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     

Neural response to bird’s own song and tutor song in the zebra finch field L and caudal mesopallium

Neurons in the song system nuclei of songbirds exhibit a strong preference for the sound of the birds own song relative to that of conspecific songs. This selectivity is observed in the high vocal center and the nucleus interface of the nidopallium, two song nuclei that receive input from the birds auditory system. To investigate the role of the auditory system in generating the selective responses observed in the song system, we recorded auditory responses in the zebra finch primary auditory forebrain, field L, and in a secondary auditory area, the caudal mesopallium. Field L and caudal mesopallium project directly or indirectly to the high vocal center and nucleus interface of the nidopallium and are presumed to provide substantial auditory input to the song system. We found that, on average, neurons in field L and caudal mesopallium did not show positive selective responses for the birds own song or tutor song relative to conspecific song. Moreover, there were no particular sub-areas in the auditory telencephalon that were relatively more selective than the average. The selectivity for the birds own song would therefore be restricted to song nuclei and would arise in one processing step, potentially found at the interface between the auditory and the song systems.Abbreviations BOS birds own song - CM caudal mesopallium (older term: caudal hyperstriatum ventrale or cHV) - Con conspecific song - HVC high vocal center - LMAN lateral magnocellular nucleus of the anterior nidopallium (older term: lateral magnocellular nucleus of the anterior neostriatum) - LPS pallial-subpallial lamina (older term: lamina medularis dorsalis or LMD) - NCM caudal medial nidopallium (older term: caudo-medial neostriatum) - NIf nucleus interface of the nidopallium (older term: nucleus interface of the neostriatum) - RA robust nucleus of arcopallium (older term: robust nucleus of the archistriatum) - Rev reverse BOS - Revorder reverse order of BOS - Uva nucleus uvaeformis of the thalamusNew avian brain terminology has been used in this paper (). Older terms are given in parentheses in the list of abbreviations     

Small molecule analysis and imaging of fatty acids in the zebra finch song system using time-of-flight-secondary ion mass spectrometry

Fatty acids are central to brain metabolism and signaling, but their distributions within complex brain circuits have been difficult to study. Here we applied an emerging technique, time-of-flight secondary ion mass spectrometry (ToF-SIMS), to image specific fatty acids in a favorable model system for chemical analyses of brain circuits, the zebra finch (Taeniopygia guttata). The zebra finch, a songbird, produces complex learned vocalizations under the control of an interconnected set of discrete, dedicated brain nuclei 'song nuclei'. Using ToF-SIMS, the major song nuclei were visualized by virtue of differences in their content of essential and non-essential fatty acids. Essential fatty acids (arachidonic acid and docosahexaenoic acid) showed distinctive distributions across the song nuclei, and the 18-carbon fatty acids stearate and oleate discriminated the different core and shell subregions of the lateral magnocellular nucleus of the anterior nidopallium. Principal component analysis of the spectral data set provided further evidence of chemical distinctions between the song nuclei. By analyzing the robust nucleus of the arcopallium at three different ages during juvenile song learning, we obtain the first direct evidence of changes in lipid content that correlate with progression of song learning. The results demonstrate the value of ToF-SIMS to study lipids in a favorable model system for probing the function of lipids in brain organization, development and function.     

Auditory-vocal cholinergic pathway in the songbird brain

Male zebra finches learn to imitate a tutor's song through auditory and motor learning. The two main song control nuclei in the zebra finch forebrain, the higher vocal center (HVC) and the robust nucleus of the archistriatum (RA), receive cholinergic innervation from the ventral paleostriatum (VP) of the basal forebrain which may play a key role in song learning. By injecting neuroanatomical tracers, we found a topographically segregated pathway from nucleus ovoidalis (Ov) to VP that in turn projects in a topographic fashion to HVC and RA. Ov is a major relay in the main ascending auditory pathway. The results suggest that the cholinergic neurons in the VP responsible for song learning are regulated by auditory information from the Ov.     

