Seasonal changes in astrocytes parallel neuronal plasticity in the song control area HVc of the canary

In the song control area HVc of the canary, intercellular dye‐coupling among astrocytes was studied by intracellular injection of neurobiotin into identified single astrocytes. Injection of individual astrocytes into acute slices resulted in dye spread to neighboring astrocytes, covering a sphere of up to 1 mm in diameter. The astrocytic nature of the dye‐coupled cells was verified by double labeling of neurobiotin‐filled cells with antisera for the astrocytic filament proteins GFAP or vimentin. The similarity in the number of dye‐coupled cells and the total number of astrocytes labeled by immunocytochemical markers indicate that dye‐coupling is specific for astrocytes and labels almost the entire local astrocytic population. Within the major nucleus for vocal control (HVc), approximately 25% more astroglial cells were present than in the surrounding forebrain tissue. There is no apparent hindrance of dye spread at the border of the HVc. The density of dye‐coupled astrocytes and the expression of cytoskeletal filament proteins differed markedly between the reproductive period in spring and the quiescent period in autumn. While vimentin is the major astroglial filament in autumn, GFAP is strongly expressed in spring. The density of dye‐coupled astrocytes reveals a marked increase in the reproductive period, followed by a reduction in autumn. The data indicate that the astrocytic population in the avian forebrain undergoes significant changes coincident with the known functional changes in the vocal control nuclei during periods of song production. GLIA 27:88–100, 1999. © 1999 Wiley‐Liss, Inc.     

Differential expression pattern and steroid hormone sensitivity of SNAP-25 and synaptoporin mRNA in the telencephalic song control nucleus HVC of the zebra finch

Gonadal steroid hormones play an important role in the process of sexual differentiation of brain areas and behavior such as singing and song learning in songbirds. These hormones affect behavior controlling circuits on both the gross morphological and ultrastructural levels. Here we study whether the expression of genes coding for synaptic proteins is sensitive to gonadal steroid hormones and whether such altered expression coincides with changes in brain area size. We treated adult male zebra finches with the aromatase inhibitor fadrozole, to reduce estrogen synthesis and analyzed the mRNA expression of the synaptic proteins synaptoporin (SPO) and synaptosomal-associated protein 25 kDa (SNAP-25) in song control areas and surrounding tissues of adult male zebra finches. SPO and SNAP-25 are differently expressed throughout the song system. Generally, the telencephalic song nuclei expressed SNAP-25 at high intensity whereas SPO expression was area-specific. Elevated levels of SNAP-25 mRNA were present in the nucleus hyperstriatalis ventrale pars caudale (HVC) and in the robust nucleus of the archistriatum (RA). SPO mRNA was found in moderate levels in the HVC, in low levels in the lateral nucleus magnocellularis (lMAN) and Area X, and was absent in the RA. The treatment significantly increased the mRNA level of SPO in the HVC, whereas SNAP-25 expression level was not affected. These expression patterns are not explained by the decrease of HVC volume after treatment. The decreased HVC size is not area-specific but correlates with an overall reduction in size and an overall increase in cell density of the forebrain.     

Corticosterone and dehydroepiandrosterone have opposing effects on adult neuroplasticity in the avian song control system

Chronic elevations in glucocorticoids can decrease the production and survival of new cells in the adult brain. In rat hippocampus, supraphysiological doses of dehydroepiandrosterone (DHEA; a sex steroid precursor synthesized in the gonads, adrenals, and brain) have antiglucocorticoid properties. With male song sparrows (Melospiza melodia), we examined the effects of physiological doses of corticosterone, the primary circulating glucocorticoid in birds, and DHEA on adult neuroplasticity. We treated four groups of nonbreeding sparrows for 28 days with empty (control), corticosterone, DHEA, or corticosterone + DHEA implants. Subjects were injected with BrdU on days 3 and 4. In HVC, a critical song control nucleus, corticosterone and DHEA had independent, additive effects. Corticosterone decreased, whereas DHEA increased, HVC volume, NeuN⁺ cell number, and BrdU⁺ cell number. Coadministration of DHEA completely reversed the neurodegenerative effects of chronic corticosterone treatment. In an efferent target of HVC, the robust nucleus of the arcopallium (RA), DHEA increased RA volume, but this effect was blocked by coadministration of corticosterone. There were similar antagonistic interactions between corticosterone and DHEA on BrdU⁺ cell number in the hippocampus and ventricular zone. This is the first report on the effects of corticosterone treatment on the adult song control circuit, and HVC was the most corticosterone‐sensitive song nucleus examined. In HVC, DHEA is neuroprotective and counteracts several pronounced effects of corticosterone. Within brain regions that are particularly vulnerable to corticosterone, such as the songbird HVC and rat hippocampus, DHEA appears to be a potent native antiglucocorticoid. J. Comp. Neurol. 518:3662–3678, 2010. © 2010 Wiley‐Liss, Inc.     

