Cross-species conservation in the regulation of parvalbumin by perineuronal nets.

Parvalbumin (PV) neurons play an integral role in regulating neural dynamics and plasticity. Therefore, understanding the factors that regulate PV expression is important for revealing modulators of brain function. While the contribution of PV neurons to neural processes has been studied in mammals, relatively little is known about PV function in non-mammalian species, and discerning similarities in the regulation of PV across species can provide insight into evolutionary conservation in the role of PV neurons. Here we investigated factors that affect the abundance of PV in PV neurons in sensory and motor circuits of songbirds and rodents. In particular, we examined the degree to which perineuronal nets (PNNs), extracellular matrices that preferentially surround PV neurons, modulate PV abundance as well as how the relationship between PV and PNN expression differs across brain areas and species and changes over development. We generally found that cortical PV neurons that are surrounded by PNNs (PV+PNN neurons) are more enriched with PV than PV neurons without PNNs (PV-PNN neurons) across both rodents and songbirds. Interestingly, the relationship between PV and PNN expression in the vocal portion of the basal ganglia of songbirds (Area X) differed from that in other areas, with PV+PNN neurons having lower PV expression compared to PV-PNN neurons. These relationships remained consistent across development in vocal motor circuits of the songbird brain. Finally, we discovered a causal contribution of PNNs to PV expression in songbirds because degradation of PNNs led to a diminution of PV expression in PV neurons. These findings in reveal a conserved relationship between PV and PNN expression in sensory and motor cortices and across songbirds and rodents and suggest that PV neurons could modulate plasticity and neural dynamics in similar ways across songbirds and rodents.

Contribution of birds to the study of sexual differentiation of brain and behavior.

Behavioral neuroendocrinology has largely relied on mammalian models to understand the relationship between hormones and behavior, even if this discipline has historically used a larger diversity of species than other fields. Recent advances revealed the potential of avian models in elucidating the neuroendocrine bases of behavior. This paper provides a review focused mainly on the contributions of our laboratory to the study of sexual differentiation in Japanese quail and songbirds. Quail studies have firmly established the role of embryonic estrogens in the sexual differentiation of male copulatory behavior. While most sexually differentiated features identified in brain structure and physiology result from the different endocrine milieu of adults, a few characteristics are organized by embryonic estrogens. Among them, a sex difference was identified in the number and morphology of microglia which is not associated with sex differences in the concentration/expression of neuroinflammatory molecules. The behavioral role of microglia and neuroinflammatory processes requires further investigations. Sexual differentiation of singing in zebra finches is not mediated by the same endocrine mechanisms as male copulatory behavior and "direct" genetic effect, i.e., not mediated by gonadal steroids have been identified. Epigenetic contributions have also been considered. Finally sex differences in specific aspects of singing behavior have been identified in canaries after treatment of adults with exogenous testosterone suggesting that these aspects of song are differentiated during ontogeny. Integration of quail and songbirds as alternative models has thus expanded understanding of the interplay between hormones and behavior in the control of sexual differentiation.

Sex differences in song syntax and syllable diversity in testosterone-induced songs of adult male and female canaries.

BACKGROUND: Behavioral sex differences are widespread in the animal world. These differences can be qualitative (i.e., behavior present in one sex but not the other, a true sex dimorphism) or quantitative (behavior is present at a higher rate or quality in one sex compared to the other). Singing in oscine songbirds is associated with both types of differences. In canaries, female rarely sing spontaneously but they can be induced to do so by treatments with steroids. Song in these females is, however, not fully masculinized and exhibits relatively subtle differences in quality as compared with male song. We analyzed here sex differences in syllable content and syllable use between singing male and female canaries. METHODS: Songs were recorded from three groups of castrated male and three groups of photoregressed female canaries that had received Silastic™ implants filled with testosterone (T), with T plus estradiol (E2), or left empty (control). After 6 weeks of hormone treatment, 30 songs were recorded from each of the 47 subjects. Songs were segmented and each syllable was annotated. Various metrics of syllable diversity were extracted and network analysis was employed to characterize syllable sequences. RESULTS: Male and female songs were characterized by marked sex differences related to syllable use. Compared to females, males had a larger syllable-type repertoire and their songs contained more syllable types. Network analysis of syllable sequences showed that males follow more fixed patterns of syllable transitions than females. Both sexes, however, produced song of the same duration containing the same number of syllables produced at similar rates (numbers per second). CONCLUSIONS: Under the influence of T, canaries of both sexes are able to produce generally similar vocalizations that nevertheless differ in specific ways. The development of song during ontogeny appears to be a very sophisticated process that is presumably based on genetic and endocrine mechanisms but also on specific learning processes. These data highlight the importance of detailed behavioral analyses to identify the many dimensions of a behavior that can differ between males and females.

