Effect of Intraseptal Vasotocin and Vasoactive Intestinal Polypeptide Infusions on Courtship Song and Aggression in the Male Zebra Finch (Taeniopygia guttata)

The present experiments were conducted to test the hypothesis that septal arginine vasotocin (AVT) and vasoactive intestinal polypeptide (VIP) modulate directed song (a courtship behaviour) and aggression in male zebra finches ( Taeniopygia guttata ). Subjects were surgically fitted with a guide cannula directed at the septum. Following recovery they were tested for aggression and directed song following infusions of AVT, its antagonist (anti-vasopressin, AVP), and saline volume control. Infusion of the AVT antagonist significantly reduced all three aggressive behaviours measured (pecks, beak fences and chases); and AVT infusion significantly facilitated beak fencing. Vasoactive intestinal polypeptide treatment significantly reduced pecking. No treatment produced a change in directed song. Comparison with findings in mammals suggests that modulation of aggression by septal AVT (or AVP) is evolutionarily conserved in vertebrates, but modulation of aggression by VIP has not previously been reported for any vertebrate.     

Enhanced fos expression in the zebra finch (Taeniopygia guttata) brain following first courtship

Young zebra finch males that court a female for the first time develop a stable preference for the females of that species. On the neuronal level, consolidation of the imprinted information takes place. Here we demonstrate that first courtship or being chased around in the cage leads to enhanced fos expression in forebrain areas implicated in learning and imprinting in zebra finch males compared with birds reared in isolation or in the aviary. Two of the forebrain areas highly active during first courtship (as demonstrated by the 14C-2-deoxyglucose technique), the imprinting locus latral neo/hyperstriatum ventrale (LNH) and the secondary visual area hyperstriatum accessorium/dorsale (HAD), demonstrate enhanced fos expression. Two other imprinting-related areas, the medial neo/hyperstriatum ventrale (MNH) and archistriatum/neostriatum caudale (ANC), do show c-fos induction; however, the areas are not congruous with those demarcated by the 2-DG autoradiographic studies. Additional telencephalic areas include the olfactory lobe, the information storage site lobus parolfactorius (LPO), the memory site hippocampus, the auditory caudomedial neostriatum implicated in the strength of song learning, and the caudolateral neostriatum, which is comparable to the mammalian prefrontal cortex. In addition, c-fos is induced by first courtship and chasing in neurosecretory cell groups of the preoptic area and hypothalamus associated with the repertoire of sexual behavior and stress or enhanced arousal. Enhanced fos expression is also observed in brainstem sources of specific (noradrenergic, catecholaminergic) and nonspecific (reticular formation) activating pathways with inputs to higher brain areas implicated in the imprinting process. Birds reared in isolation or alternatively in the aviary with social and sexual contact to conspecifics showed attenuated or no fos expression in most of the above-mentioned areas. First courtship and chasing both lead to enhanced uptake of 2-DG in the four imprinting areas, as well as subsequent changes in spine density-an anatomical manifestation of the imprinting process. fos expression in the imprinting and other telencephalic, preoptic, hypothalamic, and mesencephalic brain regions indicates processing of stimuli originating from exposure (like chasing) and the analysis of stimuli in a behaviorally relevant, sexually explicit context (like first courtship). c-fos induction in these brain areas indicates its involvement in the triggering of neural changes that accompany the learning process of imprinting, leading eventually to alterations in dendritic spine density in the zebra finch.     

Differential expression of the immediate early genes FOS and ZENK following auditory stimulation in the juvenile male and female zebra finch

