Canary Brain Research Articles

Sex-and Region-Dependent Expression of the Autism-Linked ADNP Correlates with Social- and Speech-Related Genes in the Canary Brain

The activity-dependent neuroprotective protein (ADNP) syndrome is an autistic-like disorder, instigated by mutations in ADNP. This syndrome is characterized by developmental delays, impairments in speech, motor function, abnormal hearing, and intellectual disabilities. In the Adnp-haploinsufficient mouse model, many of these impediments are evident, appearing in a sex-dependent manner. In zebra finch songbird (ZF; Taeniopygia guttata), an animal model used for song/language studies, ADNP mRNA most robust expression is observed in the cerebrum of young males, potentially corroborating with male ZF exclusive singing behavior and developed cerebral song system. Herein, we report a similar sex-dependent ADNP expression profile, with the highest expression in the cerebrum (qRT-PCR) in the brain of another songbird, the domesticated canary (Serinus canaria domestica). Additional analyses for the mRNA transcripts of the ADNP regulator, vasoactive intestinal peptide (VIP), sister gene ADNP2, and speech-related Forkhead box protein P2 (FoxP2) revealed multiple sex and brain region-dependent positive correlations between the genes (including ADNP). Parallel transcript expression patterns for FoxP2 and VIP were observed alongside specific FoxP2 increase in males compared with females as well as VIP/ADNP2 correlations. In spatial view, a sexually independent extensive form of expression was found for ADNP in the canary cerebrum (RNA in situ hybridization). The songbird cerebral mesopallium area stood out as a potentially high-expressing ADNP tissue, further strengthening the association of ADNP with sense integration and auditory memory formation, previously implicated in mouse and human.

Development of Perineuronal Nets during Ontogeny Correlates with Sensorimotor Vocal Learning in Canaries.

Songbirds are a powerful model to study vocal learning given that aspects of the underlying behavioral and neurobiological mechanisms are analogous in many ways to mechanisms involved in speech learning. Perineuronal nets (PNNs) represent one of the mechanisms controlling the closing of sensitive periods for vocal learning in the songbird brain. In zebra finches, PNN develop around parvalbumin (PV)-expressing interneurons in selected song control nuclei during ontogeny and their development is delayed if juveniles are deprived of a tutor. However, song learning in zebra finches takes place during a relatively short period of development, and it is difficult to determine whether PNN development correlates with the end of the sensory or the sensorimotor learning period. Canaries have a longer period of sensorimotor vocal learning, spanning over their first year of life so that it should be easier to test whether PNN development correlates with the end of sensory or sensorimotor vocal learning. Here, we quantified PNN around PV-interneurons in the brain of male canaries from hatching until the first breeding season and analyzed in parallel the development of their song. PNN development around PV-interneurons specifically took place and their number reached its maximum around the end of the sensorimotor learning stage, well after the end of sensory vocal learning, and correlated with song development. This suggests that PNN are specifically involved in the termination of the sensitive period for sensorimotor vocal learning.

Testosterone stimulates perineuronal nets development around parvalbumin cells in the adult canary brain in parallel with song crystallization

Perineuronal nets (PNN) of the extracellular matrix are dense aggregations of chondroitin-sulfate proteoglycans that usually surround fast-spiking parvalbumin-expressing inhibitory interneurons (PV). The development of PNN around PV appears specifically at the end of sensitive periods of visual learning and limits the synaptic plasticity in the visual cortex of mammals. Seasonal songbirds display a high level of adult neuroplasticity associated with vocal learning, which is regulated by fluctuations of circulating testosterone concentrations. Seasonal changes in testosterone concentrations and in neuroplasticity are associated with vocal changes between the non-breeding and breeding seasons. Increases in blood testosterone concentrations in the spring lead to the annual crystallization of song so that song becomes more stereotyped. Here we explore whether testosterone also regulates PNN expression in the song control system of male and female canaries. We show that, in both males and females, testosterone increases the number of PNN and of PV neurons in the three main telencephalic song control nuclei HVC, RA (nucleus robustus arcopallialis) and Area X and increases the PNN localization around PV interneurons. Singing activity was recorded in males and quantitative analyses demonstrated that testosterone also increased male singing rate, song duration and song energy while decreasing song entropy. Together, these data suggest that the development of PNN could provide the synaptic stability required to maintain the stability of the testosterone-induced crystallized song. This provides the new evidence for a role of PNN in the regulation of adult seasonal plasticity in seasonal songbirds.