Male canaries normally sing complex songs at high rate while females only rarely sing very simple songs. Testosterone induces active singing in both male and female canaries, but female song is still not fully masculinized by these treatments even if song duration does not differ between the sexes. We analyzed the syllable repertoire and the sequence of use for different syllables in canaries of both sexes treated with testosterone or testosterone supplemented with estradiol. Compared to females, males had a larger syllable-type repertoire and their songs contained more syllable types. Syllable transitions were also more fixed in males. Sex differences in adult singing of canaries are thus a complex mixture of differences that result from the different endocrine condition of males and females (and are thus partially reversed by administration of exogenous testosterone) and of more stable differences that presumably develop during the ontogenetic process under the influence of endocrine and genetic differences and of differential learning processes. Canary song thus represents an outstanding model system to analyze the interaction between nature and nurture in the acquisition of a sophisticated learned behavior as well as the mechanisms controlling sex differences in vocal learning and production.

Steroid-dependent plasticity in the song control system: Perineuronal nets and HVC neurogenesis.

The vocal control nucleus HVC in songbirds has emerged as a widespread model system to study adult brain plasticity in response to changes in the hormonal and social environment. I review here studies completed in my laboratory during the last decade that concern two aspects of this plasticity: changes in aggregations of extracellular matrix components surrounding the soma of inhibitory parvalbumin-positive neurons called perineuronal nets (PNN) and the production/incorporation of new neurons. Both features are modulated by the season, age, sex and endocrine status of the birds in correlation with changes in song structure and stability. Causal studies have also investigated the role of PNN and of new neurons in the control of song. Dissolving PNN with chondroitinase sulfate, a specific enzyme applied directly on HVC or depletion of new neurons by focalized X-ray irradiation both affected song structure but the amplitude of changes was limited and deserves further investigations.

Effects of the depletion of neural progenitors by focal X-ray irradiation on song production and perception in canaries.

The song control nucleus HVC of songbirds has emerged as a widespread model system to study adult neurogenesis and the factors that modulate the incorporation of new neurons, including seasonal state, sex differences or sex steroid hormone concentrations. However, the specific function of these new neurons born in adulthood remains poorly understood. We implemented a new procedure based on focal X-ray irradiation to deplete neural progenitors in the ventricular zone adjacent to HVC and study the functional consequences. A 23 Gy dose depleted by more than 50 percent the incorporation of BrdU in neural progenitors, a depletion that was confirmed by a significant decrease in doublecortin positive neurons. This depletion of neurogenesis significantly increased the variability of testosterone-induced songs in females and decreased their bandwidth. Expression of the immediate early gene ZENK in secondary auditory areas of the telencephalon that respond to song was also inhibited. These data provide evidence that new neurons in HVC play a role in both song production and perception and that X-ray focal irradiation represents an excellent tool to advance our understanding of adult neurogenesis.

Does the syrinx, a peripheral structure, constrain effects of sex steroids on behavioral sex reversal in adult canaries?

We previously confirmed that effects of testosterone (T) on singing activity and on the volume of brain song control nuclei are sexually differentiated in adult canaries: females are limited in their ability to respond to T as males do. Here we expand on these results by focusing on sex differences in the production and performance of trills, i.e., rapid repetitions of song elements. We analyzed >42,000 trills recorded over a period of 6 weeks from 3 groups of castrated males and 3 groups of photoregressed females that received Silastic™ implants filled with T, T plus estradiol or left empty as control. Effects of T on the number of trills, trill duration and percent of time spent trilling were all stronger in males than females. Irrespective of endocrine treatment, trill performance assessed by vocal deviations from the trill rate versus trill bandwidth trade-off was also higher in males than in females. Finally, inter-individual differences in syrinx mass were positively correlated with specific features of trills in males but not in females. Given that T increases syrinx mass and syrinx fiber diameter in males but not in females, these data indicate that sex differences in trilling behavior are related to sex differences in syrinx mass and syrinx muscle fiber diameter that cannot be fully suppressed by sex steroids in adulthood. Sexual differentiation of behavior thus reflects organization not only of the brain but also of peripheral structures.