Maximized song learning of juvenile male zebra finches following BDNF expression in the HVC

During song learning, vocal patterns are matched to an auditory memory acquired from a tutor, a process involving sensorimotor feedback. Song sensorimotor learning and song production of birds is controlled by a set of interconnected brain nuclei, the song control system. In male zebra finches, the beginning of the sensorimotor phase of song learning parallels an increase of the brain‐derived neurotrophic factor (BDNF) in just one part of the song control system, the forebrain nucleus HVC. We report here that transient BDNF‐mRNA upregulation in the HVC results in a maximized copying of song syllables. Each treated bird shows motor learning to an extent similar to that of the selected best learners among untreated zebra finches. Because this result was not found following BDNF overexpression in the target areas of HVC within the song system, HVC‐anchored mechanisms are limiting sensorimotor vocal learning.     

Testosterone‐induced neuroendocrine changes in the medial preoptic area precede song activation and plasticity in song control nuclei of female canaries

Testosterone plays a key role in the control of seasonal changes in singing behavior and its underlying neural circuitry. After administration of exogenous testosterone, song quality and song control nuclei volumes change over the course of weeks, but song rate increases within days. The medial preoptic nucleus (POM) controls sexual motivation and testosterone action in POM increases sexually motivated singing. In this study, we investigated the time course of testosterone action in the song control nuclei and POM, at the gross anatomical and cellular level. Photosensitive female canaries were injected with BrdU to label newborn neurons. One day later they were transferred to a long‐day photoperiod and implanted with testosterone‐filled or empty implants. Brains and blood were collected 1, 2, 9 or 21 days later. Testosterone increased POM volume within 1 day, whereas the volume of song control nuclei increased significantly only on day 21 even if a trend was already observed for HVC on day 9. The density of newborn neurons in HVC, labeled by Bromodeoxyuridine (BrdU) and doublecortin, was increased by testosterone on days 9 and 21 although a trend was already detectable on day 2. In POM, testosterone increased the number and size of aromatase‐immunoreactive neurons already after 1 day. This rapid action of testosterone in POM supports its proposed role in controlling singing motivation. Although testosterone increased the number of newborn neurons in HVC rapidly (9, possibly 2 days), it is unlikely that these new neurons affect singing behavior before they mature and integrate into functional circuits.     

Ultrastructural characterization of synaptic terminals formed on newly generated neurons in a song control nucleus of the adult canary forebrain

The fine structure of synaptic terminals contacting neurons generated in the forebrain of adult male canaries was investigated by autoradiography and electron microscopy. The procedure for labeling the new neurons included pretreating adult canaries with 3H-thymidine and sacrificing them 23-45 days later. Neurons were identified as newly generated by the presence of 3H-thymidine in the cell nucleus. The new neurons in the nucleus hyperstriatum ventralis, pars caudalis (HVc) were identified by autoradiography and light microscopy and examined with electron microscopy. Several types of synaptic terminals contacted the cell body and proximal dendrites of the newly formed neurons. Synaptic junctions were formed by terminals that contained spherical, agranular vesicles, large dense-core vesicles and spherical, agranular vesicles, and pleomorphic or flattened synaptic vesicles. Terminals that contained spherical vesicles were most often associated with asymmetric synaptic densities, and terminals that contained pleomorphic or flattened vesicles formed symmetric junctions. New neurons were also contacted by small terminals that contained few vesicles and had little pre- or postsynaptic density associated with the junction; these terminals may be a special type or may be in the process of developing their synaptic contact with the new neuron. In addition, rare terminals that appeared to be degenerating or to contain debris from other degenerating neural elements contacted new neurons. In summary, these data indicate that the new neurons, which are known to be inserted into existing neural networks, receive synaptic input from at least three different sources.     

Lateral asymmetries and testosterone-induced changes in the gross morphology of the hypoglossal nucleus in adult canaries.

