In most vertebrate species, male sexual behavior is activated by the action of testosterone on the preoptic area and in many cases this behavioral activation requires transformation of the steroid into estradiol and 5α-dihydrotestosterone. These metabolites, either alone or in synergy, act at the cellular level to produce changes in neuronal function that mediate behavioral changes. Variation in plasma testosterone concentrations does not simply correlate in a positive manner with the degree of behavioral activation. For example, in many species, testosterone cannot activate male-typical sexual behaviors in females. This sex difference in the behavioral consequences of testosterone results from organizational effects of sex steroids that irreversibly differentiate the brain during ontogeny so that male brains will respond in adulthood to testosterone while female brains will not. Behavioral effects of testosterone are largely mediated by changes in the transcription of specific proteins, including enzymes that synthesize or catabolize neurotransmitters and their receptors. These changes in protein expression in turn produce changes in neurotransmission in a defined neuronal circuitry that supports behavior expression. There are more recently described effects of steroids that do not involve protein transcription but rather involve actions at the neuronal membrane or direct changes in intracellular signaling cascades that also seem to contribute to the control of male sexual behavior. These principles seem to apply to a wide range of non-mammalian vertebrate species though a number of specialized reproductive strategies that could be associated with exceptions to these general rules have also been described.

Mate choice is a critical decision-making process, having lasting impacts on an individual’s time, energy, and reproductive success. Across songbirds, females are generally assumed to prefer higher song rates, greater complexity, and higher quality performances; however, there is growing evidence implicating syllable level details in songbird communication. Here, we build on our previous psychoacoustic results to ask whether female zebra finches use the kinds of syllable level details that they are capable of hearing. Female zebra finches produce calls during male songs as a component of courtship. These calls can be leveraged to explore how females assess and interact with male songs. To test whether syllable level details are behaviorally relevant in a courtship context, we quantified female call responses to manipulated songs in four experiments. First, we validated that our playback procedure elicited robust calling responses from females (Exp 1). Next, we found that females decreased calling to songs where syllables were spectro-temporally reversed (REVERSAL), but did not respond differently if the syllable order was manipulated (SHUFFLED). Females also modulated their calling when experimental songs were composed of natural rendition-to-rendition variation in song syllables (RENDITION) relative to songs consisting of a single repeated rendition (FIXED) (Exp 2). Furthermore, we found that females decreased calling responses even when only a portion of syllables were spectro-temporally reversed (Exp 4). Across these experiments, we also report the striking extent to which females habituated to a male’s song (Exps 3 and 4). To maximize female responses, we tried adjusting the paradigm in Exps 3 and 4 to increase female calling. However, our adjustments had minimal effects, consistent with the notion that females rapidly decreased calling in response to a given males’ stimuli. Altogether, our results contribute to growing evidence that syllable level details in birdsong are behaviorally relevant, and, perhaps more importantly, demonstrate that birds’ enhanced ability to discriminate acoustic fine structure as shown in psychophysical tests plays a role in communication.

Social relationships are essential for a wide range of species, including humans. The mechanisms of pair formation and maintenance appear to be distinct, although relatively little is known about pair maintenance compared to bond formation. A social conditioned place preference (SCPP) test, a paradigm not often used, could be used to reveal differences in the mechanisms of bond formation and maintenance. Here we used the SCPP test across two experiments to investigate how the behavioral and neuroendocrine mechanisms of social affiliation change over the course of pair bonding in the monogamous zebra finch. In our procedure, we had three 30 min phases: preconditioning occurring on Day 1, followed by conditioning and postconditioning, both on Day 2. In our first experiment, we simply asked whether courting and paired dyads would form SCPPs in this assay. We found evidence that courting, but not paired birds, formed SCPPs. This was particularly evident for courting females. Courting and paired dyads also engaged in different behaviors during the conditioning phase. Despite these effects of social context on behavior, there was no clear relationship between these behavioral differences and the formation of SCPPs for courting males or females. We next tested whether pharmacological treatment with D1 and D2 receptor agonists would alter the effects of social context on the formation of SCPPs. Specifically, we hypothesized that treatment with a D1-agonist (SKF 38393) would block the formation of SCPPs in courting dyads and that activation of D2-like receptors (quinpirole) would induce SCPPs in already established pairs. In contrast with Experiment 1, we did not see any groups forming SCPPs, but paired males, regardless of pharmacological treatment, appeared to form aversions to the conditioning side. Furthermore, there was significantly less courtship and affiliative behavior in the second experiment. Combined, this leads us to suspect that coupling the SCPP with an s.c. injection made the entire procedure too aversive to reveal any SCPP. Despite this, there were subtle effects of pharmacological manipulation on social behavior during the conditioning phase. All together, the results from our two experiments (1) suggest that there are differences in the mechanisms by which courting and paired dyads relate to their partner in novel environments, (2) highlight the differences in social interactions between courting and paired birds, and (3) leave open the possibility that dopamine signaling differentially shapes social behavior and the formation of SCPPs in courting and paired birds.

