Allele-specific cis-regulatory methylation of the gene for vasoactive intestinal peptide in white-throated sparrows.

White-throated sparrows (Zonotrichia albicollis) offer a unique opportunity to connect genotype with behavioral phenotype. In this species, a rearrangement of the second chromosome is linked with territorial aggression; birds with a copy of this "supergene" rearrangement are more aggressive than those without it. The supergene has captured the gene VIP, which encodes vasoactive intestinal peptide, a neuromodulator that drives aggression in other songbirds. In white-throated sparrows, VIP expression is higher in the anterior hypothalamus of birds with the supergene than those without it, and expression of VIP in this region predicts the level of territorial aggression regardless of genotype. Here, we aimed to identify epigenetic mechanisms that could contribute to differential expression of VIP both in breeding adults, which exhibit morph differences in territorial aggression, and in nestlings, before territorial behavior develops. We extracted and bisulfite-converted DNA from samples of the hypothalamus in wild-caught adults and nestlings and used high-throughput sequencing to measure DNA methylation of a region upstream of the VIP start site. We found that the allele inside the supergene was less methylated than the alternative allele in both adults and nestlings. The differential methylation was attributed primarily to CpG sites that were shared between the alleles, not to polymorphic sites, which suggests that epigenetic regulation is occurring independently of the genetic differentiation within the supergene. This work represents an initial step toward understanding how epigenetic differentiation inside chromosomal inversions leads to the development of alternative behavioral phenotypes.

Inside the supergene of the bird with four sexes.

The white-throated sparrow (Zonotrichia albicollis) offers unique opportunities to understand the adaptive value of supergenes, particularly their role in alternative phenotypes. In this species, alternative plumage morphs segregate with a nonrecombining segment of chromosome 2, which has been called a 'supergene'. The species mates disassortatively with respect to the supergene; that is, each breeding pair consists of one individual with it and one without it. This species has therefore been called the "bird with four sexes". The supergene segregates with a behavioral phenotype; birds with it are more aggressive and less parental than birds without it. Here, we review our efforts to identify the genes inside the supergene that are responsible for the behavioral polymorphism. The gene ESR1, which encodes estrogen receptor α, differs between the morphs and predicts both territorial and parental behavior. Variation in the regulatory regions of ESR1 causes an imbalance in expression of the two alleles, and the degree to which this imbalance favors the supergene allele predicts territorial singing. In heterozygotes, knockdown of ESR1 causes a phenotypic switch, from more aggressive to less aggressive. We recently showed that another gene important for social behavior, vasoactive intestinal peptide (VIP), is differentially expressed between the morphs and predicts territorial singing. We hypothesize that ESR1 and VIP contribute to behavior in a coordinated way and could represent co-adapted alleles. Because the supergene contains more than 1000 individual genes, this species provides rich possibilities for discovering alleles that work together to mediate life-history trade-offs and maximize the fitness of alternative complex phenotypes.

A supergene-linked estrogen receptor drives alternative phenotypes in a polymorphic songbird.

Behavioral evolution relies on genetic changes, yet few behaviors can be traced to specific genetic sequences in vertebrates. Here we provide experimental evidence showing that differentiation of a single gene has contributed to the evolution of divergent behavioral phenotypes in the white-throated sparrow, a common backyard songbird. In this species, a series of chromosomal inversions has formed a supergene that segregates with an aggressive phenotype. The supergene has captured ESR1, the gene that encodes estrogen receptor α (ERα); as a result, this gene is accumulating changes that now distinguish the supergene allele from the standard allele. Our results show that in birds of the more aggressive phenotype, ERα knockdown caused a phenotypic change to that of the less aggressive phenotype. We next showed that in a free-living population, aggression is predicted by allelic imbalance favoring the supergene allele. Finally, we identified cis-regulatory features, both genetic and epigenetic, that explain the allelic imbalance. This work provides a rare illustration of how genotypic divergence has led to behavioral phenotypic divergence in a vertebrate.

Vasoactive intestinal peptide as a mediator of the effects of a supergene on social behaviour.