The caudal portion of the hypoglossal nucleus (tracheosyringeal, nXIIts) contains the motor neurons that innervate the syrinx in songbirds. It receives projections from telencephalic and midbrain nuclei that are necessary for song production. Its neurons concentrate androgens. The present study assesses the gross morphology of the hypoglossal nucleus in canaries. In this species song is more frequent, elaborate, and stereotyped in males than in females. Adult females respond to testosterone by developing a stereotyped song that is sung frequently. Song in male canaries is much more disrupted by damage on the left side of the song system than by damage on the right. We find anatomical correlates for each of these attributes in the nXIIts. This nucleus is 83% larger in males than in females. This is caused primarily by a sex difference in neuropil volume as there is no significant sex difference in the number of neurons in nXIIts. nXIIts grows by 34% in females given testosterone as adults. It is about 8% larger on the left than on the right in males, females, and females treated with testosterone. Sex differences are also found in the rostral (lingualis) portion of nXII, which controls muscles of the tongue, but there is no effect here of adult treatment with testosterone. Comparisons of these data with earlier measures of synaptic density and morphology in nXIIts suggest that the testosterone acts on this nucleus by inducing a modest increase in synapse numbers and by altering the efficacy of synapses in nXIIts. This contrasts with the effects of testosterone on n. robustus archistriatalis, a telencephalic component of the song system in which testosterone induces massive amounts of synaptogenesis.     

Anatomical plasticity in the adult zebra finch song system

In many songbirds, vocal learning‐related cellular plasticity was thought to end following a developmental critical period. However, mounting evidence in one such species, the zebra finch, suggests that forms of plasticity common during song learning continue well into adulthood, including a reliance on auditory feedback for song maintenance. This reliance wanes with increasing age, in tandem with age‐related increases in fine motor control. We investigated age‐related morphological changes in the adult zebra finch song system by focusing on two cortical projection neuron types that 1) share a common efferent target, 2) are known to exhibit morphological and functional change during song learning, and 3) exert opposing influences on song acoustic structure. Neurons in HVC and the lateral magnocellular nucleus of the anterior nidopallium (LMAN) both project to the robust nucleus of the arcopallium (RA). During juvenile song learning and adult song maintenance, HVC promotes song syllable stereotypy, whereas LMAN promotes learning and acoustic variability. After retrograde labeling of these two cell types in adults, there were age‐related increases in dendritic arbor in HVC‐RA but not LMAN‐RA neurons, resulting in an increase in the ratio of HVC‐RA:LMAN‐RA dendritic arbor. Differential growth of HVC relative to LMAN dendrites may relate to increases in song motor refinement, decreases in the reliance of song on auditory feedback, or both. Despite this differential growth with age, both cell types retain the capacity for experience‐dependent growth, as we show here. These results may provide insights into mechanisms that promote and constrain adult vocal plasticity. J. Comp. Neurol. 520:3673–3686, 2012. © 2012 Wiley Periodicals, Inc.     

Delineation of a brain nucleus: comparisons of cytochemical, hodological, and cytoarchitectural views of the song control nucleus HVc of the adult canary

The present investigation used stable area-specific, neuronal properties instead of Nissl stain to delineate the boundaries of the nucleus hyperstriatalis caudal c (HVc) in the telencephalon of the adult male canary. Immunocytochemical procedures combined with retrograde tracing labeled a large population of perennial long-projecting neurons that contain estrogen receptors in the canary HVc. The HVc area defined by the distribution of these neurons was congruent with the HVc area defined in Nissl-stained sections during the breeding period. The HVc area defined in Nissl-stained preparations showed an extensive seasonal change in size, confirming previous results (Nottebohm: Science, 214:1368-1370, '81). In contrast, the HVc area defined by the distribution of the estrogen receptor containing long-projection neurons showed little or no seasonal change in size. Because these neurons are permanent, the HVc seems to be of rather constant size year round. The internal morphology of the HVc, however, undergoes seasonal alterations, which are reflected in changes in size of the HVc area distinguishable in Nissl-stained sections. The combination of cytoarchitectural criteria of Nissl-stained preparations with area-specific cytochemical and hodological markers to delineate the boundaries of a brain nucleus might give new insights in the partitioning and neuronal plasticity of brain areas.     

