Hormone accumulation in song regions of the canary brain

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

Hormone accumulation in song regions of the canary brain.

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

Effects of early song experience on song preferences and song control and auditory brain regions in female house finches (Carpodacus mexicanus)

We examined the effects of song tutoring on adult song preferences, volume of song-control brain regions, and activity of auditory brain regions in female house finches (Carpodacus mexicanus). Hand-reared females were tutored with local songs, foreign songs, or no song. We then examined adult song preferences, determined the Nissl-defined volume of the song-control nuclei HVc, Area X, and RA, and compared the number of cells immunoreactive for Zenk protein in the auditory regions NCM and cmHV, following playback of songs heard early in life (Tutor/Playback Match) versus not heard (Tutor/Playback Nonmatch). All hand-reared birds exhibited preferences for locally recorded song over foreign or heterospecific song. We found no difference in the volume of song-control nuclei among the three groups. As well, we found no difference in the number of Zenk immunoreactive cells in NCM and cmHV between females in the Tutor/Playback Match group and females in the Tutor/Playback Nonmatch group. Isolate-reared birds showed greater Zenk immunoreactivity following song playback than either tutored group. Thus, early auditory experience may not play a role in adult geographic song preferences, suggesting that genetic factors can lead to preferences for songs of local dialects. Song tutoring did not influence the size of song-control regions nor Zenk induction levels following song playback, suggesting that early experience with particular songs does not influence Zenk expression. However, overall greater activation in isolate females in auditory areas suggests that exposure to song early in life may increase the selectivity of Zenk activation to song playback in auditory areas.     

Left hypoglossal dominance in the control of canary and white-crowned sparrow song

Summary The syrinx of songbirds includes two separate sound sources, the internal tympaniform membranes (ITM), which form the medial wall of each bronchus. The performance of each ITM is controlled by the muscles of that syringeal half. In the canarySerinus canarius, hypoglossal fibers reaching the syrinx via the tracheosyringealis branch of the hypoglossus are responsible for sound modulation. The muscles controlling the performance of the left syringeal half are innervated solely by the left tracheosyringealis; those controlling the right syringeal half are innervated only by the right tracheosyringealis. In the canary and white-crowned sparrow (Zonotrichia leucophrys) a great majority of song elements disappears after section of the left tracheosyringealis, yet remains intact after section of the right one. This phenomenon, earlier described in the chaffinch (Nottebohm, 1970, 1971, 1972) and confirmed in the white-throated sparrow (Lemon, 1973), has been called left hypoglossal dominance. Left hypoglossal dominance occurs in canaries with small or large song repertoires. It occurs in chronically deafened canaries that never had access to their own auditory feedback; it also occurs in birds that had the right or left cochlea removed at an early age. To this extent, left hypoglossal dominance seems to emerge in the individual as a motor phenomenon.We wish to thank Betsy Manning for all the time and effort she spent recording the song of our birds. We are also indebted to Professor Peter Marler, Rockefeller University, for letting us include in our study several birds which he reared in noise and which formed part of an earlier experiment (Marler et al., 1973). Our research was supported by NIH grant MH 18343.     

Seasonal changes in patterns of gene expression in avian song control brain regions

Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by altering the hormonal and photoperiodic conditions in seasonally-breeding Gambel's white-crowned sparrows and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. We found that 363 genes varied by more than 1.5 fold (>log(2) 0.585) in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity.     

The effect of male song on an estrogen-dependent behavior pattern in the female canary (Serinus canarius)

Previous experiments showed that the length of the photoperiod influences the effectiveness of exogenous estrogen in inducing nest-building behavior in ovariectomized female canaries. Long photoperiods affect this aspect of reproductive behavior in two ways. They influence estrogen output by the classical hypothalamus-pituitary-gonad route, and they allow expression of estrogen-dependent behavior. The question arises, do external sitmuli that affect reproductive development, other than the photoperiod, also influence the effectiveness of exogenous estrogen?In this study tape-recorded male song was found to affect the amount of estrogen-induced nest-building of ovariectomized canaries on three different photoperiods. Females exposed to male canary song built more actively than the controls on all day lengths, but budgerigar song was without effect.     

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

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

Testosterone treatment increases the metabolic capacity of adult avian song control nuclei

In songbirds, the size of brain nuclei that control song learning and production change seasonally. These changes are mainly controlled by seasonal changes in plasma testosterone (T) concentration. One hypothesis to explain why it may be adaptive for these areas to regress in the fall is that this would decrease the metabolic demand of maintaining a large song system when singing is reduced or absent. We used a marker for cellular metabolism to examine birds with regressed song nuclei and compared them to birds whose song nuclei were induced to grow by administration of exogenous T. Photorefractory male Gambel's white-crowned sparrows were captured during their autumnal migration and kept in outdoor aviaries on a natural photoperiod. We implanted birds with Silastic capsules containing T or with empty implants. Three weeks later the birds were sacrificed. We assayed the brains for cytochrome oxidase (CO) activity and measured the volume of four song nuclei: HVc, RA, 1MAN, and area X. All four nuclei increased in volume in response to T treatment. T treatment increased the metabolic capacity of area X, HVc, and RA relative to surrounding tissue but had no effect on the metabolic capacity of 1MAN. These results support the hypothesis that song nuclei are more metabolically active under the influence of T than they are when plasma T levels are low.     