Supergenes, or linked groups of alleles that are inherited together, present excellent opportunities to understand gene-behaviour relationships. In white-throated sparrows (Zonotrichia albicollis), a supergene on the second chromosome associates with a more aggressive and less parental phenotype. This supergene includes the gene for vasoactive intestinal peptide (VIP), a neuropeptide known to play a causal role in both aggression and parental behaviour. Here, using a free-living population, we compared the levels of VIP mRNA between birds with and without the supergene. We focused on the anterior hypothalamus and infundibular region, two brain regions containing VIP neurons known to play a causal role in aggression and parental behaviour, respectively. First, we show that the supergene enhances VIP expression in the anterior hypothalamus and that expression positively predicts vocal aggression independently of genotype in both sexes. Next, we show that the supergene reduces VIP expression in the infundibular region, which suggests reduced secretion of prolactin, a pro-parental hormone. Thus, the patterns of VIP expression in these two regions are consistent with the enhanced aggression and reduced parental behaviour of birds with the supergene allele. Our results illustrate mechanisms by which elements of genomic architecture, such as supergenes, can contribute to the evolution of alternative behavioural phenotypes.

A chromosomal inversion predicts the expression of sex steroid-related genes in a species with alternative behavioral phenotypes.

In white-throated sparrows, a chromosomal rearrangement has led to alternative phenotypes that differ in sex steroid-dependent behaviors. The rearrangement has captured the genes estrogen receptor alpha and 5-alpha reductase, making these genes strong candidates for mediating the behavioral phenotypes. We report here that of the two genes, expression of estrogen receptor alpha mRNA differs between the morphs and predicts behavior to a much greater extent than does expression of 5-alpha reductase mRNA. Differentiation of estrogen receptor alpha, therefore, is likely more important for the behavioral phenotypes. We also found that in some brain regions, the degree to which testosterone treatment affects the expression of steroid-related genes depends strongly on morph. A large morph difference in estrogen receptor alpha mRNA expression in the amygdala appears to be independent of plasma testosterone; this difference persists during the non-breeding season and is detectable in nestlings at post-hatch day seven. The latter result suggests a substrate for organizational effects of hormones during development.

Rapid effects of 17ß-estradiol on aggressive behavior in songbirds: Environmental and genetic influences.

Contribution to Special Issue on Fast effects of steroids. 17ß-estradiol (E2) has numerous rapid effects on the brain and behavior. This review focuses on the rapid effects of E2 on aggression, an important social behavior, in songbirds. First, we highlight the contributions of studies on song sparrows, which reveal that seasonal changes in the environment profoundly influence the capacity of E2 to rapidly alter aggressive behavior. E2 administration to male song sparrows increases aggression within 20 min in the non-breeding season, but not in the breeding season. Furthermore, E2 rapidly modulates several phosphoproteins in the song sparrow brain. In particular, E2 rapidly affects pCREB in the medial preoptic nucleus, in the non-breeding season only. Second, we describe studies of the white-throated sparrow, which reveal how a genetic polymorphism may influence the rapid effects of E2 on aggression. In this species, a chromosomal rearrangement that includes ESR1, which encodes estrogen receptor α (ERα), affects ERα expression in the brain and the ability of E2 to rapidly promote aggression. Third, we summarize studies showing that aggressive interactions rapidly affect levels of E2 and other steroids, both in the blood and in specific brain regions, and the emerging potential for steroid profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS). Such studies of songbirds demonstrate the value of an ethologically informed approach, in order to reveal how steroids act rapidly on the brain to alter naturally-occurring behavior.

Rapid effects of estradiol on aggression depend on genotype in a species with an estrogen receptor polymorphism.

The white-throated sparrow (Zonotrichia albicollis) represents a powerful model in behavioral neuroendocrinology because it occurs in two plumage morphs that differ with respect to steroid-dependent social behaviors. Birds of the white-striped (WS) morph engage in more territorial aggression than do birds of the tan-striped (TS) morph, and the TS birds engage in more parenting behavior. This behavioral polymorphism is caused by a chromosomal inversion that has captured many genes, including estrogen receptor alpha (ERα). In this study, we tested the hypothesis that morph differences in aggression might be explained by differential sensitivity to estradiol (E2). We administered E2 non-invasively to non-breeding white-throated sparrows and quantified aggression toward a conspecific 10 min later. E2 administration rapidly increased aggression in WS birds but not TS birds, consistent with our hypothesis that differential sensitivity to E2 may at least partially explain morph differences in aggression. To query the site of E2 action in the brain, we administered E2 and quantified Egr-1 expression in brain regions in which expression of ERα is known to differ between the morphs. E2 treatment decreased Egr-1 immunoreactivity in nucleus taeniae of the amygdala, but this effect did not depend on morph. Overall, our results support a role for differential effects of E2 on aggression in the two morphs, but more research will be needed to determine the neuroanatomical site of action.