Expression of brain lipid binding protein in the brain of the adult canary and its implications for adult neurogenesis

Brain lipid binding protein (BLBP), a member of the fatty acid binding protein family, is expressed at high levels in the mammalian central nervous system during development, but not in adulthood. Because the brain of adult birds continues to show significant levels of neurogenesis, we thought it likely that BLBP expression would also be present. We used a polyclonal antibody against BLBP to study the presence of this protein in the adult canary brain. This antibody stained 1) fibers and perikarya of radial cells in the telencephalon; 2) Bergmann glia in the cerebellum; 3) astrocytes; 4) tanicytes in the walls of the third ventricle; 5) the neuropil of certain forebrain and brainstem regions, including nuclei of the song system; and 6) some migrating cells in the telencephalon. This anatomical distribution suggests that BLBP plays a role in the neuronal migration and synaptic reorganization of adult avian brain. J. Comp. Neurol. 385:415–426, 1997. © 1997 Wiley-Liss, Inc.     

Seasonal changes in the size of the avian song control nucleus HVC defined by multiple histological markers

Bird song is controlled by a discrete network of brain nuclei. The size of several song control nuclei changes seasonally in many seasonally breeding songbird species. Reports of seasonal changes in the size of song nuclei have relied primarily on Nissl stains to define the borders of these regions. Recent studies found that the size of the song nucleus higher vocal center (HVC) in male canaries did not change seasonally when its borders were defined by histological markers other than Nissl staining. We used three labels to define the borders of the HVC in male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii): Nissl staining, the distribution of acetylcholinesterase-positive neuropil, and the distribution of neurons projecting to another song nucleus, area X. The HVC was larger in males exposed to a breeding photoperiod and testosterone concentrations than in males exposed to a nonbreeding photoperiod and testosterone concentrations, regardless of which of these three methods was used to define the borders of the HVC. This result suggests that seasonal changes in the Nissl-defined borders of the HVC reflect changes in the distribution of physiologically relevant markers of the nucleus and are not merely artifacts of the Nissl-staining method. J. Comp. Neurol. 381:253-261, 1997. © 1997 Wiley-Liss, Inc.     

Lesions to the medial preoptic nucleus affect immediate early gene immunolabeling in brain regions involved in song control and social behavior in male European starlings

The medial preoptic nucleus (POM) is a brain region outside of the song control system of songbirds. It has been implicated in song production, sexual motivation, and the integration of both sensory and hormonal information with appropriate behavioral responses. The POM is well positioned neuroanatomically to interact with multiple regions involved in song, social behavior, and motivation. However, little is known about the brain regions with which the POM directly or indirectly communicates to influence song. To gain insight into the neuronal circuits normally activated in association with POM activity during male song, we compared activity within multiple brain regions using immunolabeling for protein products of immediate early genes (IEGs) zenk (aka egr-1 ) and c-fos (indirect markers of neuronal activity) in sham and POM-lesioned male European starlings ( Sturnus vulgaris ). As compared to sham lesions, POM lesions disrupted song and interest in a nest box, and females responded less to POM-lesioned males. POM lesions reduced numbers of IEG-labeled cells and disrupted correlations between numbers of IEG-labeled cells and song within several song control, limbic, hypothalamic and midbrain regions. These results are consistent with the possibility that the POM integrates activity among nuclei involved in song control, social behavior and motivational state that work in concert to promote sexually motivated communication.     

Masculinisation of the Zebra Finch Song System: Roles of Oestradiol and the Z‐chromosome Gene Tubulin‐Specific Chaperone Protein A

Robust sex differences in brain and behaviour exist in zebra finches. Only males sing, and forebrain song control regions are more developed in males. The factors driving these differences are not clear, although numerous experiments have shown that oestradiol (E₂) administered to female hatchlings partially masculinises brain and behaviour. Recent studies suggest that an increased expression of Z‐chromosome genes in males (ZZ; females: ZW) might also play a role. The Z‐gene tubulin‐specific chaperone A (TBCA) exhibits increased expression in the lateral magnocellular nucleus of the anterior nidopallium (LMAN) of juvenile males compared to females; TBCA+ cells project to the robust nucleus of the arcopallium (RA). In the present study, we investigated the role of TBCA and tested hypotheses with respect to the interactive or additive effects of E₂ and TBCA. We first examined whether E₂ in hatchling zebra finches modulates TBCA expression in the LMAN. It affected neither the mRNA, nor protein in either sex. We then unilaterally delivered TBCA small interfering (si)RNA to the LMAN of developing females treated with E₂ or vehicle and males treated with the aromatase inhibitor, fadrozole, or its control. In both sexes, decreasing TBCA in LMAN reduced RA cell number, cell size and volume. It also decreased LMAN volume in females. Fadrozole in males increased LMAN volume and RA cell size. TBCA siRNA delivered to the LMAN also decreased the projection from this brain region to the RA, as indicated by anterograde tract tracing. The results suggest that TBCA is involved in masculinising the song system. However, because no interactions between the siRNA and hormone manipulations were detected, TBCA does not appear to modulate effects of E₂ in the zebra finch song circuit.     