Intraspecific variation in testosterone-induced neuroplasticity in two canary strains

Temperate zone songbird species, such as the canary (Serinus canaria), can serve as model systems to investigate adult seasonal plasticity in brain and behavior. An increase in day length, experienced by canaries in the early spring stimulates gonadal recrudescence and an associated increase in circulating testosterone concentrations. This increase in plasma testosterone results in marked morphological changes in well-defined neural circuitry regulating reproductive behaviors including birdsong as well as behavioral changes such as increases in song length and complexity. An obvious measure of plasticity in neural morphology can be assessed via changes in brain nuclei volume and testosterone actions on a number of cellular features including the integration and incorporation of new neurons in the adult canary brain. Previous work in our lab suggests that there may be systematic intraspecific variability within canaries in testosterone-induced adult neuroplasticity. For example, the song nucleus HVC increases in size in response to testosterone in male canaries but we found that males of the American Singer strain exhibited minimal and variable responses as compared to other canary strains such as the Border canary strain, which is thought to be closer to wild type canaries. In this study, we systematically compared the effects of testosterone on the volume of song nuclei and the number of new neurons as assessed with the neurogenesis marker doublecortin in American Singer and Border canaries. We found more pronounced testosterone-induced neuroplasticity in the Border strain than the American Singer. These data suggest that the process of selection for certain strain phenotypes is also associated with significant changes in hormone-regulated brain plasticity.

Male song quality modulates c-Fos expression in the auditory forebrain of the female canary

In canaries, specific phrases of male song (sexy songs, SS) that are difficult to produce are especially attractive for females. Females exposed to SS produce more copulation displays and deposit more testosterone into their eggs than females exposed to non-sexy songs (NS). Increased expression of the immediate early genes c-Fos or zenk (a.k.a. egr-1) has been observed in the auditory forebrain of female songbirds hearing attractive songs. C-Fos immunoreactive (Fos-ir) cell numbers were quantified here in the brain of female canaries that had been collected 30min after they had been exposed for 60min to the playback of SS or NS or control white noise. Fos-ir cell numbers increased in the caudomedial mesopallium (CMM) and caudomedial nidopallium (NCM) of SS birds as compared to controls. Song playback (pooled SS and NS) also tended to increase average Fos-ir cell numbers in the mediobasal hypothalamus (MBH) but this effect did not reach full statistical significance. At the individual level, Fos expression in CMM was correlated with its expression in NCM and in MBH but also with the frequency of calls that females produced in response to the playbacks. These data thus indicate that male songs of different qualities induce a differential metabolic activation of NCM and CMM. The correlation between activation of auditory regions and of the MBH might reflect the link between auditory stimulation and changes in behavior and reproductive physiology.

Evaluating the predictive value of doublecortin as a marker for adult neurogenesis in canaries (Serinus canaria)

Doublecortin (DCX) is an important microtubule-associated protein involved in the migration of young neurons into the cortical layers of the brain during early human development. The continued expression of DCX in brain areas with protracted neuron recruitment has promoted this endogenous protein as a popular indirect tool to monitor adult neurogenesis in a variety of species. However, little is known about its possible involvement in other cellular processes and a thorough validation of DCX as a quantitative measure for neurogenesis is generally lacking. Here we investigated the relationship between DCX expression and neuron recruitment in the brains of adult canaries (Serinus canaria), a species well-known for its adult neurogenesis. We examined the age and functional state of DCX-labeled cells by using mitotic and neuron-specific markers, retrograde tracings, and immediate early gene colocalizations. Although DCX expression was high in brain areas implicated in adult neurogenesis, DCX-expressing neurons were also abundant in regions that do not recruit new neurons. Moreover, DCX expression was observed in adult, active neurons, differentiated projection neurons, and birth-dated neurons of up to 1 year of age. Season and testosterone treatment affected DCX expression in two song control nuclei, HVC and Area X, but did not correlate with known patterns of neuron recruitment. Together, these results demonstrate that DCX expression is not exclusive to young migrating neurons, and does not predict neuron recruitment equally throughout the canary brain. Therefore, DCX labeling needs careful validation for each brain region separately in each species analyzed when used to quantify adult neurogenesis.