Polymorphisms in sex steroid receptors: From gene sequence to behavior.

Sex steroid receptors have received much interest as potential mediators of human behaviors and mental disorders. Candidate gene association studies have identified about 50 genetic variants of androgen and estrogen receptors that correlate with human behavioral phenotypes. Because most of these polymorphisms lie outside coding regions, discerning their effect on receptor function is not straightforward. Thus, although discoveries of associations improve our ability to predict risk, they have not greatly advanced our understanding of underlying mechanisms. This article is intended to serve as a starting point for psychologists and other behavioral biologists to consider potential mechanisms. Here, I review associations between polymorphisms in sex steroid receptors and human behavioral phenotypes. I then consider ways in which genetic variation can affect processes such as mRNA transcription, splicing, and stability. Finally, I suggest ways that hypotheses about mechanism can be tested, for example using in vitro assays and/or animal models.

Estrogen Receptor Alpha as a Mediator of Life-History Trade-offs.

Trade-offs between competitive and parental strategies often are mediated by sex steroids. The mechanisms underlying steroid signaling and metabolism may therefore serve as targets of disruptive selection that leads to alternative behavioral phenotypes. White-throated sparrows exhibit two color morphs that differ in both competitive and parental behavior; white-striped (WS) birds engage in more territorial singing, whereas tan-striped (TS) birds provision nestlings more often. Although WS birds have higher levels of plasma testosterone (T) and estradiol than do TS birds, experimental equalization of these hormones does not abolish morph differences in singing. Neural sensitivity to sex steroids may differ between the morphs because the gene for estrogen receptor alpha (ERα) has been captured by a chromosomal rearrangement found only in the WS birds. We recently showed that expression of this gene differs between the morphs and may drive the behavioral polymorphism. First, the ERα promoter region contains fixed polymorphisms that affect transcription efficiency in vitro. Second, in a free-living population, local expression of ERα depends strongly on morph and predicts both territorial singing and parental provisioning. Differential ERα expression is particularly striking in the medial amygdala; WS birds have three times more ERα mRNA than do TS birds. This difference persists during the non-breeding season and is unaffected by exogenous T treatment. Finally, preliminary data generated by RNA-seq confirm that ERα expression in MeA is both differentially expressed and correlated with territorial singing. Together, these results suggest that ERα may be a target of disruptive selection that leads to alternative behavioral strategies. Our future directions include a more detailed analysis of the ERα promoter regions to determine the molecular basis of differential expression as well as gene network analyses to identify genes connected to ERα.

Estrogen receptor α polymorphism in a species with alternative behavioral phenotypes.

The evolution of behavior relies on changes at the level of the genome; yet the ability to attribute a behavioral change to a specific, naturally occurring genetic change is rare in vertebrates. In the white-throated sparrow (Zonotrichia albicollis), a chromosomal polymorphism (ZAL2/2(m)) is known to segregate with a behavioral phenotype. Individuals with the ZAL2(m) haplotype engage in more territorial aggression and less parental behavior than individuals without it. These behaviors are thought to be mediated by sensitivity to sex steroids, and the chromosomal rearrangement underlying the polymorphism has captured a prime candidate gene: estrogen receptor 1 (ESR1), which encodes estrogen receptor α (ERα). We therefore hypothesized that the behavioral effects of the ZAL2(m) rearrangement are mediated by polymorphism in ESR1. We report here that (i) the ESR1 promoter region contains fixed polymorphisms distinguishing the ZAL2(m) and ZAL2 alleles; (ii); those polymorphisms regulate transcription efficiency in vitro and therefore potentially do the same in vivo (iii); the local expression of ERα in the brain depends strongly on genotype in a free-living population; and (iv) ERα expression in the medial amygdala and medial preoptic area may fully mediate the effects of genotype on territorial aggression and parenting, respectively. Thus, our study provides a rare glimpse of how a chromosomal polymorphism has affected the brain and social behavior in a vertebrate. Our results suggest that in this species, differentiation of ESR1 has played a causal role in the evolution of phenotypes with alternative life-history strategies.