A sensorimotor area in the songbird brain is required for production of vocalizations in the song learning period of development

Sensory feedback is essential for acquiring and maintaining complex motor behaviors, including birdsong. In zebra finches, auditory feedback reaches the song control circuits primarily through the nucleus interfacialis nidopalii (Nif), which provides excitatory input to HVC (proper name)—a premotor region essential for the production of learned vocalizations. Despite being one of the major inputs to the song control pathway, the role of Nif in generating vocalizations is not well understood. To address this, we transiently inactivated Nif in late juvenile zebra finches. Upon Nif inactivation (in both hemispheres or on one side only), birds went from singing stereotyped zebra finch song to uttering highly variable and unstructured vocalizations resembling sub‐song, an early juvenile song form driven by a basal ganglia circuit. Simultaneously inactivating Nif and LMAN (lateral magnocellular nucleus of the anterior nidopallium), the output nucleus of a basal ganglia circuit, inhibited song production altogether. These results suggest that Nif is required for generating the premotor drive for song. Permanent Nif lesions, in contrast, have only transient effects on vocal production, with song recovering within a day. The sensorimotor nucleus Nif thus produces a premotor drive to the motor pathway that is acutely required for generating learned vocalizations, but once permanently removed, the song system can compensate for its absence. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1213–1225, 2016     

HVC microlesions do not destabilize the vocal patterns of adult male zebra finches with prior ablation of LMAN

The songs of adult male zebra finches (Taeniopygia guttata) arise by an integration of activity from two neural pathways that emanate from the telencephalic nucleus HVC (proper name). One pathway descends directly from HVC to the vocal premotor nucleus RA (the robust nucleus of the arcopallium) whereas a second pathway descends from HVC into a basal ganglia circuit (the anterior forebrain pathway, AFP) that also terminates in RA. Although HVC neurons that project directly to RA outnumber those that contribute to the AFP, both populations are distributed throughout HVC. Thus, partial ablation (microlesion) of HVC should damage both pathways in a proportional manner. We report here that bilateral HVC microlesions in adult male zebra finches produce an immediate loss of song stereotypy from which birds recover, in some cases within 3 days. The contribution of the AFP to the onset of song destabilization was tested by ablating the output nucleus of this circuit (LMAN, the lateral magnocellular nucleus of the anterior nidopallium) prior to bilateral HVC microlesions. Song stereotypy was largely unaffected. Together, our findings suggest that adult vocal production involves nonproportional integration of two streams of neural activity with opposing effects on song--HVC's direct projection to RA underlies production of stereotyped song whereas the AFP seems to facilitate vocal variation. However, the rapid recovery of song in birds with HVC microlesions alone suggests the presence of dynamic corrective mechanisms that favor vocal stereotypy.     

Anatomically Discrete Sex Differences in Neuroplasticity in Zebra Finches as Reflected by Perineuronal Nets

Large morphological sex differences in the vertebrate brain were initially identified in song control nuclei of oscines. Besides gross differences between volumes of nuclei in males and females, sex differences also concern the size and dendritic arborization of neurons and various neurochemical markers, such as the calcium-binding protein parvalbumin (PV). Perineuronal nets (PNN) of the extracellular matrix are aggregates of different compounds, mainly chondroitin sulfate proteoglycans, that surround subsets of neurons, often expressing PV. PNN develop in zebra finches song control nuclei around the end of the sensitive period for song learning and tutor deprivation, known to delay the end of the song learning sensitive period, decreases the numbers of PNN in HVC. We demonstrate here the existence in zebra finches of a major sex difference (males > females) affecting the number of PNN (especially those surrounding PV-positive cells) in HVC and to a smaller extent the robust nucleus of the arcopallium, RA, the two main nuclei controlling song production. These differences were not present in Area X and LMAN, the lateral magnocellular nucleus of the anterior nidopallium. A dense expression of material immunoreactive for chondroitin sulfate was also detected in several nuclei of the auditory and visual pathways. This material was often organized in perineuronal rings but quantification of these PNN did not reveal any sex difference with the exception that the percentage of PNN surrounding PV-ir cells in the dorsal lateral mesencephalic nucleus, MLd, was larger in females than in males, a sex difference in the opposite direction compared to what is seen in HVC and RA. These data confirm and extend previous studies demonstrating the sex difference affecting PNN in HVC-RA by showing that this sex difference is anatomically specific and does not concern visual or auditory pathways.     

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.     

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.     