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.     

Lateralization of syringeal function during song production in the canary

The canary (Serinus canaria) vocal organ, the syrinx, has two separate sound sources, one in the cranial end of each bronchus. Previous investigations of whether song syllables are produced unilaterally or bilaterally have provided two contradictory results, as one researcher suggested that almost all syllables are produced by the left side of the syrinx alone, whereas another researcher suggested that both sides contribute similarly to all syllables. Our experiments, which involved unilateral bronchus plugging followed later by denervation of the ipsilateral syringeal muscles, attempted to resolve this disagreement. The males with right bronchus plugs, singing on the left side of the syrinx alone, produced nearly normal songs, whereas the birds with left bronchus plugs, singing on the right side, sang quite poorly. Interpretation of these data is difficult because it is not clear how syringeal function would be affected if the airflow rate through the intact side is increased above normal, nor is it known if the bird can compensate for bronchus occlusion. Nonetheless, we suggest that in male canaries most syllables are normally sung by the left side alone, with some syllables being produced by the right side alone and some being sung by both sides together. Right nerve section had little effect on the right-bronchus-plugged males' ability to sing, but the repertoires of the left-plugged males were altered after left nerve section, indicating the possibility that signals carried by the left nerve exert an influence on the contralateral side.     

The energy cost of song in the canary, Serinus canaria

Although sound production requires energy, it has been unclear how much singing increases metabolic rate in passerine birds. We measured the rate of oxygen consumption of two breeds of canary that sang inside a respirometry chamber. Metabolic rate increased with the proportion of time that birds spent singing. Average metabolic rate during singing at 15-20°C was 1.05-1.07 times that of standing quietly in the same temperature range or 2.2-2.6 times basal metabolic rate (BMR). Whether an increase in metabolic rate during song of this order would represent a fitness cost to free-living passerine birds would depend upon the circumstances. Singing rather than perching during the day would raise metabolic rate only slightly. Singing at night or at dawn, instead of sleeping with a metabolic rate closer to BMR, would cause a greater increase in metabolism. Birdsong could act as a condition-dependent signal, since birds that are easily able to achieve energy balance could afford the cost of singing, but those close to their energy limits might not. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.      

Estrogens and non-estrogenic ovarian influences combine to promote the recruitment and decrease the turnover of new neurons in the adult female canary brain

The higher vocal center (HVC) of the songbird forebrain exhibits persistent neurogenesis in adulthood, particularly in a region of the mediocaudal neostriatum that is associated with a subventricular layer of estrogen receptive cells. We asked whether estrogens might influence adult neurogenesis, by assessing the effect of ovariectomy on HVC neuronal production in the adult female canary. Fifteen 1-year-old females were separated into groups of ovariectomized, estradiol-replaced ovariectomized, and gonadally intact birds. To label dividing cells and their progency, the birds were given [³H]thymidine for 8 days, killed 32 days later, and their brains autoradiographed. A significant rise was noted in the number of HVC neurons per section in estradiol-treated birds relative to the untreated control birds. The number of [³H]-thymidine-labeled HVC neurons was also higher in the estrogen-treated birds; however, the neuronal labeling index (LI) did not vary as a function of estradiol replacement, as the total number of HVC neurons rose in parallel with the added new neurons. In contrast, the neuronal LI did rise as a result of ovariectomy, and this ovariectomy-associated increase in the LI was not reversed by estradiol. Among non-neuronal cell types, the endothelial LI was higher in estrogen-treated birds than in their untreated counterparts, suggesting estrogen-associated angiogenesis. Radioimmunoassay confirmed that serum estradiol was reduced in the castrated birds. Since estrogen appeared to promote the survival of [³H]thymidine⁺ neurons, we next sought to determine whether estrogen acted directly on the newly generated neurons, or rather indirectly through an intermediary cell population. To this end, we asked whether the new neurons or their precursors expressed estrogen receptor immunoreactivity (ER-IR). Five adult male canaries were given [³H]thymidine for periods ranging from 2 to 28 days, killed at varying times up to 3 weeks therafter, then probed for ER-IR and autoradiographed. [³H]thymidine⁺ cells displayed no detectable ER-IR within their first 4 weeks of postmitotic life. Rather, during migration from the ventricular zone (VZ), the new neurons traversed a layer of mitotically quiescent, ER⁺ subventricular cells. Double labeling for ER-IR and cell-type selective antigens confirmed that these ER⁺ cells were neurons. These results indicate that the early survival of new neurons in the adult songbird HVC is promoted by estrogen, and may be mediated by the estrogen-stimulated paracrine release of neurotrophic agents by ER-IR subventricular neurons. Our data suggest that estrogen's promotion of neuronal survival may operate concurrently with an estrogen-independent ovarian suppression of neuronal mitogenesis.     