Does the syrinx, a peripheral structure, constrain effects of sex steroids on behavioral sex reversal in adult canaries?

We previously confirmed that effects of testosterone (T) on singing activity and on the volume of brain song control nuclei are sexually differentiated in adult canaries: females are limited in their ability to respond to T as males do. Here we expand on these results by focusing on sex differences in the production and performance of trills, i.e., rapid repetitions of song elements. We analyzed more than 42,000 trills recorded over a period of 6 weeks from 3 groups of castrated males and 3 groups of photoregressed females that received Silasticâ"¢ implants filled with T, T plus estradiol or left empty as control. Effects of T on the number of trills, trill duration and percent of time spent trilling were all stronger in males than females. Irrespective of endocrine treatment, trill performance assessed by vocal deviations from the trill rate versus trill bandwidth trade-off was also higher in males than in females. Finally, inter-individual differences in syrinx mass were positively correlated with trill production in males but not in females. Given that T increases syrinx mass and syrinx fiber diameter in males but not in females, these data indicate that sex differences in trilling behavior are related to sex differences in syrinx mass and syrinx muscle fiber diameter that cannot be fully reversed by sex steroids in adulthood. Sexual differentiation of behavior thus reflects organization not only of the brain but also of peripheral structures.

Testosterone treatment reveals marked sex differences in song diversity and syllable syntax in adult canaries.

Background. Behavioral sex differences are widespread in the animal world. These differences can be qualitative (i.e., behavior present in one sex but not the other, a true sex dimorphism) or quantitative (behavior is present at a higher rate or quality in one sex compared to the other). Singing in oscine songbirds is associated with both types of differences. In canaries, female rarely sing spontaneously but they can be induced to do so by treatments with steroids. Song in these females is however not fully masculinized and exhibits relatively subtle differences in quality as compared with male song. We analyzed here sex differences in syllable content and syllable use between singing male and female canaries. Methods. Songs were recorded from 3 groups of castrated male and 3 groups of photoregressed female canaries that had received Silasticâ"¢ implants filled with testosterone (T), with T plus estradiol (E2), or left empty (control). After 6 weeks of hormone treatment, 30 songs were recorded from each of the 47 subjects. Songs were segmented and each syllable was annotated. Various metrics of syllable diversity were extracted and network analysis was employed to characterize syllable sequences. Results. Male and female songs were characterized by marked sex differences related to syllable use. Compared to females, males had a larger syllable type repertoire and their songs contained more syllable types. Network analysis of syllable sequences showed that males follow more fixed patterns of syllable transitions than females. Both sexes however produced song of the same duration containing the same number of syllables produced at similar rates (numbers per second). Conclusions. Under the influence of T canaries of both sexes are able to produce generally similar vocalizations that nevertheless differ in specific ways. The development of song during ontogeny appears to be a very sophisticated process that is presumably based on genetic and endocrine mechanisms but also on specific learning processes. These data highlight the importance of detailed behavioral analyses in order to identify the many dimensions of a behavior that can differ between males and females.

Cross-species conservation in the regulation of parvalbumin by perineuronal nets.

Parvalbumin (PV) neurons play an integral role in regulating neural dynamics and plasticity. Therefore, understanding the factors that regulate PV expression is important for revealing modulators of brain function. While the contribution of PV neurons to neural processes has been studied in mammals, relatively little is known about PV function in non-mammalian species, and discerning similarities in the regulation of PV across species can provide insight into evolutionary conservation in the role of PV neurons. Here we investigated factors that affect the abundance of PV in PV neurons in sensory and motor circuits of songbirds and rodents. In particular, we examined the degree to which perineuronal nets (PNNs), extracellular matrices that preferentially surround PV neurons, modulate PV abundance as well as how the relationship between PV and PNN expression differs across brain areas and species and changes over development. We generally found that cortical PV neurons that are surrounded by PNNs (PV+PNN neurons) are more enriched with PV than PV neurons without PNNs (PV-PNN neurons) across both rodents and songbirds. Interestingly, the relationship between PV and PNN expression in the vocal portion of the basal ganglia of songbirds (Area X) differed from that in other areas, with PV+PNN neurons having lower PV expression compared to PV-PNN neurons. These relationships remained consistent across development in vocal motor circuits of the songbird brain. Finally, we discovered a causal contribution of PNNs to PV expression in songbirds because degradation of PNNs led to a diminution of PV expression in PV neurons. These findings reveal a conserved relationship between PV and PNN expression in sensory and motor cortices and across songbirds and rodents and suggest that PV neurons could modulate plasticity and neural dynamics in similar ways across songbirds and rodents.

