Sarah A. Heimovics

Rapid Effects of Aggressive Interactions on Aromatase Activity and Oestradiol in Discrete Brain Regions of Wild Male White-Crowned Sparrows

Testosterone is critical for the activation of aggressive behaviours. In many vertebrate species, circulating testosterone levels rapidly increase after aggressive encounters during the early or mid‐breeding season. During the late breeding season, circulating testosterone concentrations did not change in wild male white‐crowned sparrows after an aggressive encounter and, in these animals, changes in local neural metabolism of testosterone might be more important than changes in systemic testosterone levels. Local neural aromatisation of testosterone into 17β‐oestradiol (E2) often mediates the actions of testosterone, and we hypothesised that, in the late breeding season, brain aromatase is rapidly modulated after aggressive interactions, leading to changes in local concentrations of E2. In the present study, wild male white‐crowned sparrows in the late breeding season were exposed to simulated territorial intrusion (STI) (song playback and live decoy) or control (CON) for 30 min. STI significantly increased aggressive behaviours. Using the Palkovits punch technique, 13 brain regions were collected. There was high aromatase activity in several nuclei, although enzymatic activity in the CON and STI groups did not differ in any region. E2 concentrations were much higher in the brain than the plasma. STI did not affect circulating levels of E2 but rapidly reduced E2 concentrations in the hippocampus, ventromedial nucleus of the hypothalamus and bed nucleus of the stria terminalis. Unexpectedly, there were no correlations between aromatase activity and E2 concentrations in the brain, nor were aromatase activity or brain E2 correlated with aggressive behaviour or plasma hormone levels. This is one of the first studies to measure E2 in microdissected brain regions, and the first study to do so in free‐ranging animals. These data demonstrate that social interactions have rapid effects on local E2 concentrations in specific brain regions.

D1-like dopamine receptor density in nuclei involved in social behavior correlates with song in a context-dependent fashion in male European starlings

Research in songbirds shows that singing behavior is regulated by both brain areas involved in vocal behavior as well as those involved in social behavior. Interestingly, the precise role of these regions in song can vary as a function of the social, environmental and breeding context. To date, little is known about the neurotransmitters underlying such context-dependent regulation of song. Dopamine (DA) modulates highly motivated, goal-directed behaviors (including sexually motivated song) and emerging data implicate DA in the context-dependent regulation of singing behavior. This study was performed to begin to examine whether differences in DA receptors may underlie, in part, context-dependent differences in song production. We used autoradiographic procedures to label D1-like and D2-like DA receptors to examine the relationship between DA receptor density and singing behavior in multiple contexts in male European starlings (Sturnus vulgaris). Within a breeding context (when testosterone (T) was high), D1-like receptor density in the medial preoptic nucleus (POM) and midbrain central gray (GCt) negatively correlated with song used to attract a female. Additionally in this context, D1-like receptor density in POM, GCt, medial bed nucleus of the stria terminalis (BSTm), and lateral septum (LS) negatively correlated with song likely used to defend a nest box. In contrast, in a non-breeding context (when T was low), D1-like receptor density in POM and LS positively correlated with song used to maintain social flocks. No relationships were identified between song in any context and D2-like receptor densities. Differences in the brain regions and directional relationships between D1-like receptor binding and song suggest that dopaminergic systems play a region and context-specific role in song. These data also suggest that individual variation in singing behavior may, in part, be explained by individual differences in D1-like receptor density in brain regions implicated in social behavior.

Evidence that dopamine within motivation and song control brain regions regulates birdsong context-dependently

Vocal communication is critical for successful social interactions among conspecifics, but little is known about how the brain regulates context-appropriate communication. The neurotransmitter dopamine (DA) is involved in modulating highly motivated, goal-directed behaviors (including sexually motivated singing behavior), and emerging data suggest that the role of DA in vocal communication may differ depending on the context in which it occurs. To address this possibility, relationships between immunolabeled tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamine synthesis) and song produced within versus outside of a breeding context were explored in male European starlings (Sturnus vulgaris). Immunocytochemistry for dopamine beta-hydroxylase (DBH; the enzyme that converts DA to norepinephrine) was also performed to provide insight into whether relationships between song and TH immunoreactivity reflected dopaminergic or noradrenergic neurotransmission. Measures of TH and DBH were quantified in song control regions (HVC, Area X, robust nucleus of the acropallium) and regions implicated in motivation (medial preoptic nucleus (POM), ventral tegmental area (VTA), and midbrain central gray). In Area X, POM, and VTA measures of TH correlated with song produced within, but not outside of a breeding context. DBH in these regions did not correlate with song in either context. Together, these data suggest DA in both song control and motivation brain regions may be more tightly linked to the regulation of highly goal-directed, sexually motivated vocal behavior.