Pronounced sexual dimorphism is generally assumed to evolve through sexual selection for elaborate male traits. However, there is increasing evidence that sexual dimorphism in traits such as birdsong may also evolve through loss of elaboration in females, but the evolutionary drivers underlying this process are obscure. Here we analyse ecological and natural history traits for over 1300 songbird species and show that increased female song incidence and elaboration are most directly associated with year-round territoriality, biparental care, and large body size. Phylogenetic path analysis indicates that mating system and breeding latitude primarily have indirect effects on female song evolution. Stable, tropical life histories and mating systems with biparental care promote female song, whereas evolutionary transitions to migration, reduced territoriality, and loss of male care led to losses or reductions of female song incidence. Our analyses provide a comprehensive framework for studying the drivers of sex differences and similarities in birdsong and reveal novel interactions among natural history and sexual selection pressures that have been hypothesized to independently shape elaborate traits.

This short essay is based on the presentation I gave at the meeting of the Society of Behavioral Neuroendocrinology held in Tours, France in June 2023 when I received the Lehrman award for career achievement. I conducted my PhD at the Institute of Animal Behavior that was founded by Lehrman at Rutgers University. Although I never met Lehrman I was mentored by several scientists who had worked with him. I therefore decided to organize my essay about the Lehrman award around the enduring legacy of Lehrman's work as it influenced my career. Four aspects of the scientific legacy of Daniel Lehrman are distinguished and I discuss how these insights affected my own research program. A concise summary of the research I conducted in collaboration with PhD students and postdocs that relates to these topics is then presented. This research that involves several avian species includes studies of how behavior can influence the endocrine physiology and behavior of receivers of a particular signal. The cloning of the gonadotropin-releasing hormone (GnRH) gene in songbirds and analysis of the environmental factors influencing its expression demonstrated how the GnRH neuronal system is a key link between the perception of behavior and changes in endocrine physiology. Finally, there is a review of studies of the experimental analysis of the brain sites where testosterone acts to control the motivation to sing and the quality of song in songbirds that follow directly in the Lehrman line of work on how steroids regulate avian reproductive behaviors.

Highly sensitive in situ hybridization procedures (RNAScope) were used to quantify the expression of three dopamine receptors (Drd1, Drd2, and Drd3) in two song control nuclei (HVC and the Area X of the basal ganglia) that are known to receive dopaminergic inputs and in the periaqueductal gray (PAG) of male and female canaries. Both sexes were treated with testosterone to ensure they would sing actively. We also determined the excitatory versus inhibitory phenotype of the cells expressing these receptors as well as their activation following a period of song production. The three receptor types were identified in each brain area, with the exception of Drd3 in Area X. The density of cells expressing each receptor varied as a function of receptor type and brain area. Surprisingly few sex differences were detected; they do not seem to explain the sex differences in testosterone-induced song. Overall, the density of Drd-positive cells was much lower in PAG than in the two song control nuclei. In HVC, the majority of cells expressing the three receptor subtypes were VGlut2-positive, whereas colocalization with Vglut2 occurred in few cells in Area X and in an intermediate proportion of cells in PAG. The number of inhibitory cells expressing dopamine receptors was limited. Most dopaminoceptive cells in Area X did not express either excitatory or inhibitory markers. Finally, cellular activation during singing behavior, as measured by the expression of Egr1, was observed in cells expressing each of the three dopamine receptor subtypes, except Drd3 in the PAG.

Birdsong is an excellent system for studying complex vocal signaling in both males and females. Historically, most research in captivity has focused only on male song. This has left a gap in our understanding of the environmental, neuroendocrine, and mechanistic control of female song. Here, we report the overall acoustic features, repertoire, and stereotypy of both male and female Red-Cheeked Cordon Bleus (Uraeginthus bengalus) (RCCBs) songs in the lab. We found few sex differences in the acoustic structure, song repertoire, and song stereotypy of RCCBs. Both sexes had similar song entropy, peak frequency, and duration. Additionally, individuals of both sexes sang only a single song type each and had similar levels of song and syllable stereotypy. However, we did find that female RCCBs had higher song bandwidth but lower syllable repertoires. Finally, and most strikingly, we found highly individualistic songs in RCCBs. Each individual produced a stereotyped and unique song with no birds sharing song types and very few syllable types being shared between birds of either sex. We propose that RCCBs represent a promising species for future investigations of the acoustic sex differences in song in a lab environment, and also for understanding the evolutionary driving forces behind individualistic songs.