The brains of adult zebra finches (Taeniopygia guttata) are tuned to the songs of conspecifics. In adult males, the caudomedial neostriatum (NCM) responds to zebra finch song, and in adult females the NCM and hippocampus (HP) are active following exposure to zebra finch song more than other auditory stimuli. The caudal hyperstriatum ventrale (cHV) in both sexes also responds to song, but in females not as selectively as the NCM and HP. While much is known about the adult perceptual circuit, less is known about its development. The present study exposed d30 male and female zebra finches to conspecific or heterospecific song, tones or silence, and examined the densities of FOS- and ZENK-immunoreactive nuclei in the NCM, cHV and HP. Significant interactions existed between sex and auditory stimulus condition for both immediate early genes, but they were in opposite directions. That is, across the three regions, FOS-immunoreactive neurons were increased in females following presentation of conspecific songs; males did not show an effect of stimulus exposure. In contrast, the density of ZENK-positive neurons was increased in males, but not females, following zebra finch song exposure. The FOS results demonstrate that some neural responses required for song perception may develop earlier in females than males; data on ZENK induction suggest the opposite. Overall, differences in juvenile immediate early gene activation suggest either that males and females employ divergent neural mechanisms for song perception or that the developmental trajectories leading to common neural responses differ.     

Hormone concentrating cells in vocal control and other areas of the brain of the zebra finch ( Poephila guttata).

Using the autoradiographic method in the zebra finch (poephila guttata), areas of the brain were identified which contain cells which accumulate testosterone. Among these areas are the caudal nucleus of the hyperstriatum ventrale, nucleus intercollicularis of the midbrain, and the tracheosyringeal portion of the hypoglossal nucleus of the medulla (nXIIts). These three are known to control or influence androgen dependent song and other vocalizations of passeriform birds, and nXIIts is composed of the motoneurons innervating the vocal (syringeal) muscles. Other areas containing hormone-concentrating cells are the medial preoptic area, nucleus periventricularis magnocellularis of the hypothalamus, dorsal infundibular layers, dorsomedial thalamus, lateral septum, magnocellular nucleus of the anterior neostriatum, periventricular medial neostriatum, nucleus taeniae of the archistriatum, and ventral paleostriatum augmentatum. Accumulation by cells in the preoptic area, hypothalamus, and limbic forebrain is consistent with a general vertebrate pattern of distribution of brain cells which accumulate sex steroids. Some of these same areas may be involved in the control of androgen dependent events such as courtship, copulation, aggression, and feedback regulation of the hypophysis.     

Androgen effects on the development of the zebra finch song system.

In adult zebra finches, males sing and females do not. This behavioral sex difference is associated with sex differences in brain regions that control singing. Treatment of hatchling females with androgen causes permanent masculinization of neuron number in the robust nucleus of the archistriatum (RA), a brain region involved in song. We have re-examined the possible permanent effects of androgen on the zebra finch song system by treating male and female hatchlings with Silastic implants containing 50 or 200 micrograms 5 alpha-dihydrotestosterone (DHT) or 200 micrograms of the antiandrogen flutamide. Birds were sacrificed as adults and their brains prepared for histological analysis of the vocal control nuclei. Some DHT-treated females also received testosterone in adulthood. DHT had small, but significant effects on several attributes of the female song system, including increases of neuron number in RA and neuron size of RA and lateral magnocellular nucleus of the neostriatum (IMAN). DHT had no significant effects in males. Flutamide did not demasculinize any measures of the male song system but hypermasculinized volume and neuron number in RA. These data confirm studies that report few masculinizing effects of androgen on the female zebra finch song system. Nevertheless, the modest effects of DHT leave open the possibility that masculinization of the song system requires an action of androgen, perhaps in synergy with estrogen.     

Lack of sexual dimorphism in steroid accumulation in vocal control brain regions of duetting song birds

Tritiated testosterone was injected into bay wrens (Thryothorus nigricapillus), a neotropical species in which the female sings a complex song in intricately timed vocal duets with males. Autoradiographic analysis indicated that male and female wrens have the same proportion of cells labeled by testosterone or its metabolites in two brain regions involved in song: the caudal nucleus of the ventral hyperstriatum (HVc) and the magnocellular nucleus of the anterior neostriatum (MAN). This contrasts with the zebra finch, a species in which only the males sing: a considerably greater proportion of male zebra finch cells in HVc and MAN are labeled than in females. This suggests that female birds that produce complex vocalizations have evolved neural song control systems that are extremely similar to those of males in steroid hormone sensitivity.     