Rapid and Widespread Effects of 17β-Estradiol on Intracellular Signaling in the Male Songbird Brain: A Seasonal Comparison

Across vertebrate species, 17β-estradiol (E(2)) acts on the brain via both genomic and nongenomic mechanisms to influence neuronal physiology and behavior. Nongenomic E(2) signaling is typically initiated by membrane-associated estrogen receptors that modulate intracellular signaling cascades, including rapid phosphorylation of ERK. Phosphorylated ERK (pERK) can, in turn, rapidly phosphorylate tyrosine hydroxylase (TH) and cAMP response element-binding protein (CREB). Recent data suggest that the rapid effects of E(2) on mouse aggressive behavior are more prominent during short photoperiods (winter) and that acute aromatase inhibition reduces songbird aggression in winter only. To date, seasonal plasticity in the rapid effects of E(2) on intracellular signaling has not been investigated. Here, we compared the effects of acute (15 min) E(2) treatment on pERK, pTH, and pCREB immunoreactivity in male song sparrows (Melospiza melodia) pretreated with the aromatase inhibitor fadrozole during the breeding and nonbreeding seasons. We examined immunoreactivity in 14 brain regions including portions of the song control system, social behavior network, and the hippocampus (Hp). In both seasons, E(2) significantly decreased pERK in nucleus taeniae of the amygdala, pTH in ventromedial hypothalamus, and pCREB in mesencephalic central gray, robust nucleus of the arcopallium, and caudomedial nidopallium. However, several effects were critically dependent upon season. E(2) decreased pERK in caudomedial nidopallium in the breeding season only and decreased pCREB in the medial preoptic nucleus in the nonbreeding season only. Remarkably, E(2) decreased pERK in Hp in the breeding season but increased pERK in Hp in the nonbreeding season. Together, these data demonstrate that E(2) has rapid effects on intracellular signaling in multiple regions of the male brain and also demonstrate that rapid effects of E(2) can be profoundly different across the seasons.

Measurement of steroid concentrations in brain tissue: methodological considerations

It is well recognized that steroids are synthesized de novo in the brain (neurosteroids). In addition, steroids circulating in the blood enter the brain. Steroids play numerous roles in the brain, such as influencing neural development, adult neuroplasticity, behavior, neuroinflammation, and neurodegenerative diseases such as Alzheimer’s disease. In order to understand the regulation and functions of steroids in the brain, it is important to directly measure steroid concentrations in brain tissue. In this brief review, we discuss methods for the detection and quantification of steroids in the brain. We concisely present the major advantages and disadvantages of different technical approaches at various experimental stages: euthanasia, tissue collection, steroid extraction, steroid separation, and steroid measurement. We discuss, among other topics, the potential effects of anesthesia and saline perfusion prior to tissue collection; microdissection via Palkovits punch; solid phase extraction; chromatographic separation of steroids; and immunoassays and mass spectrometry for steroid quantification, particularly the use of mass spectrometry for “steroid profiling.” Finally, we discuss the interpretation of local steroid concentrations, such as comparing steroid levels in brain tissue with those in the circulation (plasma vs. whole blood samples; total vs. free steroid levels). We also present reference values for a variety of steroids in different brain regions of adult rats. This brief review highlights some of the major methodological considerations at multiple experimental stages and provides a broad framework for designing studies that examine local steroid levels in the brain as well as other steroidogenic tissues, such as thymus, breast, and prostate.

Breeding-context-dependent relationships between song and cFOS labeling within social behavior brain regions in male European starlings (Sturnus vulgaris)

Male European starlings (Sturnus vulgaris) sing throughout the year, but the social factors that motivate singing behavior differ depending upon the context in which song is produced. In a non-breeding context (when testosterone concentrations are low), starlings form large, mixed-sex flocks and song is involved in flock cohesion and perhaps maintenance of social hierarchies. In contrast, in a breeding context (when testosterone concentrations are high), male song plays a direct role in mate attraction. How the nervous system ensures that song production occurs in an appropriate context in response to appropriate stimuli is not well understood. The song control system regulates song production, learning, and, to some extent, perception; however, these nuclei do not appear to regulate the social context in which song is produced. A network of steroid hormone sensitive nuclei of the basal forebrain and midbrain regulates social behavior. The present study used the immediate early gene cFOS to explore possible involvement of these regions in context-dependent song production. Numbers of cFOS-labeled cells in the medial bed nucleus of the stria terminalis, anterior hypothalamus, and ventromedial nucleus of the hypothalamus related positively only to song produced in a breeding context. In contrast, numbers of cFOS-labeled cells in three zones of the lateral septum related positively only to song produced in a non-breeding context. Taken together, these data suggest differential regulation of male starling song by social behavior nuclei depending upon the breeding context in which it is produced.