Delayed development of song control nuclei in the zebra finch is related to behavioral development

The postnatal development of two visual areas (nucleus rotundus and ectostriatum) and two song control areas (hyperstriatum ventrale pars caudale, HVc, and nucleus robustus archistriatalis, RA) of the zebra finch brain was followed from birth to adulthood. The following parameters were investigated: (1) neuron size, (2) volume of the brain nuclei, and (3) myelination of axons. The nucleus rotundus, the diencephalic station of the tectofugal pathway, exhibits the fastest development: rotundal neurons reach their maximum size at 20 days of age; the volume of this structure reaches adult size at the same time. The process of myelination begins between day 5 and day 10 and is completed at 40 days of age. A similar temporal sequence of development is seen in the ectostriatum, except myelination starts some days later. Thus the development of these visual areas is completed at 40 days. In contrast, the development of the song control nuclei is delayed. Neurons in RA and HVc grow steadily up to 40 days of age, attaining a size larger than that observed in adults. Whereas the volume of HVc increases until day 40 and remains stable thereafter, RA volume increases until day 70 and evidences a decrease thereafter. It is not until postnatal day 20 (RA) and day 40 (HVc) that the myelination process starts in the song control areas. Adult myelin density is achieved by 70 days in RA and by 100 days in HVc. It can be demonstrated that the development of the visual system parallels the development of visual performance of the birds. Delayed growth of song control nuclei coincides with development of song.     

Differential molecular profiles of astrocytes in degeneration and re‐innervation after sensory deafferentation of the adult rat cochlear nucleus

Ablating the cochlea causes total sensory deafferentation of the cochlear nucleus. Over the first postoperative week, degeneration of the auditory nerve and its synaptic terminals in the cochlear nucleus temporally overlaps with its re‐innervation by axon collaterals of medial olivocochlear neurons. At the same time, astrocytes increase in size and density. We investigated the time courses of the expression of ezrin, polysialic acid, matrix metalloprotease‐9 and matrix metalloprotease‐2 within these astrocytes during the first week following cochlear ablation. All four proteins are known to participate in degeneration, regeneration, or both, following injury of the central nervous system. In a next step, stereotaxic injections of kainic acid were made into the ventral nucleus of the trapezoid body prior to cochlear ablation to destroy the neurons that re‐innervate the deafferented cochlear nucleus by axon collaterals developing growth‐associated protein 43 immunoreactivity. This experimental design allowed us to distinguish between molecular processes associated with degeneration and those associated with re‐innervation. Under these conditions, astrocytic growth and proliferation showed an unchanged deafferentation‐induced pattern. Similarly, the distribution and amount of ezrin and matrix metalloprotease‐9 in astrocytes after cochlear ablation developed in the same way as under cochlear ablation alone. In sharp contrast, the astrocytic expression of polysialic acid and matrix metalloprotease‐2 normally invoked by cochlear ablation collapsed when re‐innervation of the cochlear nucleus was inhibited by lesioning medial olivocochlear neurons with kainic acid. In conclusion, re‐innervation, including axonal growth and synaptogenesis, seems to prompt astrocytes to recompose their molecular profile, paving the way for tissue reorganisation after nerve degeneration and loss of synaptic contacts.     

Intrauterine proximity to male fetuses affects the morphology of the sexually dimorphic nucleus of the preoptic area in the adult rat brain

Previous studies on polytocous rodents have revealed that the fetal intrauterine position influences its later anatomy, physiology, reproductive performance and behavior. To investigate whether the position of a fetus in the uterus modifies the development of the brain, we examined whether the structure of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of rat brains accorded to their intrauterine positions. Brain sections of adult rats gestated between two male fetuses (2M) and between two female fetuses (2F) in the uterus were analysed for their immunoreactivity to calbindin-D28k, which is a marker of the SDN-POA. The SDN-POA volume of the 2M adult males was greater than that of the 2F adult males, whereas the SDN-POA volume of the 2M and 2F adult females showed no significant difference. This result indicated that contiguous male fetuses have a masculinizing effect on the SDN-POA volume of the male. To further examine whether the increment of SDN-POA volume in adulthood was due to exposure to elevated steroid hormones during fetal life, concentrations of testosterone and 17beta-estradiol in the brain were measured with 2M and 2F fetuses during gestation, respectively. On gestation day 21, the concentrations of testosterone and 17beta-estradiol in the brain were significantly higher in the 2M male rats as compared with the 2F male rats. The results suggested that there was a relationship between the fetal intrauterine position, hormone transfer from adjacent fetuses and the SDN-POA volume in adult rat brains.     