Photoperiodic control of singing behavior and reproductive physiology in male Fife fancy canaries.

Temperate-zone birds display marked seasonal changes in reproductive behaviors and the underlying hormonal and neural mechanisms. These changes were extensively studied in canaries (Serinus canaria) but differ between strains. Fife fancy male canaries change their reproductive physiology in response to variations in day length but it remains unclear whether they become photorefractory (PR) when exposed to long days and what the consequences are for gonadal activity, singing behavior and the associated neural plasticity. Photosensitive (PS) male birds that had become reproductively competent (high song output, large testes) after being maintained on short days (SD, 8 L:16D) for 6 months were divided into two groups: control birds remained on SD (SD-PS group) and experimental birds were switched to long days (16 L:8D) and progressively developed photorefractoriness (LD-PR group). During the following 12 weeks, singing behavior (quantitatively analyzed for 3 × 2 hours every week) and gonadal size (repeatedly measured by CT X-ray scans) remained similar in both groups but there was an increase in plasma testosterone and trill numbers in the LD-PR group. Day length was then decreased back to 8 L:16D for LD-PR birds, which immediately induced a cessation of song, a decrease in plasma testosterone concentration, in the volume of song control nuclei (HVC, RA and Area X), in HVC neurogenesis and in aromatase expression in the medial preoptic area. These data demonstrate that Fife fancy canaries readily respond to changes in photoperiod and display a pattern of photorefractoriness following exposure to long days that is associated with marked changes in brain and behavior.

The neural distribution of the avian homologue of oxytocin, mesotocin, in two songbird species, the zebra finch and the canary: A potential role in song perception and production.

The avian homologue of oxytocin (OT), formerly called mesotocin, influences social behaviors in songbirds and potentially song production. We sought to characterize the distribution of OT peptide in the brain of two songbird species: canaries (Serinus canaria) and zebra finches (Taeniopygia guttata). To visualize OT, we performed immunocytochemistry using an antibody previously shown to identify OT in avian species. In both canaries and zebra finches, dense OT-ir perikarya were located in the paraventricular nucleus (PVN), preoptic area (POA), supraoptic nucleus (SON), and medial bed nucleus of the stria terminalis (BNSTm). We also observed morphologically distinct OT-ir cells scattered throughout the mesopallium. OT-ir fibers were observed in the PVN, ventral medial hypothalamus (VMH), periaqueductal gray (PAG), intercollicular nucleus (ICo), and ventral tegmental area (VTA). We also observed punctate OT-ir fibers in the song control nucleus HVC. In both male and female canaries, OT-ir fibers were present in the lateral septum (LS), but innervation was greater in males. We did not observe this sex difference in zebra finches. Much of the OT staining observed is consistent with general distributions within the vertebrate hypothalamus, indicating a possible conserved function. However, some extra-hypothalamic distributions, such as perikarya in the mesopallium, may be specific to songbirds and play a role in song perception and production. The presence of OT-ir fibers in HVC and song control nuclei projecting dopaminergic regions provides anatomical evidence in support of the idea that OT can influence singing behavior-either directly via HVC or indirectly via the PAG, VTA, or POA.

Treatment with androgens plus estrogens cannot reverse sex differences in song and the song control nuclei in adult canaries.