Anatomically discrete sex differences and enhancement by testosterone of cell proliferation in the telencephalic ventricle zone of the adult canary brain

Previous work in songbirds has suggested that testosterone increases neuronal recruitment and survival in HVC but does not affect neuronal proliferation in the ventricular zone and that males and females have similar rates of proliferation except at discrete locations. Many of these conclusions are however based on limited data or were inferred indirectly. Here we specifically tested the effects of testosterone on cellular proliferation in the ventricular zone of both male and female adult canaries. We implanted adult birds of both sexes with testosterone or empty implants for 1 week and injected them with BrdU. One day later, we collected their brains and quantified BrdU-positive cells in the ventricular zone (VZ) at different rostro-caudal levels of the brain, ranging from the level where the song nucleus Area X occurs through the caudal extent of HVC. Proliferation in the dorsal part of the VZ was low and unaffected by sex or testosterone treatment. In the ventral part of the VZ, females had more proliferating cells than males, but only at rostral levels, near Area X. Also in the ventral part of the VZ, testosterone increased proliferation in birds of both sexes, but only in the mid- to caudal-VZ, caudal to the level of Area X, around the septum and HVC. We thus demonstrate here that there is both an effect of testosterone and possibly a more subtle effect of sex on cellular proliferation in the adult songbird brain, and that these effects are specific to different levels of the brain.

The Hippocampus of Food-Storing Birds

when we examined sections from our control birds we saw something very striking. The hippocampus was clearly much larger in chickadees than shown in the atlas. Where the canary atlas showed a smooth curve as the dorsal surface of the brain travelled medially and descended between the hemispheres, the chickadees had a large bump. Previous work by many researchers on the songbird brain had prepared us for the idea that regions of the bird brain could vary between species. Song control nuclei vary in relative size between species and it seemed a likely hypothesis that food-storing birds, which can remember the spatial location of thousands of scattered food caches, might have a hippocampus that was bigger than the hippocampus of non-storing birds. With help from the Long Point Bird Observatory in Ontario, Anthony and I began collecting food-storing and nonstoring birds, sectioning the brains and measuring the relative size of the hippocampus. I contacted my former postdoctoral supervisor at Oxford, John Krebs, and John proposed that his team would collect European species in a parallel project. In Canada, Anthony and I, with help from George Wallace and the banders at Long Point, were able to collect individuals of 23 species from 3 food-storing families (chickadees, nuthatches and jays) and 10 non-food-storing families and subfamilies (wrens, kinglets, thrushes, mimids, starlings, warblers, cardinals, buntings, finchIn 1984–1985, Anthony Vaccarino and I performed a number of experiments on the effects of hippocampal lesions on the accuracy of cache recovery in food-storing black-capped chickadees [Sherry and Vaccarino, 1989]. Like most research, the genesis of this project involved many people. I presented a poster on memory for cache sites at the Neurobiology of Learning and Memory meeting in Irvine, Calif. in 1984 and Larry Squire pointed out to me that the obvious next experiment was to test for hippocampal involvement. I mentioned this idea to my friend Michael Leon at the same meeting, noting that I had no experience doing neurosurgery on 11-gram passerine birds and Michael, with his usual aplomb said that was no problem, there were colleagues at my own institution, the University of Toronto, like Alison Fleming, who could easily teach me the surgical skills. Alison introduced me to Anthony, who was working in Alison’s lab as an undergraduate, and we were on our way. We found that hippocampal lesions severely disrupted the ability of chickadees to find their caches. Fortunately for us, the birds continued to make caches and search for them, they just searched in the wrong places. This work formed part of Anthony’s undergraduate thesis and we published the paper in Behavioral Neuroscience in 1989 [Sherry and Vaccarino, 1989]. We had used the Stokes, Leonard and Nottebohm canary brain atlas [Stokes et al., 1974] as a guide to lesion placement but Published online: August 26, 2011