Seasonal change in neuron size and spacing but not neuronal recruitment in a basal ganglia nucleus in the avian song control system

Neural plasticity in the song control system of seasonally breeding songbirds accompanies seasonal changes in singing behavior. The volume of Area X, a song control nucleus that forms a portion of the avian basal ganglia, is 75% larger in the spring than it is in the fall. The neuronal basis of the seasonal plasticity in Area X is largely unknown, however. We examined neuronal attributes of Area X in wild adult male song sparrows (Melospiza melodia) captured during the spring and the fall after being implanted for 30 days with osmotic pumps containing [3H]thymidine. We measured the volume of Area X from thionin-stained sections, and neuronal density and number, and average area of the soma from sections labeled with an antibody against Hu, a neuron-specific protein. We sampled two neuron classes: "small" neurons that were most likely striatal-like spiny neurons and "large" neurons, which most likely included pallidal-like projection neurons. We also analyzed seasonal patterns of neuronal recruitment to Area X. The average area of the soma and neuronal spacing for both neuronal classes were greater in breeding birds. There was no difference in total neuron number for both neuronal classes between seasons. The average area of the soma and density and number of newly recruited neurons did not vary across seasons. These results demonstrate that seasonal plasticity in Area X includes changes in neuron size and neuronal density, but not changes in the rate at which new neurons are recruited.     

Characterization of projections from a sexually dimorphic motor nucleus in the spinal cord of adult green anoles

Male green anoles possess two copulatory organs (hemipenes), which are independently controlled by bilateral muscles: the transversus penis (TPN) and retractor penis magnus (RPM). Adult females do not possess hemipenes or either of the two related muscles. Motoneurons projecting to the TPN lie in spinal segments trunk 17 and sacral 1 (T17-S1). Overall, motoneurons in this region are larger and more numerous in males than females. The present studies were designed to determine 1) whether motoneurons projecting to the RPM are located in the same sexually dimorphic nucleus, 2) other targets of T17-S1 motoneurons, and 3) the approximate proportion of motoneurons projecting to each muscle. In Study 1, unilateral injection of the retrograde tracer Fast Blue (FB) into RPMs and simultaneous unilateral injection of either Cholera Toxin-fluorescein (CT-FITC) or Diamidino Yellow into TPNs revealed that RPM and TPN motoneurons are indeed interdigitated in T17-S1. In Study 2, FB was used to characterize other targets of this nucleus in both males and females. In adult males, projections to four muscles accounted for 96% of the T17-S1 motoneurons: the TPN, RPM, caudifemoralis (CF), and cloacal sphincter (SC). In adult females, projections to the CF and SC comprised 70% of this nucleus. These data demonstrate that the T17-S1 nucleus is a mixed spinal nucleus that has projections to muscles present in both sexes, as well as those present only in males and specialized for male copulatory behavior.     

Accumulation of estrogen in a vocal control brain region of a duetting song bird

Tritiated estradiol (E) was injected into bay wrens (Trhyothorus nigricapillus), a tropical in which females sing complex vocal duets with males. Autoradiographic analysis revealed that males and females have equal proportions of cells labeled by estradiol or its metabolites (E target cells) in a telencephalic region involved in song: the caudal nucleus of the ventral hyperstriatum (HVc). Other forebrain song regions failed to show labeling by E. E or its metabolites were accumulated, however, by cells in the midbrain song region ICo (the intercollicular nucleus) and in hypothalamic regions. This pattern of accumulation in the song systems differs from that observed in a previous study in which bay wrens were injected with tritiated testosterone (T); T or its metabolites were accumulated by cells in HVc, RA (robust nucleus of the archistriatum), MAN (magnocellular nucleus of the neostriatum), ICo, and nXII (hypoglossal nucleus). Such comparison suggests that cells in HVc have different steroid accumulation properties from those in other song regions. Bay wrens differ from zebra finches (Poephilia guttata), in which HVc contains very few E target cells. The wrens are more similar to canaries (Serinus canarius), because both species have E target cells in HVc, and females of both species are able to sing. The interspecies comparison raises the question of whether the ability of HVc cells to accumulate E or its metabolites in both species constitutes a precondition for the bisexual potential for song production.