Estradiol-dependent modulation of serotonergic markers in auditory areas of a seasonally breeding songbird.

Because no organism lives in an unchanging environment, sensory processes must remain plastic so that in any context, they emphasize the most relevant signals. As the behavioral relevance of sociosexual signals changes along with reproductive state, the perception of those signals is altered by reproductive hormones such as estradiol (E2). We showed previously that in white-throated sparrows, immediate early gene responses in the auditory pathway of females are selective for conspecific male song only when plasma E2 is elevated to breeding-typical levels. In this study, we looked for evidence that E2-dependent modulation of auditory responses is mediated by serotonergic systems. In female nonbreeding white-throated sparrows treated with E2, the density of fibers immunoreactive for serotonin transporter innervating the auditory midbrain and rostral auditory forebrain increased compared with controls. E2 treatment also increased the concentration of the serotonin metabolite 5-HIAA in the caudomedial mesopallium of the auditory forebrain. In a second experiment, females exposed to 30 min of conspecific male song had higher levels of 5-HIAA in the caudomedial nidopallium of the auditory forebrain than birds not exposed to song. Overall, we show that in this seasonal breeder, (a) serotonergic fibers innervate auditory areas; (b) the density of those fibers is higher in females with breeding-typical levels of E2 than in nonbreeding, untreated females; and (c) serotonin is released in the auditory forebrain within minutes in response to conspecific vocalizations. Our results are consistent with the hypothesis that E2 acts via serotonin systems to alter auditory processing.

Estradiol-dependent catecholaminergic innervation of auditory areas in a seasonally breeding songbird.

A growing body of evidence suggests that gonadal steroids such as estradiol (E2) alter neural responses not only in brain regions associated with reproductive behavior but also in sensory areas. Because catecholamine systems are involved in sensory processing and selective attention, and because they are sensitive to E2 in many species, they may mediate the neural effects of E2 in sensory areas. Here, we tested the effects of E2 on catecholaminergic innervation, synthesis and activity in the auditory system of white-throated sparrows, a seasonally breeding songbird in which E2 promotes selective auditory responses to song. Non-breeding females with regressed ovaries were held on a winter-like photoperiod and implanted with silastic capsules containing either no hormone or E2. In one hemisphere of the brain, we used immunohistochemistry to quantify fibers immunoreactive for tyrosine hydroxylase or dopamine beta-hydroxylase in the auditory forebrain, thalamus and midbrain. E2 treatment increased catecholaminergic innervation in the same areas of the auditory system in which E2 promotes selectivity for song. In the contralateral hemisphere we quantified dopamine, norepinephrine and their metabolites in tissue punches using HPLC. Norepinephrine increased in the auditory forebrain, but not the midbrain, after E2 treatment. We found that evidence of interhemispheric differences, both in immunoreactivity and catecholamine content that did not depend on E2 treatment. Overall, our results show that increases in plasma E2 typical of the breeding season enhanced catecholaminergic innervation and synthesis in some parts of the auditory system, raising the possibility that catecholamines play a role in E2-dependent auditory plasticity in songbirds.

Estradiol-dependent modulation of auditory processing and selectivity in songbirds.

The steroid hormone estradiol plays an important role in reproductive development and behavior and modulates a wide array of physiological and cognitive processes. Recently, reports from several research groups have converged to show that estradiol also powerfully modulates sensory processing, specifically, the physiology of central auditory circuits in songbirds. These investigators have discovered that (1) behaviorally-relevant auditory experience rapidly increases estradiol levels in the auditory forebrain; (2) estradiol instantaneously enhances the responsiveness and coding efficiency of auditory neurons; (3) these changes are mediated by a non-genomic effect of brain-generated estradiol on the strength of inhibitory neurotransmission; and (4) estradiol regulates biochemical cascades that induce the expression of genes involved in synaptic plasticity. Together, these findings have established estradiol as a central regulator of auditory function and intensified the need to consider brain-based mechanisms, in addition to peripheral organ dysfunction, in hearing pathologies associated with estrogen deficiency.

