse effects of early stress exposure in rodents. However, little is known about this effect in songbirds. Therefore, we took a novel approach to investigate if housing condition can modify the effects of early Cort exposure on adult song in male zebra finches. We manipulated early housing conditions to include breeding in large communal flight cages (FC; standard housing condition; with mixed-sex and mix-aged birds) versus individual breeding cages (IBC, one male–female pair with small, IBC-S, or large clutches, IBC-L) in post-hatch Cort treated male birds. We found that Cort treated birds from IBC-S have higher overall song learning scores (between tutor and pupil) than from FC but there is no difference between these groups in the No-Cort treated birds. When examining the effects of Cort within each housing condition, overall song learning scores decreased in Cort treated birds from flight cages but increased in birds from IBC-S compared to controls. Likewise, the total number of syllables and syllable types increased significantly in Cort treated birds from IBC-S, but decreased in FC-reared birds though this effect was not statistically significant. These findings suggest that the effects of early Cort treatment on learned features of song depend on housing condition.     

Antiandrogen blocks estrogen-induced masculinization of the song system in female zebra finches

Song behavior and the neural song system that serves it are sexually dimorphic in zebra finches. In this species, males sing and females normally do not. The sex differences in the song system include sex differences in the proportion of neurons that express androgen receptors, which is higher in specific brain regions of males. Estradiol (E2) administered in early development profoundly masculinizes the song system of females, including the proportion of neurons expressing androgen receptors. We examined whether or not the expression of these androgen receptors was causally related to the E2-induced masculinization of this system by co-administering Flutamide, which blocks androgen action at the receptor, along with E2 at hatching. E2 alone had its usual masculinizing effect on the female song system, measured in adulthood: increasing the size of song nuclei, the size of neurons in HVC, RA, and 1MAN, and the number of neurons in HVC. E2's masculinizing action, however, was significantly diminished on all measures by co-administering Flutamide. Indeed, females receiving both E2 and Flutamide were never significantly more masculine than controls on any measure. Flutamide alone had no effect. Our results strongly suggest that the activation of androgen receptors is necessary for the E2-induced masculinization of the song system in females.     

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.