Adult treatments with testosterone (T) do not activate singing behavior nor promote growth of song control nuclei to the same extent in male and female canaries (Serinus canaria). Because T acts in part via aromatization into an estrogen and brain aromatase activity is lower in females than in males in many vertebrates, we hypothesized that this enzymatic difference might explain the sex differences seen even after exposure to the same amount of T. Three groups of castrated males and 3 groups of photoregressed females (i.e., with quiescent ovaries following exposure to short days) received either 2 empty 10 mm silastic implants, one empty implant and one implant filled with T or one implant filled with T plus one with estradiol (E2). Songs were recorded for 3 h each week for 6 weeks before brains were collected and song control nuclei volumes were measured in Nissl-stained sections. Multiple measures of song were still different in males and females following treatment with T. Co-administration of E2 did not improve these measures and even tended to inhibit some measures such as song rate and song duration. The volume of forebrain song control nuclei (HVC, RA, Area X) and the rate of neurogenesis in HVC was increased by the two steroid treatments, but remained significantly smaller in females than in males irrespective of the endocrine condition. These sex differences are thus not caused by a lower aromatization of the steroid; sex differences in canaries are probably organized either by early steroid action or by sex-specific gene regulation directly in the brain.

Role of aromatase in distinct brain nuclei of the social behaviour network in the expression of sexual behaviour in male Japanese quail.

In male Japanese quail, brain aromatase is crucial for the hormonal activation of sexual behaviour, but the sites producing neuro-oestrogens that are critical for these behaviours have not been completely identified. This study examined the function of aromatase expressed in several nuclei of the social behaviour network on a measure of sexual motivation known as the frequency of rhythmic cloacal sphincter movements (RCSM) and on copulatory behaviour. Sexually experienced castrated males chronically treated with testosterone were stereotaxically implanted with the aromatase inhibitor vorozole (VOR), or cholesterol as control, and tested for sexual behaviour. In experiment 1, males were implanted in the medial preoptic nucleus (POM) with VOR, a manipulation known to reduce the expression of copulatory behaviour. This experiment served as positive control, but also showed that VOR implanted in the dorsomedial or lateral portions of the POM similarly inhibits male copulatory behaviour compared to control implants. In experiments 2 to 4, males received stereotaxic implants of VOR in the periaqueductal gray (PAG), the nucleus taeniae of the amygdala (TnA) and the ventromedial nucleus of the hypothalamus (VMN), respectively. Sexual behaviour was affected only in individuals where VOR was implanted in the PAG: these males displayed significantly lower frequencies of cloacal contact movements, the last step of the copulatory sequence. Inhibition of aromatase in the TnA and VMN did not alter copulatory ability. Overall, RCSM frequency remained unaffected by VOR regardless of implantation site. Together, these results suggest that neuro-oestrogens produced in the POM contribute the most to the control of male copulatory behaviour, while aromatase expressed in the PAG might also participate to premotor aspects of male copulatory behaviour.

Rapid changes in brain estrogen concentration during male sexual behavior are site and stimulus specific.

Classically, estrogens regulate male sexual behavior through effects initiated in the nucleus. However, neuroestrogens, i.e., estrogens locally produced in the brain, can act within minutes via membrane-initiated events. In male quail, rapid changes in brain aromatase activity occur after exposure to sexual stimuli. We report here that local extracellular estrogen concentrations measured by in vivo microdialysis increase during sexual interactions in a brain site- and stimulus-specific manner. Indeed, estrogen concentrations rose within 10 min of the initiation of sexual interaction with a female in the medial preoptic nucleus only, while visual access to a female led to an increase in estrogen concentrations only in the bed nucleus of the stria terminalis. These are the fastest fluctuations in local estrogen concentrations ever observed in the vertebrate brain. Their site and stimulus specificity strongly confirm the neuromodulatory function of neuroestrogens on behavior.

Membrane-initiated actions of sex steroids and reproductive behavior: A historical account.

It was assumed for a long time that sex steroids are activating reproductive behaviors by the same mechanisms that produce their morphological and physiological effects in the periphery. However during the last few decades an increasing number of examples were identified where behavioral effects of steroids were just too fast to be mediated via changes in DNA transcription. This progressively forced behavioral neuroendocrinologists to recognize that part of the effects of steroids on behavior are mediated by membrane-initiated events. In this review we present a selection of these early data that changed the conceptual landscape and we provide a summary the different types of membrane-associated receptors (estrogens, androgens and progestagens receptors) that are playing the most important role in the control of reproductive behaviors. Then we finally describe in more detail three separate behavioral systems in which membrane-initiated events have clearly been established to contribute to behavior control.