Neural correlates of singing behavior in male zebra finches (Taeniopygia guttata)

This study examined the relationship between the volumes of four song control nuclei: the high vocal center (HVC), the lateral part of the magnocellular nucleus of the anterior neostriatum (lMAN), Area X, and the robust nucleus of the archistriatum (RA), as well as syrinx mass, with several measures of song output and song complexity in male zebra finches (Taeniopygia guttata). Male zebra finches' songs were recorded in standardized recording sessions. The syrinx and brain were subsequently collected from each bird. Volumes of the song control nuclei were reconstructed by measuring the cross-sectional area of serial sections. Syrinx mass was positively correlated with RA volume. The volume of lMAN was negatively related to element repertoire size and the number of elements per phrase. We found no other correlations between brain and behavioral measures. This study, combined with others, indicates that the evidence for a general relationship among songbirds between HVC volume and song complexity is equivocal. There are clear species differences in this brain-behavior correlation. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 421–430, 1998     

The distribution of tyrosine hydroxylase in the canary brain: demonstration of a specific and sexually dimorphic catecholaminergic innervation of the telencephalic song control nuclei

Singing and the processing of auditory information related to song can be affected by experimental manipulations of catecholamine activity in the brain of zebra finches. We investigated, by immunocytochemistry in the brain of male and female canaries, the distribution of tyrosine hydroxylase (TH), the rate-limiting step in the synthesis of catecholamines. Fibers immunoreactive for TH (TH-ir) were particularly abundant in the lobus parolfactorius, the paleostriatum primitivum, and the nucleus septalis lateralis. A high density of TH-ir basket-like structures was observed in the caudomedial neostriatum, an area involved in song perception and recognition. In most males, a high density of TH-ir fibers outlined the telencephalic song control nuclei including the high vocal center, the nucleus robustus archistriatalis, the nucleus interfascialis, the lateral and medial parts of the magnocellular nucleus of the anterior neostriatum, and area X of the lobus parolfactorius. The higher density of fibers immunoreactive for TH in these nuclei, compared with the surrounding telencephalon, supports the notion that the morphological evolution of the song control nuclei was accompanied by a neurochemical specialization. This specific innervation of the song control regions was, in general, not found in females. The specific presence of high densities of TH-ir fibers in the song system of male canaries and the sex difference of this innervation provide anatomical evidence in support of the claim that dopamine and/or norepinephrine play important roles in the modulation of song learning and production.     

Steroid accumulation in song nuclei of a sexually dimorphic duetting bird,the rufous and white wren

This study tested the hypothesis that the relative proportion of neurons that are hormone sensitive in avian song control nuclei is related to the basic motor ability to sing, whereas the absolute number of such neurons is related to the complexity of song behavior. Either [³H]testosterone (T) or estradiol (E₂) was injected into male and female rufous and white wrens (Thryothorus rufalbus), a tropical species in which females sing duets with males but have smaller song repertoires than males. Autoradiographic analysis indicated that there were no sex differences in the proportions of T or E₂ target cells in two song nuclei: the high vocal center (HVC) and the lateral portion of the magnocellular nucleus of the anterior neostriatum (IMAN). The density of labeled cells per unit volume of tissue did not differ between the sexes in either song nucleus. Males have larger song nuclei, however, which is consistent with their more complex song behavior, and therefore have a greater total number of hormone-sensitive neurons in these regions than do females. Comparison of these results with measures of hormone accumulation in zebra finches, canaries, and bay wrens supports the hypothesis presented. © 1996 John Wiley & Sons, Inc.     

鸣禽发声学习行为的神经生物学机制

鸣禽发声学习的控制系统主要由一条直接神经通路和一条辅助神经通路组成,由前脑控制发声学习的最高中枢ＨＶＣ、旁嗅叶的Ｘ区和巨细胞核外侧部（ｌＭＡＮ）组成的辅助通路,对鸟类发声学习行为的发育和调制具有重要作用。发声控制系统中神经元类型、数量及再生与更替、神经组构及其重组、神经介质和受体的分布等差异,决定了鸣禽在发声学习行为表现的差异以及性双态性。本文对近年鸟类控制发声学习行煌神经生物学机制的进展作了较为     

Neurotensin and neurotensin receptor 1 mRNA expression in song‐control regions changes during development in male zebra finches