Identification,distribution, and developmental changes of a melatonin binding site in the song control system of the zebra finch

In many avian species, singing is a circadian or seasonal behavior that appears to be widely dependent on gonadal steroid hormones. To explore the possibility of a further hormone-dependent vocal control mechanism driven by the action of melatonin, we examined the binding of iodinated melatonin (IMEL) in the vocal control network of adult and juvenile (22- and 40-day-old) zebra finches. IMEL binding areas of the zebra finch brain were localized and characterized by using quantitative in vitro autoradiography. In the vocal control system, dense IMEL binding sites were restricted to the nucleus hyperstriatalis ventrale, pars caudalis (HVC). The binding of IMEL to the HVC and to visual areas, e.g., the ectostriatum and the optic tectum, was saturable and showed a single class of high-affinity binding sites with binding affinities (K_ds) in the range of 5–20 pM. Competition experiments with various indols and IMEL showed that the IMEL binding site in the zebra finch brain has properties similar to the high-affinity melatonin receptor described in the chicken, in the house sparrow, and in the mammalian brain and retina. Similar to the zebra finch HVC, the HVC of other songbirds, e.g., male canaries and male house sparrows, has the most intense IMEL binding of all areas of the vocal control network. The IMEL binding in the forebrain vocal control areas of the zebra finch, but not that in the visual processing areas, was sexually dimorphic in correlation with the sexually dimorphic neuroanatomy of the forebrain vocal control areas. In the HVC, there is a developmental increase in the maximal number of binding sites for IMEL and in the protein content, so that the adult phenotype of dense IMEL binding develops between day 40 and day 80. The distribution and developmental pattern of IMEL binding in the song system suggests that melatonin has a role in the motor control of singing. Melatonin binding sites in HVC could link HVC-based song control to circadian and circannual changes in the photoperiod independent of gonadal steroids. © 1996 Wiley-Liss, Inc.     

Distribution of language‐related Cntnap2 protein in neural circuits critical for vocal learning

Variants of the contactin associated protein‐like 2 (Cntnap2) gene are risk factors for language‐related disorders including autism spectrum disorder, specific language impairment, and stuttering. Songbirds are useful models for study of human speech disorders due to their shared capacity for vocal learning, which relies on similar cortico‐basal ganglia circuitry and genetic factors. Here we investigate Cntnap2 protein expression in the brain of the zebra finch, a songbird species in which males, but not females, learn their courtship songs. We hypothesize that Cntnap2 has overlapping functions in vocal learning species, and expect to find protein expression in song‐related areas of the zebra finch brain. We further expect that the distribution of this membrane‐bound protein may not completely mirror its mRNA distribution due to the distinct subcellular localization of the two molecular species. We find that Cntnap2 protein is enriched in several song control regions relative to surrounding tissues, particularly within the adult male, but not female, robust nucleus of the arcopallium (RA), a cortical song control region analogous to human layer 5 primary motor cortex. The onset of this sexually dimorphic expression coincides with the onset of sensorimotor learning in developing males. Enrichment in male RA appears due to expression in projection neurons within the nucleus, as well as to additional expression in nerve terminals of cortical projections to RA from the lateral magnocellular nucleus of the nidopallium. Cntnap2 protein expression in zebra finch brain supports the hypothesis that this molecule affects neural connectivity critical for vocal learning across taxonomic classes. J. Comp. Neurol. 522:169–185, 2014. © 2013 Wiley Periodicals, Inc.     

Sex differences in the vocal motor pathway of the zebra finch revealed by real-time optical imaging technique

Male zebra finches sing, whereas female zebra finches do not. To elucidate the neural mechanisms underlying sexual dimorphism in song behavior, the spatio-temporal properties of neural activity in the vocal motor pathway of the zebra finch were examined in sliced brain preparations using a real-time optical recording technique. Electrical stimulation to higher vocal center (HVC) fibers induced within 20 ms neural activities in the robust nucleus of the archistriatum (RA) of both male and female finches, although the amplitude was smaller and the latency was greater in females than in males. Bicuculline, a GABA(A) receptor antagonist, induced a robust activity in female RA, but had little effect in males. This suggests that neural connections from HVC to RA in female zebra finches are inhibited by GABAergic inputs. The results provide first evidence that an inhibitory neurotransmitter is involved in the sex difference in the motor vocal pathway of song birds.     