A customizable 3-dimensional digital atlas of the canary brain in multiple modalities

Songbirds are well known for their ability to learn their vocalizations by imitating conspecific adults. This uncommon skill has led to many studies examining the behavioral and neurobiological processes involved in vocal learning. Canaries display a variable, seasonally dependent, vocal behavior throughout their lives. This trait makes this bird species particularly valuable to study the functional relationship between the continued plasticity in the singing behavior and alterations in the anatomy and physiology of the brain. In order to optimally interpret these types of studies, a detailed understanding of the brain anatomy is essential. Because traditional 2-dimensional brain atlases are limited in the information they can provide about the anatomy of the brain, here we present a 3-dimensional MRI-based atlas of the canary brain. Using multiple imaging protocols we were able to maximize the number of detectable brain regions, including most of the areas involved in song perception, learning, and production. The brain atlas can readily be used to determine the stereotactic location of delineated brain areas at any desirable head angle. Alternatively the brain data can be used to determine the ideal orientation of the brain for stereotactic injections, electrophysiological recordings, and brain sectioning. The 3-dimensional canary brain atlas presented here is freely available and is easily adaptable to support many types of neurobiological studies, including anatomical, electrophysiological, histological, explant, and tracer studies.

Expression of reelin, its receptors and its intracellular signaling protein, Disabled1 in the canary brain: Relationships with the song control system

Songbirds produce learned vocalizations that are controlled by a specialized network of neural structures, the song control system. Several nuclei in this song control system demonstrate a marked degree of adult seasonal plasticity. Nucleus volume varies seasonally based on changes in cell size or spacing, and in the case of nucleus HVC and area X on the incorporation of new neurons. Reelin, a large glycoprotein defective in reeler mice, is assumed to determine the final location of migrating neurons in the developing brain. In mammals, reelin is also expressed in the adult brain but its functions are less well characterized. We investigated the relationships between the expression of reelin and/or its receptors and the dramatic seasonal plasticity in the canary (Serinus canaria) brain. We detected a broad distribution of the reelin protein, its mRNA and the mRNAs encoding for the reelin receptors (VLDLR and ApoER2) as well as for its intracellular signaling protein, Disabled1. These different mRNAs and proteins did not display the same neuroanatomical distribution and were not clearly associated, in an exclusive manner, with telencephalic brain areas that incorporate new neurons in adulthood. Song control nuclei were associated with a particular specialized expression of reelin and its mRNA, with the reelin signal being either denser or lighter in the song nucleus than in the surrounding tissue. The density of reelin-immunoreactive structures did not seem to be affected by 4 weeks of treatment with exogenous testosterone. These observations do not provide conclusive evidence that reelin plays a prominent role in the positioning of new neurons in the adult canary brain but call for additional work on this protein analyzing its expression comparatively during development and in adulthood with a better temporal resolution at critical points in the reproductive cycle when brain plasticity is known to occur.

The microtubule-associated protein doublecortin is broadly expressed in the telencephalon of adult canaries

The protein doublecortin (DCX) is expressed in post-mitotic migrating and differentiating neurons in the developing vertebrate brain and, as a part of the microtubule machinery, is required for neuronal migration. DCX expression is generally maximal during embryonic and early post-natal life but decreases markedly and almost disappears in older animals in parallel with the major decrease or cessation of neurogenesis. In several seasonally breeding songbird species such as canaries, the volume of several song control nuclei in the brain varies seasonally such that the largest nuclei are observed in the late spring and early summer. This variation is based on changes in cell size, dendritic branching, and, in nucleus HVC, on the incorporation of neurons newly born in adulthood. Because songbirds maintain an active neurogenesis and neuronal incorporation in their telencephalon throughout their lives, we investigated here the distribution of DCX-immunoreactive (ir) structures in the brain of adult male canaries. Densely stained DCX-ir cells were found exclusively in parts of the telencephalon that are known to incorporate new neurons in adulthood, in particular the nidopallium. Within this brain region, the boundaries of the song control nucleus HVC could be clearly distinguished from surrounding structures by a higher density of DCX-ir structures. In most telencephalic areas, about two thirds of these cells displayed a uni- or bipolar fusiform morphology suggesting they were migrating neurons. The rest of the DCX-ir cells in the telencephalon were larger and had a round multipolar morphology. No such staining was found in the rest of the brain. The broad expression of DCX specifically in adult brain structures that exhibit the characteristic of active incorporation of new neurons suggests that DCX plays a key role in the migration of new neurons in the brain of adult songbirds as it presumably does during ontogeny.