Learned vocalizations are important for communication in some vertebrate taxa. The neural circuitry for the learning and production of vocalizations is well known in songbirds, many of which learn songs initially during a critical period early in life. Dopamine is essential for motor learning, including song learning, and dopamine‐related measures change throughout development in song‐control regions such as HVC, the lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, and the robust nucleus of the arcopallium (RA). In mammals, the neuropeptide neurotensin strongly interacts with dopamine signaling. This study investigated a potential role for the neurotensin system in song learning by examining how neurotensin (Nts) and neurotensin receptor 1 (Ntsr1) expression change throughout development. Nts and Ntsr1 mRNA expression was analyzed in song‐control regions of male zebra finches in four stages of the song learning process: pre‐subsong (25 days posthatch; dph), subsong (45 dph), plastic song (60 dph), and crystallized song (130 dph). Nts expression in LMAN during the subsong stage was lower compared to other time points. Ntsr1 expression was highest in HVC, Area X, and RA during the pre‐subsong stage. Opposite and complementary expression patterns for the two genes in song nuclei and across the whole brain suggest distinct roles for regions that produce and receive Nts. The expression changes at crucial time points for song development are similar to changes observed in dopamine studies and suggest Nts may be involved in the process of vocal learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 671–686, 2018     

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)     

雌性斑胸草雀发育和成体期发声核团LMAN中神经元数量的高水平维持

鸟类的发声和发声学习涉及处于脑中不同水平的相互联系的一些发声核团。在很多鸟类中, 仅雄性鸣啭, 例如在斑胸草雀(Taeniogygia guttata) 中, 雌雄个体间存在显著的性差异, 雌性发声系统和发声核团中的神经元数量显著少于雄性。推测这种性差异也可能存在于弓状皮质前部大细胞外侧核(LMAN), 但有关这方面的报道还很少。为探讨雌雄鸣禽脑中神经元数量发育的规律, 我们应用半薄切片对斑胸草雀发育不同阶段的神经元数量和密度的变化进行了数量分析。结果表明, 在全部实验组中, 发育10 d和成体雌性LMAN中神经元的密度和神经元数量均无明显变化, 神经元的数量维持在一个较高水平。在雄鸟中, 我们发现在发育早期神经元的数量有极为显著的减少, 这与已有的报道, 即应用非类固醇技术获得的结果相似。LMAN中神经元数量的减少(57%) 一直持续到成体。因此, 成年雌鸟LMAN中的全部神经元数量是雄鸟的4 倍(P<0 001)。推测雌鸟脑中存在较多的神经元数量可能与其在发育和成体期间需维持适当的神经网络有关, 这种神经构筑对于动物种间识别等功能可能具有特殊意义。     

Early condition, song learning, and the volume of song brain nuclei in the zebra finch (Taeniopygia guttata)

Songbirds are an important model system for the study of the neurological bases of song learning, but variation in song learning accuracy and adult song complexity remains poorly understood. Current models of sexual selection predict that signals such as song must be costly to develop or maintain to constitute honest indicators of male quality. It has been proposed that reductions of nestling condition during song development might limit the expression of song learning. Adult song could thus act as an indicator of early stress as only males that enjoy good condition during development could learn accurately and sing long songs or large repertoires. We tested this hypothesis in the zebra finch by modifying early condition through cross-fostering chicks to small, medium, and large broods. Song learning was very accurate and was found to reflect very closely tutor song characteristics and to depend on the number of males in the tutoring group. Although the brood size manipulation strongly affected several measures of nestling condition and adult biometry, we found no relationship between early condition and song learning scores or song characteristics. Similarly, brain mass and high vocal center (HVC), robust nucleus of the arcopallium (RA), and lateral magnocellular nucleus of the anterior nidopallium (LMAN) volumes did not covary with nestling condition and growth measurements. We found no significant relationship between song repertoire size and HVC and RA volumes, although there was a nonsignificant trend for HVC to increase with increasing proportion of learnt elements in a song. In conclusion, the results provide no evidence for song learning to be limited by nestling condition during the period of nutritional dependence from the parents in this species.     

Effects of castration and testosterone treatment on the activity of testosterone-metabolizing enzymes in the brain of male and female zebra finches