C-fos induction in forebrain areas of two different visual pathways during consolidation of sexual imprinting in the zebra finch (Taeniopygia guttata)

Two forebrain areas in the hyperpallium apicale and in the lateral nidopallium of isolated male zebra finches are highly active (2-deoxyglucose technique) on exposure to females for the first time, that is first courtship. These areas also demonstrate enhanced neuronal plasticity when screened with c-fos immunocytochemistry. Both are areas involved in the processing of visual information conveyed by the two major visual pathways in birds, strengthening our hypothesis that courtship in the zebra finch is a visually guided behaviour. First courtship and chased birds show enhanced c-fos induction in the hyperpallial area, which could represent neuronal activity reflecting changes in the immediate environment. The enhanced expression of fos in first courtship birds in lateral nidopallial neurons indicates imminent long-lasting changes at the synaptic level that form the substrate for imprinting, a stable form of learning in birds.     

Distribution of GABA-like immunoreactivity in the song system of the zebra finch

GABA-like immunoreactivity (GABA-LIR) was mapped in the male and female zebra finch song system using a polyclonal antibody to GABA. GABA-LIR was found throughout the song system in neurons and neuropil of the robust nucleus of the archistriatum (RA), the higher vocal center (HVC), Area X, the magnocellular nucleus of the neostriatum (MAN), and the dorsomedial portion of the nucleus intercollicularis (DM of ICo). Puncta present in the lateral division of MAN (1MAN) may be local interneurons since the only known afferents of 1MAN are from the dorsolateral nucleus of the anterior thalamus (DLM), which did not appear to have any cell bodies with GABA-LIR. Distinct and dense puncta with GABA-LIR were present in DLM, and may be projections from Area X/lobus parolfactorius (LPO). Dramatic sex differences in GABA-LIR distribution were found. Females did not appear to have any GABA-LIR above background in either RA of HVC. Females also did not appear to have a distinct Area X, although they did have many small, lightly staining cell bodies in the corresponding LPO. The distribution of GABA-LIR and sex differences in its distribution suggests that GABAergic neurons may play a role in the acquisition and/or production of song in the zebra finch.     

An interspecific comparison using immunofluorescence reveals that synapse density in the avian song system is related to sex but not to male song repertoire size

Immunofluorescent labeling of synaptic vesicle protein 2 (SV2) and confocal microscopy were employed to assess the role of synapse density in the functioning of the avian song system. Synapse density in premotor nuclei HVC and RA was measured, in both sexes of two species characterized by male-only singing behavior: the zebra finch Taeniopygia guttata, which sings a single, stereotyped song, and the Carolina wren Thryothorus ludovicianus, which sings a large repertoire of different songs. Multiple levels of analyses demonstrate overall similarity of synapse density between nuclei HVC and RA, suggesting that synapse density is regulated uniformly across these regions within individuals. Male zebra finches and male Carolina wrens have equivalent synapse densities, suggesting a common pattern of masculinized development despite dramatic behavioral differences. Female Carolina wrens have synaptic density similar to that of males of both species, while female zebra finches exhibit greater synaptic densities in both regions than do male zebra finches or both sexes of wrens. Prior reports implicate testosterone as a regulator of synapse density in this system; sex differences in circulating or neural testosterone may contribute to the sexual dimorphism of synapse density observed here. Interspecific comparison of song system synapse density in nonsinging females suggests that synapse density in female songbirds may be a particularly labile trait.     

Effect of social factors on the development of PKC expression in songbird brain

Vocal learning by songbirds is affected by a song template and social factors. Normal male zebra finches with a song template and social interactions with other birds showed a transient increase of protein kinase C (PKC)-like immunoreactivity in the song control nuclei during a sensitive period of song learning. However, auditory isolation, which prevents the development of a song template induced an abnormal PKC development, which depended on the social interactions with other birds. In conclusion, social experience during an early sensitive period of song learning in the zebra finch is essential for the normal development of PKC expression in the robust nucleus of the arcopallium (RA), a premotor nucleus related to vocal plasticity.     