Abstract Pronounced sexual dimorphism is thought to evolve through sexual selection for elaborate male traits. Increasing evidence suggests that sexual dimorphism in traits such as birdsong may also evolve through loss of elaboration in females, but the evolutionary drivers underlying this are obscure. Here we analyse ecological and natural history traits of over 1300 songbird species and show that increased female song incidence and elaboration are most directly associated with year-round territoriality, biparental care, and large body size. Moreover, phylogenetic path analysis indicates that mating system and breeding latitude have only indirect effects. Stable, tropical life histories and mating systems with biparental care promote female song, whereas evolutionary transitions to migration, reduced territoriality, and loss of male care led to losses of female song. Our results provide the first comprehensive framework to understand the drivers of sex differences in birdsong and reveal novel interactions among natural history, social, and sexual selection pressures in the evolution of avian song in both sexes.

The auditory sensitivity of a small songbird, the red-cheeked cordon bleu, was measured using the standard methods of animal psychophysics. Hearing in cordon bleus is similar to other small passerines with best hearing in the frequency region from 2 to 4 kHz and sensitivity declining at the rate of about 10 dB/octave below 2 kHz and about 35 dB/octave as frequency increases from 4 to 9 kHz. While critical ratios are similar to other songbirds, the long-term average power spectrum of cordon bleu song falls above the frequency of best hearing in this species.

During breeding when testosterone concentrations are high, male songbirds that are open-ended vocal learners like canaries (Serinus canaria) tend to produce a stable, stereotyped song that facilitates mate attraction or territory defense. Outside breeding contexts, song becomes more variable. The neuroendocrine mechanisms controlling this vocal variability across seasons are not entirely clear. We tested whether androgen signaling within the lateral magnocellular nucleus of the anterior nidopallium (LMAN), a cortical-like brain region of the vocal control system known as a vocal variability generator, plays a role in seasonal vocal variability. We first characterized song in birds housed alone on a short day (SD) photoperiod, which simulates non-breeding conditions. Then, cannulae filled with the androgen receptor (AR) blocker flutamide or left empty as control were implanted bilaterally in LMAN. Birds were then transferred to long days (LD) to simulate the breeding season and song was analyzed again. Blocking AR in LMAN increased acoustic variability of song and the acoustic variability of syllables. However, blocking AR in LMAN did not impact the variability of syllable usage nor their sequencing in LD birds, song features that are controlled by androgen signaling in a somatosensory brain region of the vocal control system called HVC. These findings highlight the multifactorial, non-redundant actions of steroid hormones in controlling complex social behaviors such as birdsong. They also support the hypothesis that LMAN is a key brain area for the effects of testosterone on song plasticity both seasonally in adults and during the song crystallization process at sexual maturity.

Recent psychophysical experiments have shown that zebra finches (Taeniopygia guttata-a songbird) are surprisingly insensitive to syllable sequence changes in their species-specific motifs while budgerigars (Melopsittacus undulatus-a psittacine) do much better when tested on exactly the same sounds. This is unexpected since zebra finch males learn the order of syllables in their songs when young and sing the same song throughout adulthood. Here we probe the limits of this species difference by testing birds on an order change involving just two syllables, hereafter called bi-syllable phrases. Results show budgerigars still perform better than zebra finches on an order change involving just two syllables. An analysis of response latencies shows that both species respond to an order change in a bi-syllable motif at the onset of the first syllable rather than listening to the entire sequence before responding. Additional tests with one syllable omitted or doubled, or with white noise bursts substituted for syllables, indicate that the first syllable in the sequence has a dominant effect on subsequent discrimination of changes in a bi-syllable pattern. These results are surprising in that zebra finch males sing their full motif syllable sequence with a high degree of stereotypy throughout life, suggesting that this consistency in production may not rely on perceptual mechanisms for processing syllable order in adulthood. Budgerigars, on the other hand, are quite sensitive to bi-syllable order changes, an ability that may be related to useful information being encoded in the sequence of syllables in their natural song. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

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.

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.