Recently, we described the distribution of testosterone-metabolizing enzymes (i.e., aromatase, 5 alpha- and 5 beta-reductases) in the zebra finch (Taeniopygia guttata) brain using a sensitive radioenzyme assay combined to the Palkovits punch method. A number of sex-differences in the activity of these enzymes were observed especially in nuclei of the song-control system. The hormonal controls of these differences have now been analyzed by gonadectomizing birds of both sexes and by giving them a replacement therapy with silastic implants of testosterone (T). Five nuclei of the song system (Area X [X], nucleus magnocellularis of the anterior neostriatum [MAN], nucleus robustus archistriatalis [RA], nucleus intercollicularis [ICo], hyperstriatum ventrale, pars caudalis [HVc]) and three preoptic-hypothalamic areas (preoptic anterior [POA], periventricular magnocellular nucleus [PVM], and posterior medial hypothalamic nucleus [PMH]) were studied as well as other limbic and control non-steroid-sensitive areas. The activity of the 5 alpha-reductase was higher in males than in females for the five song-control nuclei and was not affected by the hormonal treatments. The overall activity of this enzyme was not sexually dimorphic in POA and PVM. It was higher in males than in females in intact birds only, and was reduced by gonadectomy and enhanced by T. The activity of the 5 beta-reductase was higher in females than in males in all nuclei of the song system and in POA, but was not influenced by the changes in T level. Both sex and treatment effects were observed in the control of aromatase. The production of estrogens was dimorphic (females greater than males) in RA and PMH. It was increased by T in POA, PVM, and PMH, and also in RA. These data show that some of the sex differences in T-metabolizing enzymes result from the exposure to different levels of T in adulthood (e.g., 5 alpha-reductase in POA and PVM or aromatase in PVM), whereas others persist even if birds are exposed to the same hormonal conditions. These are presumably the result of organizational effects of steroids. The steroid modulation of the aromatase might be related directly to the activation of sexual, aggressive, and nest-building behaviors, whereas the stable dimorphism in 5 alpha- and 5 beta-reductase observed in the nuclei of the song system might be one of the neurochemical bases of the sex differences in the vocal behavior of the zebra finch.     

Sex differences in the telencephalic song control circuitry in Bengalese finches (Lonchura striata var. domestica)

Bengalese finches, Lonchura striata, are extremely sexually dimorphic in their singing behavior; males sing complex songs, whereas females do not sing at all. This study describes the developmental differentiation of the brain song system in Bengalese finches. Nissl staining was used to measure the volumes of four telencephalic song nuclei: Area X, HVC, the robust nucleus of the arcopallium (RA), and the lateral portion of the magnocellular nucleus of the anterior nidopallium (LMAN). In juveniles (circa 35 days old), Area X and the HVC were well developed in males, while they were absent or not discernable in females. The RA was much larger in males but barely discernable in females. In males, the volumes of Area X and the RA increased further into adulthood, but that of the HVC remained unchanged. The LMAN volume was greater in juveniles than in adults, and there was no difference in the LMAN volume between the sexes. The overall tendency was similar to that described in zebra finches, except for the volume of the RA, where the degree of sexual dimorphism is larger and the timing of differentiation occurs earlier in Bengalese finches. Motor learning of the song continues until day 90 in zebra finches, but up to day 120 in Bengalese finches. Earlier neural differentiation and a longer learning period in Bengalese finches compared with zebra finches may be related to the more elaborate song structures of Bengalese finches.     

Sexual Differences in Cell Loss during the Post-Hatch Development of Song Control Nuclei in the Bengalese Finch

Birdsongs and the regions of their brain that control song exhibit obvious sexual differences. However, the mechanisms underlying these sexual dimorphisms remain unknown. To address this issue, we first examined apoptotic cells labeled with caspase-3 or TUNEL in Bengalese finch song control nuclei - the robust nucleus of the archopallium (RA), the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the high vocal center (HVC) and Area X from post-hatch day (P) 15 to 120. Next, we investigated the expression dynamics of pro-apoptotic (Bid, Bad and Bax) and anti-apoptotic (Bcl-2 and Bcl-xL) genes in the aforementioned nuclei. Our results revealed that the female RA at P45 exhibited marked cell apoptosis, confirmed by low densities of Bcl-xL and Bcl-2. Both the male and female LMAN exhibited apoptotic peaks at P35 and P45, respectively, and the observed cell loss was more extensive in males. A corresponding sharp decrease in the density of Bcl-2 after P35 was observed in both sexes, and a greater density of Bid was noted at P45 in males. In addition, we observed that RA volume and the total number of BDNF-expressing cells decreased significantly after unilateral lesion of the LMAN or HVC (two areas that innervate the RA) and that greater numbers of RA-projecting cells were immunoreactive for BDNF in the LMAN than in the HVC. We reasoned that a decrease in the amount of BDNF transported via HVC afferent fibers might result in an increase in cell apoptosis in the female RA. Our data indicate that cell apoptosis resulting from different pro- and anti-apoptotic agents is involved in generating the differences between male and female song control nuclei.