Protein and lipid oxidative damage and complex I content are lower in the brain of budgerigar and canaries than in mice. Relation to aging rate

What are the mechanisms determining the rate of animal aging? Of the two major classes of endothermic animals, bird species are strikingly long-lived compared to mammals of similar body size and metabolic rate. Thus, they are ideal models to identify longevity-related characteristics not linked to body size or low metabolic rates. Since oxidative stress seems to be related to the basic aging process, we measured specific markers of different kinds of oxidative damage to proteins, like glutamic and aminoadipic semialdehydes (GSA and AASA, specific protein carbonyls), Nɛ-(carboxyethyl)lysine (CEL), Nɛ-(carboxymethyl)lysine (CML), and Nɛ-(malondialdehyde)lysine (MDAL), as well as mitochondrial Complex I content and amino acid and membrane fatty acyl composition, in the brain of short-lived mice (maximum life span [MLSP] 3.5 years) compared with those of long-lived budgerigar 'parakeets' (MLSP, 21 years) and canaries (MLSP, 24 years). The brains of both bird species had significantly lower levels of compounds formed as a result of oxidative (GSA and AASA), glycoxidative (CEL and CML), and lipoxidative (CML and MDAL) protein modifications, as well as a lower levels of mitochondrial complex I protein. Although it is known that fatty acid unsaturation is lower in many tissues of long-lived compared to short-lived mammals, this is not true in the particular case of brain. In agreement with this, we also found that the brain tissue of bugerigars and canaries contains no fewer double bonds than that of mice. Amino acid composition analyses revealed that bird proteins have a significantly lower content of His, Leu and Phe, as well as, interestingly, of methionine, whereas Asp, Glu, Ala, Val, and Lys contents were higher than in the mammals. These results, together with those previously described in other tissues of pigeons (MLSP, 35 years) compared to rats (MLSP, 4 years), indicate that oxidative damage to proteins, lipids and mitochondrial DNA are lower in birds (very long-lived species) than in short-lived mammals of similar body size. The lower degree of oxidative modification of bird brain proteins was not due to decreases in the target amino acids (lysine for CEL, CML, MDAL, and AASA; and arg and pro for GSA), since these were present in bird brain proteins at higher or similar levels than in those of mice. These results are consistent with the possibility that decreases in oxidative protein modification are caused at least in part by the low rate of mitochondrial oxygen radical generation in these birds, as in all long-lived homeothermic vertebrates investigated so far.

Sex differences in the distribution of the steroid receptor coactivator SRC-1 in the song control nuclei of male and female canaries

The steroid receptor coactivator SRC-1 modulates ligand-dependent transactivation of several nuclear receptors, including the receptors for sex steroid hormones. The distribution of SRC-1 transcripts was analyzed here by in situ hybridization in coronal sections through the brain of male and female canaries. A broad but heterogeneous distribution of SRC-1 transcripts was observed with high numbers of densely labeled cells being present in many steroid-sensitive areas including the medial preoptic nucleus, several hypothalamic nuclei, five song control nuclei (HVc, the lateral and medial portion of the magnocellular nucleus of the anterior neostriatum, area X and the nucleus uvaeformis) and several catecholaminergic areas (area ventralis of Tsai, substantia nigra, locus coeruleus). The volume of two song control nuclei, HVc and area X were reconstructed based on the boundaries of the cell groups exhibiting a denser SRC-1 expression as compared to the surrounding areas. Sex differences in the expression of SRC-1 were also detected in several song control nuclei. In particular, the volume of HVc based on the high density of SRC-1 expression was significantly larger in males than in females. The effect of steroids on the song control system could be, at least in part, indirect and result from a modulation by steroids of the catecholaminergic inputs to the song control nuclei. The presence of the steroid receptor coactivator SRC-1 in the telencephalic song control nuclei and in the catecholaminergic cell groups that innervate the song system supports the idea that SRC-1 expression could play an active role in the control of singing behavior by modulating estrogen and androgen receptor action at both locations.