Birdsong is a relatively well-studied behavior, both due to its importance as a model for vocal production learning and as an intriguing complex social behavior. Until the last few decades, work on birdsong focused almost exclusively on males. However, it is now widely accepted that female song not only exists, but is fairly common throughout the oscine passerines. Despite this, and the large number of researchers who have begun exploring female song in the field, researchers in the lab have been slow to adopt model species with female song. Studying female song in the lab is critical for our understanding of sex-specific factors in the physiology controlling this fascinating behavior. Additionally, as a model for vocal production learning in humans, understanding the mechanistic and neuroendocrine control of female song is clearly important. In this study, we examined the red-cheeked cordon bleu (RCCB), an Estrildid finch species with extensive female song. Specifically, we found that there were no significant sex differences in circulating levels of testosterone and progesterone, nor in song production rate. There were no significant differences in cell densities in the three nuclei of the song control system we examined. Additionally, the volume of the robust nucleus of the arcopallium was not significantly different and we report the smallest sex difference in HVC yet published in a songbird. Finally, we demonstrated similar levels of motor driven immediate early gene expression in both males and females after song production.

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. 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. 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). 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.

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.

A species' success during the invasion of new areas hinges on an interplay between the demographic processes common to invasions and the specific ecological context of the novel environment. Evolutionary genetic studies of invasive species can investigate how genetic bottlenecks and ecological conditions shape genetic variation in invasions, and our study pairs two invasive populations that are hypothesized to be from the same source population to compare how each population evolved during and after introduction. Invasive European starlings (Sturnus vulgaris) established populations in both Australia and North America in the 19th century. Here, we compare whole-genome sequences among native and independently introduced European starling populations to determine how demographic processes interact with rapid evolution to generate similar genetic patterns in these recent and replicated invasions. Demographic models indicate that both invasive populations experienced genetic bottlenecks as expected based on invasion history, and we find that specific genomic regions have differentiated even on this short evolutionary timescale. Despite genetic bottlenecks, we suggest that genetic drift alone cannot explain differentiation in at least two of these regions. The demographic boom intrinsic to many invasions as well as potential inversions may have led to high population-specific differentiation, although the patterns of genetic variation are also consistent with the hypothesis that this infamous and highly mobile invader adapted to novel selection (e.g., extrinsic factors). We use targeted sampling of replicated invasions to identify and evaluate support for multiple, interacting evolutionary mechanisms that lead to differentiation during the invasion process.

Social relationships, affiliative social attachments, are important for many species. The best studied types of relationships are monogamous pair bonds. However, it remains unclear how generalizable models of pair bonding are across types of social attachments. Zebra finches are a fascinating system to explore the neurobiology of social relationships because they form various adult bonds with both same- and opposite-sex partners. To test whether different bonds are supported by a single brain network, we quantified individuals' neuroendocrine state after either 24 h or 2 weeks of co-housing with a novel same- or opposite-sex partner. We defined neuroendocrine state by the expression of 22 genes related to 4 major signaling pathways (dopamine, steroid, nonapeptide, and opioid) in six brain regions associated with affiliation or communication [nucleus accumbens (NAc), nucleus taeniae of the amygdala (TnA), medial preoptic area (POM), and periaqueductal gray (PAG), ventral tegmental area, and auditory cortex]. Overall, we found dissociable effects of social contexts (same- or opposite-sex partnerships) and duration of co-housing. Social bonding impacted the neuroendocrine state of four regions in males (NAc, TnA, POM, and PAG) and three regions in females (NAc, TnA, and POM). Monogamous pair bonding specifically appeared to impact male NAc. However, the patterns of gene expression in zebra finches were different than has previously been reported in mammals. Together, our results support the view that there are numerous mechanisms regulating social relationships and highlight the need to further our understanding of how social interactions shape social bonds.

Glia maturation factor beta (GMFB) was identified as a growth and differentiation factor acting on neurons as well as glia. We investigated the expression of GMFB during 56 days after cryogenic brain injury, using immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and enzyme immunoassay. Immunohistochemical analysis demonstrated that the GFAP-positive astrocytes around the lesion expressed GMFB protein, peaking 14 days after injury. Weak astrocytic expression of GMFB-immunoreactivity was seen in sham-operated animal brains. Cryogenic injury (CI) induced GMFB mRNA in the lesioned side after 7 days with a maximum at 14 days. Western blotting revealed the induction of GMFB protein starting 1 day after injury, and continuing until 14 days after injury. In the enzyme immunoassay, GMFB protein concentration peaked 14 days after injury in extracts from the injured side of the brain, whereas in serum it peaked 1 day after injury. These data indicate that the expression of GMFB increased in the astrocytes around the lesioned area after cortical cryogenic brain injury. These findings may provide new insight into GMFB function in pathological conditions following brain injury.