Topography of estradiol-modulated genomic responses in the songbird auditory forebrain.

Sex steroids facilitate dramatic changes in behavioral responses to sociosexual signals and are increasingly implicated in the sensory processing of those signals. Our previous work demonstrated that in female white-throated sparrows, which are seasonal breeders, genomic responses in the auditory forebrain are selective for conspecific song over frequency-matched tones only when plasma estradiol (E2) reaches breeding levels. Here, we sought to map this E2-dependent selectivity in the best-studied area of the auditory forebrain, the caudomedial nidopallium (NCM). Nonbreeding females with low endogenous levels of E2 were treated with E2 or a placebo and exposed to conspecific song, tones, or no sound playback. Immunoreactive protein product of the immediate early gene zenk (egr-1) was then quantified within seven distinct subregions, or domains, of NCM. We report three main findings: (1) regardless of hormone treatment, the zenk response is significantly higher in dorsal than in ventral NCM, and higher in medial than in lateral NCM; (2) E2-dependent selectivity of the response is limited to the rostral and medial domains of NCM; in the more caudal domains, song induces more zenk expression than tones regardless of hormone treatment; (3) even when no sound stimuli were presented, E2 treatment significantly increased zenk expression in the rostral, but not the caudal, domains of NCM. Together, the latter two findings suggest that E2-dependent plasticity in NCM is concentrated in rostral NCM, which is hodologically and neurochemically distinct from caudal NCM. Activity in rostral NCM may therefore be seasonally regulated in this species.

Behavioral phenotypes persist after gonadal steroid manipulation in white-throated sparrows.

White-throated sparrows (Zonotrichia albicollis) exhibit a behavioral polymorphism that segregates with a plumage marker. Individuals with a white stripe (WS) on the crown engage in an aggressive strategy that involves more singing, whereas individuals with a tan stripe (TS) sing less and engage in more parental care. Previous work has shown that plasma levels of gonadal steroids differ between the morphs in both sexes, suggesting a hormonal mechanism for the polymorphic behavior in this species. Here, we eliminated morph differences in plasma levels of testosterone (T) in males and estradiol (E2) in females in order to test whether morph differences in behavior would be similarly eliminated. Males and females in non-breeding condition were treated with T or E2, respectively, so that plasma levels in the treated groups were high and equal between the WS and TS morphs. We found that despite hormone treatment, WS and TS birds differed with respect to singing behavior. WS males sang more in response to song playback than did TS males, and WS females exhibited more spontaneous song than TS females. We also found that WS males gave more chip calls, which are often used in contexts of territorial aggression. Overall, these results suggest that WS birds engage in more territorial vocalization, particularly song, than do TS birds, even when T or E2 levels are experimentally equalized. This behavioral difference may therefore be driven by other factors, such as steroid metabolism, receptor expression or function, or steroid-independent neurotransmitter systems.

Estradiol modulates neural responses to song in a seasonal songbird.

Social behaviors such as courtship, parenting, and aggression depend primarily on two factors: a social signal to trigger the behavior, and a hormonal milieu that facilitates or permits it. Gonadal steroids may alter the valence or perceived context of the signal so that the same pheromone, vocalization, or visual display may elicit very different responses depending on the receiver's plasma hormone level. The neural processes underlying this phenomenon, however, are not well understood. Here, we describe how hormones modulate neural responses to social signals in female white-throated sparrows listening to recordings of male song. While manipulating levels of the ovarian steroid estradiol, we mapped and quantified sound-induced expression of the immediate early gene egr-1 in nine brain regions that constitute a social behavior network in vertebrates. In most regions of interest, hearing male song induced more expression than hearing tones or silence, and this selectivity for song was seen only in birds with estradiol levels typical of the breeding season. In females with regressed ovaries and no exogenous estradiol, neural responses were selective for song over tones only in the lateral portion of the ventromedial hypothalamus, not in the rest of the network. Because the effects of hormone treatment on neural responses are not identical in each region, the overall pattern of activation across the network changes with estradiol level and thus with season and breeding context. Our results demonstrate a possible mechanism by which gonadal steroids may alter the processing of social signals and affect social decision-making.

Activity of the hypothalamic-pituitary-gonadal axis differs between behavioral phenotypes in female white-throated sparrows (Zonotrichia albicollis).