Gonads and Singing Play Separate, Additive Roles in New Neuron Recruitment in Adult Canary Brain

New neurons are constantly added to the high vocal center (HVC) of adult male canaries, Serinus canaria. Singing and testosterone (T) are known to promote this addition, but it is not known whether either variable can act on its own and what is their effect when acting together. We studied this question by castrating adult male canaries in late summer and quantifying their song in early fall. Intact birds served as controls. A 5 d systemic treatment of two daily injections of the cell birth marker 3H-thymidine started 10 d after surgery. Twenty days after the first 3H-thymidine injection and for a period of 1 month, we quantified the singing of all birds, which were then killed. Amount of singing, syllable diversity, and song stability were similar in intacts and castrates. When castrates and intacts that sang comparable amounts were compared, the number of 3H-labeled HVC neurons was 2.6 times higher in intacts than in castrates. In castrates with plasma T levels that were undetectable, the mean amount of singing was positively related to the number of new neurons. We suggest that singing and gonadal factors promote, separately, the recruitment of new neurons and that when they exert this effect together they do so in an additive manner.

What is the adaptive role of neurogenesis in adult birds?

Neurogenesis in adult brains was widely thought to be a specialization of lower vertebrates related to life-long changes in body size (Birse et al., 1980). In mammals, it was accepted that neurogenesis is restricted to the olfactory epithelium, despite early reports of neurogenesis in the rodent hippocampus (Altman and Das, 1965; Kaplan and Hinds, 1977; Bayer et al., 1982). This dogma of rare adult neurogenesis was further challenged by the work of Goldman and Nottebohm (1983), who described extensive neurogenesis in the brain of adult canaries, a songbird species. Since then it has become obvious that adult neurogenesis is a general feature of the forebrain and hippocampus of birds and that adult neurogenesis also occurs in various mammalian brain areas such as the hippocampus, the striatum, and the cortex, even in primates and humans (for review see Alvarez-Buylla and Kirn, 1997; Eriksson et al., 1998; Goldman, 1998; Gould et al., 1999a; Kornack and Rakic, 1999; Kaplan, 2001). Thus, besides anatomical and biochemical plasticity of permanent neurons and neuronal networks, the loss and addition of neurons in adulthood might be a major adaptive

Seasonal expression of androgen receptors, estrogen receptors, and aromatase in the canary brain in relation to circulating androgens and estrogens

Songbirds have a complex neural network for learning and production of song, namely the neural song system. Several nuclei of the song system contain androgen receptors (AR), and the neostriatal nucleus HVc also contains alpha type estrogen receptors (ER). Many songbird species show seasonal changes in both song and the neural song system that are correlated with seasonal variations in the circulating levels of gonadal steroids. However, there is increasing evidence that the sensitivity of the song system to gonadal steroids also changes seasonally. This could involve changes in the expression and activity of steroid receptors and steroid-metabolizing enzymes, such as the estrogen-synthesizing enzyme aromatase (AROM). The seasonal regulation of brain AR, ER, and AROM has not been studied before in the same individual songbirds. In this work, we compared plasma levels of androgens and estrogens, the expression level of AR-, ER-, and AROM-mRNA in the telencephalon, and brain AROM activity in male canaries between autumn (November) and spring (April) periods of high singing activity. Plasma levels of androgens and estrogens were higher in April than in November. The expression level of ER in HVc was higher in November than in April. In contrast, the expression level of AROM in the caudomedial neostriatum was higher in April than in November. However, we found no seasonal differences in the level of expression of AR and the volume of HVc as delimited by AR expression. Thus, AR expression in HVc was not correlated with circulating androgen levels. This study shows that both steroid-dependent and -independent seasonal factors regulate the action of gonadal hormones on the song system. In addition, we report a new site of AROM expression in the songbird brain, the nucleus interfacialis. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 254–268, 2000

Seasonal expression of androgen receptors, estrogen receptors, and aromatase in the canary brain in relation to circulating androgens and estrogens