Telencephalic aromatase but not a song circuit in a sub-oscine passerine, the golden collared manakin (Manacus vitellinus)

In oscine passerines, the telencephalon expresses high levels of the estrogen synthetic enzyme aromatase. In contrast, forebrain aromatase is limited to low levels at discrete limbic loci in non-passerines. The function of forebrain aromatase in oscines is unknown, however, estrogen-sensitive elements of the telencephalic song circuit (an oscine characteristic) may be influenced by local aromatization. Very few studies have investigated the neuroendocrine characteristics of sub-oscine passerines. Species of this passerine sub-order are taxonomically similar to oscines, but do not appear to learn how to sing as oscines, and show no evidence of a song circuit. The neural expression of aromatase in these birds is unknown. We asked whether the golden-collared manakin, a sub-oscine, (a) showed evidence of a song circuit, and (b) expressed aromatase in the telencephalon at high levels like the zebra finch (oscine passerine) or at low levels like the quail (non-passerine). Nissl stains and immunocytochemistry for microtubule associated proteins showed no evidence of a song circuit in manakins of either sex, whereas both techniques delineate all song nuclei in the zebra finch. However, biochemical and immunocytochemical measures reveal that in the manakin, several telencephalic loci, including the hippocampus, caudomedial neostriatum, nucleus taeniae, and the lateral neostriatum express aromatase. Assays run in parallel show low to undetectable levels of aromatase in the telencephalon of the quail (nonpasserine) and abundant levels in the zebra finch (oscine passerine), suggesting a dissociation between the presence of a song circuit and forebrain aromatase expression in this sub-oscine. These data suggest that forebrain aromatase may have evolved in sub-oscine songbirds before the evolution of a song circuit and singing behavior in oscines. Alternatively, forebrain aromatase may serve functions distinct from singing behavior.     

Developmental pattern of CB1 cannabinoid receptor immunoreactivity in brain regions important to zebra finch (Taeniopygia guttata) song learning and control

Zebra finches learn song during distinct developmental stages, making them an important species for studying mechanisms underlying vocal development. Distinct interconnected forebrain regions have been identified as important to specific features of zebra finch vocal learning and production. Because prior experiments have demonstrated that late postnatal exposure to cannabinoid agonists alters zebra finch song learning, we have sought to identify brain regions likely involved in it. By using an affinity-purified polyclonal antibody directed against the zebra finch CB(1) cannabinoid receptor, we have studied staining patterns in groups of males at 25, 50, 75, and >100 days of age (adults). A general waxing and waning of staining intensity were observed over this developmental period. Distinct staining of song-related brain regions was also noted. Early establishment of staining patterns within rostral telencephalic song regions [area X and lateral magnocellular nucleus of the anterior nidopallium (lMAN)] suggests a role in auditory learning. Later establishment and maintenance in adulthood of small somata and neuropil staining within regions of rostral telencephalon [HVC and robust nucleus of the arcopallium (RA)] are consistent with a vocal motor role for cannabinoid signaling. Our results provide insight into brain regions likely responsible for cannabinoid-altered vocal learning and add to accumulating evidence supporting an important role for cannabinoid signaling in CNS development.     

Localized immediate early gene expression related to the strength of song learning in socially reared zebra finches

Recent evidence showed that exposure of tape-tutored zebra finch (Taeniopygia guttata castanotis) males to the tutor song involves neuronal activation in brain regions outside the conventional 'song control pathways', particularly the caudal part of the neostriatum (NCM) and of the hyperstriatum ventrale (CMHV). Zebra finch males were reared with a live tutor during the sensitive period for song learning. When, as adults, they were re-exposed to the tutor song, the males showed increased expression of Fos, the protein product of the immediate early gene c-fos, in the NCM and CMHV, compared with expression in two conventional 'song control nuclei', high vocal centre (HVC) and Area X. The strength of the Fos response (which is a reflection of neuronal activation) in the NCM (but not in the other three regions) correlated significantly and positively with the number of song elements that the birds had copied from the tutor song. Thus, socially tutored zebra finch males show localized neural activation in response to tutor song exposure, which correlates with the strength of song learning.     