ZENK labeling within social behavior brain regions reveals breeding context-dependent patterns of neural activity associated with song in male European starlings (Sturnus vulgaris)

In songbirds, song learning and production are regulated by the song control system. How the rest of the brain interacts with song nuclei to ensure that song is produced in an appropriate context is not yet clear. In male European starlings (Sturnus vulgaris), breeding context song is sexually motivated, whereas, non-breeding context song is more broadly socially motivated. Brain regions involved in regulating social behavior might differentially regulate starling song depending upon the context in which it is produced. Here, we compared the number of ZENK-labeled cells in song and social behavior nuclei in starlings singing in either a breeding or a non-breeding context. Numbers of ZENK-labeled cells in HVC related positively to song produced in both contexts. Interestingly, numbers of ZENK-labeled cells in one subdivision of the lateral septum (LS) related negatively to breeding context song but positively to non-breeding context song. In a subdivision of the medial bed nucleus of the stria terminalis (BSTm) ZENK labeling only related positively to non-breeding context song, whereas, in the ventromedial nucleus of the hypothalamus (VMH) ZENK labeling showed a tighter positive relationship with breeding context song. Together, these findings indicate that social behavior brain regions outside of the song control system regulate singing behavior differently depending upon whether song is sexually or more broadly socially motivated. Breeding context-dependent regulation of song by LS, BSTm, and VMH suggests that these nuclei may be central to adjusting song production so that it occurs in response to appropriate social and environmental stimuli.

Individual differences in the motivation to communicate relate to levels of midbrain and striatal catecholamine markers in male European starlings.

Individuals display dramatic differences in social communication even within similar social contexts. Across vertebrates dopaminergic projections from the ventral tegmental area (VTA) and midbrain central gray (GCt) strongly influence motivated, reward-directed behaviors. Norepinephrine is also rich in these areas and may alter dopamine neuronal activity. The present study was designed to provide insight into the roles of dopamine and norepinephrine in VTA and GCt and their efferent striatal target, song control region area X, in the regulation of individual differences in the motivation to sing. We used high pressure liquid chromatography with electrochemical detection to measure dopamine, norepinephrine and their metabolites in micropunched samples from VTA, GCt, and area X in male European starlings (Sturnus vulgaris). We categorized males as sexually motivated or non-sexually motivated based on individual differences in song produced in response to a female. Dopamine markers and norepinephrine in VTA and dopamine in area X correlated positively with sexually-motivated song. Norepinephrine in area X correlated negatively with non-sexually-motivated song. Dopamine in GCt correlated negatively with sexually-motivated song, and the metabolite DOPAC correlated positively with non-sexually-motivated song. Results highlight a role for evolutionarily conserved dopaminergic projections from VTA to striatum in the motivation to communicate and highlight novel patterns of catecholamine activity in area X, VTA, and GCt associated with individual differences in sexually-motivated and non-sexually-motivated communication. Correlations between dopamine and norepinephrine markers also suggest that norepinephrine may contribute to individual differences in communication by modifying dopamine neuronal activity in VTA and GCt.

Effects of water restriction on reproductive physiology and affiliative behavior in an opportunistically-breeding and monogamous songbird, the zebra finch

Wild zebra finches form long-term monogamous pair-bonds that are actively maintained year-round, even when not in breeding condition. These desert finches are opportunistic breeders, and breeding is highly influenced by unpredictable rainfall. Their high levels of affiliation and complex breeding patterns make zebra finches an excellent model in which to study the endocrine regulation of affiliation. Here, we compared zebra finch pairs that were provided with water ad libitum (control) or water restricted. We examined (1) reproductive physiology, (2) pair-maintenance behaviors in several contexts, and (3) circulating and brain steroid levels. In females, water restriction profoundly reduced largest ovarian follicle size, ovary size, oviduct size, and egg laying. In males, water restriction had no effect on testes size but decreased systemic testosterone levels. However, in the hypothalamus, local testosterone and estradiol levels were unaffected by water restriction in both sexes. Systemic and local levels of the androgen precursor dehydroepiandrosterone (DHEA) were also unaffected by water restriction. Lastly, in three different behavioral paradigms, we examined a variety of pair-maintenance behaviors, and none were reduced by water restriction. Taken together, these correlational data are consistent with the hypothesis that local production of sex steroids in the brain promotes the expression of pair-maintenance behaviors in non-breeding zebra finches.

Seasonal and individual variation in singing behavior correlates with alpha 2-noradrenergic receptor density in brain regions implicated in song, sexual, and social behavior

In seasonally breeding male songbirds, both the function of song and the stimuli that elicit singing behavior change seasonally. The catecholamine norepinephrine (NE) modulates attention and arousal across behavioral states, yet the role of NE in seasonally-appropriate vocal communication has not been well-studied. The present study explored the possibility that seasonal changes in alpha 2-noradrenergic receptors (α(2)-R) within song control regions and brain regions implicated in sexual arousal and social behavior contribute to seasonal changes in song behavior in male European starlings (Sturnus vulgaris). We quantified singing behavior in aviary housed males under spring breeding season conditions and fall conditions. α(2)-R were identified with the selective ligand [(3)H]RX821002 using autoradiographic methods. The densities of α(2)-R in song control regions (HVC and the robust nucleus of the arcopallium [RA]) and the lateral septum (LS) were lower in Spring Condition males. α(2)-R densities in the caudal portion of the medial preoptic nucleus (POM) related negatively to singing behavior. Testosterone concentrations were highest in Spring Condition males and correlated with α(2)-R in LS and POM. Results link persistent seasonal alterations in the structure or function of male song to seasonal changes in NE α(2)-Rs in HVC, RA, and LS. Individual differences in α(2)-R in the POM may in part explain individual differences in song production irrespective of the context in which a male is singing, perhaps through NE modification of male sexual arousal.