Songbirds have a complex neural network for learning and production of song, namely the neural song system. Several nuclei of the song system contain androgen receptors (AR), and the neostriatal nucleus HVc also contains alpha type estrogen receptors (ER). Many songbird species show seasonal changes in both song and the neural song system that are correlated with seasonal variations in the circulating levels of gonadal steroids. However, there is increasing evidence that the sensitivity of the song system to gonadal steroids also changes seasonally. This could involve changes in the expression and activity of steroid receptors and steroid-metabolizing enzymes, such as the estrogen-synthesizing enzyme aromatase (AROM). The seasonal regulation of brain AR, ER, and AROM has not been studied before in the same individual songbirds. In this work, we compared plasma levels of androgens and estrogens, the expression level of AR-, ER-, and AROM-mRNA in the telencephalon, and brain AROM activity in male canaries between autumn (November) and spring (April) periods of high singing activity. Plasma levels of androgens and estrogens were higher in April than in November. The expression level of ER in HVc was higher in November than in April. In contrast, the expression level of AROM in the caudomedial neostriatum was higher in April than in November. However, we found no seasonal differences in the level of expression of AR and the volume of HVc as delimited by AR expression. Thus, AR expression in HVc was not correlated with circulating androgen levels. This study shows that both steroid-dependent and -independent seasonal factors regulate the action of gonadal hormones on the song system. In addition, we report a new site of AROM expression in the songbird brain, the nucleus interfacialis.

BDNF Mediates the Effects of Testosterone on the Survival of New Neurons in an Adult Brain

New neurons are incorporated into the high vocal center (HVC), a nucleus of the adult canary ( Serinus canaria) brain that plays a critical role in the acquisition and production of learned song. Recruitment of new neurons in the HVC is seasonally regulated and depends upon testosterone levels. We show here that brain-derived neurotrophic factor (BDNF) is present in the HVC of adult males but is not detectable in that of females, though the HVC of both sexes has BDNF receptors (TrkB). Testosterone treatment increases the levels of BDNF protein in the female HVC, and BDNF infused into the HVC of adult females triples the number of new neurons. Infusion of a neutralizing antibody to BDNF blocks the testosterone-induced increase in new neurons. Our results demonstrate that BDNF is involved in the regulation of neuronal replacement in the adult canary brain and suggest that the effects of testosterone are mediated through BDNF.

Toward a Song Code: Evidence for a Syllabic Representation in the Canary Brain

We show that presentation of individual canary song syllables results in distinct expression patterns of the immediate-early gene ZENK in the caudomedial neostriatum (NCM) of adult canaries. Information on the spatial distribution and labeling of stained cells provides for a classification of ZENK patterns that (1) accords to the organization of stimuli into families, (2) preserves the stimuli intrafamily relationships, and (3) confers salience to natural over artificial stimuli, resulting in a nonclassical tonotopic map. Moreover, complex syllable maps cannot be reduced to any linear combinations of simple syllable maps. These properties arise from the collective response of NCM neurons to auditory stimuli, rather than from the behavior of single neurons. The syllabic representation described here may constitute an important step toward deciphering the rules of birdsong auditory representation.

Non invasive in vivo anatomical studies of the oscine brain by high resolution MRI microscopy

We describe in this paper an in vivo Magnetic Resonance Imaging (MRI) procedure that allows one to obtain three-dimensional high quality images of the entire brain of small passerine birds such as the canary with a slice thickness of 58 micron and an image resolution of 78 microns. This imaging procedure was completed in 70 min on anaesthetised birds that later recovered uneventfully and could be reused for subsequent additional imaging. To illustrate the high resolution and anatomical detail that can be achieved, examples of coronal images through the entire hypothalamus are provided in the same sectioning plane as the previously published canary brain atlas. The data set can be used to create sections in any desired plane and the entire data set can be viewed from any point of view in a volume rendered image. This provides a useful tool in understanding the three-dimensional organisation of the brain. Similar procedures can also be applied on fixed brains and might allow an even better anatomical resolution of images because time constrains no longer limit the duration of image acquisition. The non-invasive MRI technique enables to study neuroanatomical features with a high resolution and without killing the animal subjects so that measures can be obtained in a same individual both before and after an experimental treatment.