The white-throated sparrow (Zonotrichia albicollis) lends itself particularly well to investigations of neuroendocrine mechanisms of social behavior because of a behavioral polymorphism that correlates with a plumage phenotype. Roughly half of the individuals of this species exhibit a white stripe (WS) on the crown and engage in a more aggressive strategy, whereas the other half exhibit a tan stripe (TS) and assume a more parental strategy. These behavioral differences are mirrored by hormonal and neuroendocrine differences; for example, males of the WS morph have higher plasma testosterone than do TS males, and females of the TS morph have higher plasma luteinizing hormone than females of the WS morph. These differences suggest that the regulation of the hypothalamic-pituitary-gonadal (HPG) axis may differ according to morph. In this study, we compared HPG axis activity at each level by measuring (1) the number, size, and staining intensity of GnRH immunoreactive (ir) neurons; (2) plasma LH; and (3) plasma estradiol (E2) in females. We found that TS females had more GnRH-ir neurons in the septo-preoptic area of the hypothalamus than did WS females, and GnRH-ir neuronal cell bodies were larger in the WS than the TS females. There was no morph difference in the intensity of GnRH labeling. TS females had higher plasma LH, which is consistent with a previous report, and higher plasma E2. We hypothesize that the differences in GnRH-ir cell number and size are related to differences in LH and E2 secretion, and may be relevant to polymorphic social behavior.

Rapid neuroendocrine responses to auditory courtship signals.

In many species, courtship signals enhance reproductive function in the receiver. How these social signals are processed by the brain, particularly how they induce an endocrine response, is not well understood. Songbirds provide an ideal model in which to study this phenomenon because of the large existing literature on both their auditory neurobiology and the control of their reproductive physiology by environmental cues. To date, all of the relevant studies on songbirds have involved measuring the effects of male vocalizations on ovarian function over a period of weeks, a time course that precludes detailed analysis of the neuroendocrine mechanisms operating during song perception. We played recordings of conspecific male song to laboratory-housed female white-throated sparrows and quantified the resulting rapid changes in LH as well as the induction of the immediate early gene Egr-1 in the GnRH system and mediobasal hypothalamus (MBH). Hearing song for 42 min induced LH release and Egr-1 expression in the MBH, but did not alter Egr-1 expression in GnRH neurons. The time course of LH release and the pattern of Egr-1 expression together suggest that song acts as a trigger to induce GnRH release in a manner resembling photostimulation. The Egr-1 response in the MBH was qualitatively distinguishable from the responses to either photostimulation or pharmacologically induced LH release but seemed to involve overlapping neuronal populations. Song-induced Egr-1 expression in the MBH was correlated with the expression in midbrain and forebrain auditory centers, further supporting a role for the MBH in processing social information.

Estradiol modulates brainstem catecholaminergic cell groups and projections to the auditory forebrain in a female songbird.

In songbirds, hearing conspecific song induces robust expression of the immediate early gene zenk in the auditory forebrain. This genomic response to song is well characterized in males and females of many species, and is highly selective for behaviorally relevant song. In white-throated sparrows, the selectivity of the zenk response requires breeding levels of estradiol; we previously showed that in non-breeding females with low levels of plasma estradiol, the zenk response to hearing song is no different than the response to hearing frequency-matched tones. Here, we investigated the role of brainstem catecholaminergic cells groups, which project to the forebrain, in estradiol-dependent selectivity. First, we hypothesized that estradiol treatment affects catecholaminergic innervation of the auditory forebrain as well as its possible sources in the brainstem. Immunohistochemical staining of tyrosine hydroxylase revealed that estradiol treatment significantly increased the density of catecholaminergic innervation of the auditory forebrain as well as the number of catecholaminergic cells in the locus coeruleus (A6) and the ventral tegmental area (A10), both of which are known to contain estrogen receptors in songbirds. Second, we hypothesized that during song perception, catecholaminergic cell groups of the brainstem actively participate in auditory selectivity via estrogen-dependent changes in activity. We found that hearing songs did not induce the expression of zenk, a putative marker of activity, within catecholaminergic neurons in any of the cell groups quantified. Together, our results suggest that estradiol induces changes in brainstem catecholaminergic cell groups that may play a neuromodulatory role in behavioral and auditory selectivity.