Histological effects of androgen deprivation on the adult chimpanzee epididymis

Primate sperm acquire functional maturity, including vigorous forward motility and the ability to fertilize an ovum, as they transit the unique, regional microenvironment of the epididymal lumen. Several proteins secreted into this luminal fluid are epididymal-specific and androgen-dependent, and thus contribute potentially to sperm maturation. For the adult male chimpanzee, we report the effects of GnRH antagonist-induced androgen deprivation on the histology of the epithelia and interstitium composing the ductuli efferentes, ductus epididymis, proximal ductus (vas) deferens. After 21 days of androgen deprivation, epididymal tissues exhibit characteristic atrophic changes, including cellular disorganization, degradation, and loss of structures. Androgen-deprived cytoplasm is differentially and characteristically disrupted, vacuolated, and reduced in volume, resulting in decreased epithelial height and loss of stereocilia. Most principal cell nuclei appear hyperchromatic, smaller in size, more irregular in outline, and disordered in arrangement, while others appear swollen and vacuolated. Apical cells of the efferent ducts and the basal cells and microvillar borders of the ductus epididymis seem minimally affected by androgen deprivation. Such histologically differential responses suggest correspondingly that androgen is differentially essential to the maintenance of the epididymis and thus to normal functioning of the component tissues. Therefore, epididymal epithelia directly and their secretions indirectly are differentially androgen-dependent.     

Quantitative integration of genetic factors in the learning and production of canary song

Learned bird song is influenced by inherited predispositions. The canary is a model system for the interaction of genes and learning on behaviour, especially because some strains have undergone artificial selection for song. In this study, roller canaries (bred for low-pitched songs) and border canaries (whose song is higher pitched, similar to the wild-type) were interbred and backcrossed to produce 58 males that sorted into seven genetically distinct groups. All males were tutored with the same set of songs, which included both low- and high-pitched syllables. Individuals were consistent within genetic groups but differed between groups in the proportion of low- versus high-pitched syllables they learned and sang. Both sex-linked and autosomal factors affected song learning and song production, in an additive manner. Dominant Z-chromosome factors facilitated high-pitched syllable learning and production, whereas the sex-linked alleles associated with the switch to low-pitched syllables under artificial selection were largely recessive. With respect to autosomal effects, the most surprising result is that males in the same genetic group had almost identical repertoires. This result challenges two common preconceptions: that genetic changes at different loci lead to distinct phenotypic changes, and that genetic predispositions affect learning in simple and general ways. Rather, different combinations of genetic changes can be associated with the same phenotypic effect; and predispositions can be remarkably specific, such as a tendency to learn and sing one song element rather than another.     

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.     

The effects of long-term estradiol treatment on social behavior and gene expression in adult female rats

This study tested the effects of long-term estradiol (E₂) replacement on social behavior and gene expression in brain nuclei involved in the regulation of these social behaviors in adult female rats. We developed an ultrasonic vocalization (USV) test and a sociability test to examine communications, social interactions, and social preference, using young adult female cagemates. All rats were ovariectomized (OVX) and implanted with a Silastic capsule containing E₂ or vehicle, and housed in same-treatment pairs for a 3-month period. Then, rats were behaviorally tested, euthanized, and 5 nuclei in the brain's social decision-making circuit were selected for neuromolecular profiling by a multiplex qPCR method. Our novel USV test proved to be a robust tool to measure numbers and types of calls emitted by cagemates that had been reintroduced after a 1-week separation. Results also showed that E₂-treated OVX rats had profoundly decreased numbers of USV calls compared to vehicle-treated OVX rats. In a test of sociability, in which a female was allowed to choose between her cagemate or a same-treatment novel rat, we found few effects of E₂ compared to vehicle, although interestingly, rats chose the cagemate over an unfamiliar conspecific. Gene expression results revealed that the supraoptic nucleus had the greatest number of gene changes caused by E₂: Oxt, Oxtr and Avp were increased, and Drd2, Htr1a, Grin2b, and Gabbr1 were decreased, by E₂. No genes were affected in the prefrontal cortex, and 1–4 genes were changed in paraventricular nucleus (Pgr), bed nucleus of the stria terminalis (Oxtr, Esr2, Dnmt3a), and medial amygdala (Oxtr, Ar, Foxp1, Tac3). Thus, E₂ changes communicative interactions between adult female rats, together with selected expression of genes in the brain, especially in the supraoptic nucleus.     

Effect of prenatal androgen treatment on maternal behavior in the female rat

The perinatal critical period when androgen suppresses the capacity of virgin female rats to display maternal behavior in response to pups in adulthood was studied. A single direct injection of a large dose of testosterone propionate (TP) to the fetuses on Days 19 or 21 of pregnancy, but not during the neonatal period, significantly suppressed maternal responses in females. Percentages of females with anovulatory ovaries were largest in groups treated with TP within 2 days after birth. It is suggested that the androgen-sensitive period of the maternal mediating systems in the female rat exists prenatally, whereas the critical period of the systems regulating the cyclic release of ovulatory hormone is in the neonatal period.