Lesion of a higher-order song nucleus disrupts phrase level complexity in Bengalese finches

Studies in zebra finches failed to demonstrate the involvement of the NIf, a higher-order song nucleus afferent to the HVc, in the production of learned song. The song of the Bengalese finch, a related species, has a higher level of temporal organization; multiple song phrases are organized into a song. We hypothesized that the NIf might control this complexity. To test this, we bilaterally lesioned the NIf in adult male Bengalese finches. The songs of birds with multi-phrase organization changed into simpler, mono-phrase songs. This is the first demonstration of the NIfs involvement in the production of birdsong. Zebra finch songs are syntactically simple and deterministic, and this might have caused the difficulty in demonstrating the function of the NIf in zebra finches.     

Flash evoked responses in a song control nucleus of the zebra finch (Taeniopygia guttata castanotis)

The song of the zebra finch is facilitated and altered by the presence of a female. Thus, visual information should affect the song system of the bird. Visually evoked potentials can be recorded from n. hyperstriatum ventrale pars caudale (HVc). The long latency of this potential and its variability indicate several processing steps between primary sensory areas of the telencephalon and HVC. Within HVc, under these experimental conditions no interaction between acoustic and visual input could be demonstrated. However, at the dorsal border and within the shelf below HVc, visual information seems to enhance acoustically evoked potentials.     

Autoradiographic localization of nicotinic acetylcholine receptors in the brain of the zebra finch (Poephila guttata)

We have localized nicotinic acetylcholine receptors in the zebra finch brain by using three 125I-labelled ligands: alpha bungarotoxin and two monoclonal antibodies to neuronal nicotinic receptors (MAb 35 of Tzardos et al., J. Biol. Chem., 250: 8635-8645, '81; and MAb 270 of Whiting and Lindstrom: J. Neurosci. 6: 3061-3069, '86). Unfixed brains from intact adult male and female zebra finches were prepared for in vitro autoradiography. Low-resolution film autoradiograms and high-resolution emulsion autoradiograms were prepared for each of the three ligands. The major brain structures that bind all three of the ligands are hippocampus; hyperstriatum dorsalis; hyperstriatum ventralis; nucleus lentiformis mesencephali; nucleus pretectalis, some layers of the optic tectum; nucleus mesencephalicus lateralis; pars dorsalis; locus ceruleus; and all cranial motor nuclei except nucleus nervi hypoglossi. The major structures labelled only by [125I]-alpha bungarotoxin binding included hyperstriatum accessorium and the nuclei: preopticus medialis, medialis hypothalami posterioris, semilunaris, olivarius inferior, and the periventricular organ. Of the song control nuclei, nucleus magnocellularis of the anterior neostriatum; hyperstriatum ventralis, pars caudalis; nucleus intercollicularis; and nucleus hypoglossus were labelled. The binding patterns of the two antibodies were similar to one another but not identical. Both labelled nucleus spiriformis lateralis and nucleus geniculatus lateralis, pars ventralis especially heavily and also labelled the nucleus habenula medialis; nucleus subpretectalis; nucleus isthmi, pars magnocellularis; nucleus reticularis gigantocellularis; nucleus reticularis lateralis; nucleus tractus solitarii; nucleus vestibularis dorsolateralis; nucleus vestibularis lateralis; nucleus descendens nervi trigemini; and the deep cerebellar nuclei. Lobus parolfactorius and nucleus vestibularis medialis were labelled by only MAb 270, whereas only MAb 35 labelled nucleus laminaris and the medial and lateral pontine nuclei. These data extend previous reports of cholinergic participation in the song system (Ryan and Arnold: J. Comp. Neurol. 202: 211-219, '81) to suggest that the zebra finch song system may contain several closely related nicotinic receptors. In several brain nuclei it appeared that certain anatomical portions of a nucleus or a certain class of neurons were specifically labelled. Furthermore, in certain cases, the labelling appeared to be clustered around Nissl-stained cell nuclei, thus suggesting that the receptors are concentrated on or in somata.