Luke Remage-Healey

Brain estrogens rapidly strengthen auditory encoding and guide song preference in a songbird

Higher cognitive function depends on accurate detection and processing of subtle features of sensory stimuli. Such precise computations require neural circuits to be modulated over rapid timescales, yet this modulation is poorly understood. Brain-derived steroids (neurosteroids) can act as fast signaling molecules in the vertebrate central nervous system and could therefore modulate sensory processing and guide behavior, but there is no empirical evidence for this possibility. Here we report that acute inhibition of estrogen production within a cortical-like region involved in complex auditory processing disrupts a songbird’s ability to behaviorally respond to song stimuli. Identical manipulation of local estrogen levels rapidly changes burst firing of single auditory neurons. This acute estrogen-mediated modulation targets song and not other auditory stimuli, possibly enabling discrimination among species-specific signals. Our results demonstrate a crucial role for neuroestrogen synthesis among vertebrates for enhanced sensory encoding. Cognitive impairments associated with estrogen depletion, including verbal memory loss in humans, may therefore stem from compromised moment-by-moment estrogen actions in higher-order cortical circuits.

Rapid, Hierarchical Modulation of Vocal Patterning by Steroid Hormones

Vocal control systems have been identified in all major groups of jawed vertebrates. Although steroid hormones are instrumental in the long-term development and maintenance of neural structures underlying vocalization, it is unknown whether steroids rapidly modulate the neural activity of vocal motor systems. The midshipman fish generates advertisement and agonistic calls that mainly differ in duration. A descending midbrain pathway activates a hindbrain-spinal vocal circuit that directly establishes the discharge frequency and duration of the rhythmic vocal motor volley. This vocal motor output, which can be monitored from occipital nerve roots, directly determines the rate and duration of contraction of a pair of sonic muscles and, in turn, the fundamental frequency and duration of vocalizations. Here, we demonstrate that the duration of the vocal motor volley, or fictive vocalization, is rapidly responsive to steroid hormones, including androgens, estrogens, and glucocorticoids. These responses are consistent, in part, with a nongenomic mechanism and are steroid specific at the receptor level, suggesting the possibility of multiple membrane-bound receptor populations. We also show, using intact and semi-intact preparations, that steroids hierarchically modulate fictive vocalizations; whereas the hindbrain-spinal region is both necessary and sufficient for rapid (within 5 min) effects on duration, descending midbrain input is necessary for maintenance (up to 120 min) of these effects. The conserved nature of vertebrate vocal motor systems suggests that the neuroendocrine principles outlined in this study may be a fundamental feature of all vocal vertebrates.

Synaptocrine Signaling: Steroid Synthesis and Action at the Synapse

Sex steroids have long been recognized for their dramatic impact on brain and behavior, including rapid modulation of membrane excitability. It is a widely held perception that these molecules are largely derived from peripheral sources and lack the spatial and temporal specificity ascribed to classical neuromodulatory systems. Neuromodulatory systems, in contrast, are defined by their regulated neuronal presynaptic secretion and by their functional modulation of perisynaptic events. Here we provide evidence for regulated presynaptic estrogen synthesis and functional postsynaptic actions. These results meet all the criteria for a neuromodulatory system and shift our perception of estrogens from that of peripheral signals exclusively to include that of a signaling system intrinsic to the brain itself. We apply the term synaptocrine to describe this form of neuromodulation.

Behavioral and adrenocortical responses to mate separation and reunion in the zebra finch

The adaptive value of social affiliation has been well established. It is unclear, however, what endogenous mechanisms may mediate affiliative behavior. The Australian zebra finch (Taeniopygia guttata) breeds colonially and adults maintain lifelong pair bonds that may be disrupted in the wild due to high mortality rates. Many of its natural, social behaviors are maintained in laboratory conditions, making this species well suited for studying the mechanisms of affiliation. This study examines the behavioral and neuroendocrine responses to pair mate separation and reunion in zebra finches. We measured plasma corticosterone (CORT) and behavioral changes following separation from a pair bonded mate, and again upon reintroducing the mate or an opposite-sex cagemate. Plasma CORT concentrations were: (1). elevated during pair mate separation, even in the presence of other same-sex individuals, and (2). reduced to baseline upon reunion with the pair mate but not upon re-pairing with a new opposite-sex partner. These findings show that zebra finches exhibit hormonal responses to separation and reunion specifically with a bonded pair mate and not with other familiar conspecifics. In addition, alterations in behavior during separation and reunion are consistent with monogamous pair bond maintenance. This study presents evidence for adrenocortical involvement in avian pair bonding, and for hypothalamic-pituitary-adrenal activation in response to an ecologically relevant social stressor.

A rapid neuromodulatory role for steroid hormones in the control of reproductive behavior

The long-term transcriptional actions of steroids that shape neuronal morphology and the probability of behavioral expression are well established. More recently, attention has been focused on the role of rapid (minute-by-minute) steroid actions on neuronal mechanisms of reproductive behavior. In this review, we first consider the rapid actions of steroids on mating and copulatory behaviors in tetrapod vertebrates. Evidence for rapid effects of steroids is presented for chemoinvestigatory behavior (genital sniffing of females by male mice), lordosis (arched-back mating posture in female rats), copulatory mounting (male mice and male Japanese quail), reproductive clasping (pre-copulatory mounting in newts), and paced mating (copulation rate as determined by female rats). We then review recent studies in teleost fish that demonstrate the rapid actions of steroids on vocal patterning at two levels: (1) central pattern generators and (2) social behavior in natural environments. Thus, we propose that steroid-dependent modulation of central pattern generators can govern the overt expression of reproductive behaviors via rapid non-transcriptional mechanisms that are likely to be widespread among vertebrates.

Plasticity in brain sexuality is revealed by the rapid actions of steroid hormones.

Divergent steroid hormone profiles can shape the development of male versus female neural phenotypes, but whether they also determine differences in the short-term, neurophysiological patterning of behavior is unknown. We now show that steroid hormone-specific modulation of a vocal pattern generator (VPG) diverges between reproductive morphs in a teleost fish. Only type I male midshipman acoustically court females, whereas type II males steal fertilizations from type I males and, like females, generate only agonistic calls. The androgen 11-ketotestosterone (11kT), but not testosterone (T), rapidly (within 5 min) increases type I VPG output. As now shown, T, but not 11kT, rapidly increases VPG output in type II males and females, consistent with the predominant circulating androgen in type II males and females (T) versus type Is (11kT). Receptor and enzyme antagonists reveal an unexpected divergence in androgen- versus estrogen-dependent mechanisms in, respectively, type II males versus females. Cortisol, the main circulating glucocorticoid, also has divergent actions: suppressing versus increasing VPG output in, respectively, type II males and females versus type Is. In summary, rapid steroid action on VPG activity is uncoupled from gonadal phenotype (convergent between type II males and females), whereas the receptor-mediated mechanisms of androgen action are predicted by gonadal phenotype (both male morphs are sensitive to androgen receptor blockade, whereas females are not). A comparable mix of neuroendocrine traits may explain the widespread distribution of intrasexual behavioral phenotypes among teleosts and vertebrates in general. Moreover, the fundamental organization/activation principles that predict the steroid-dependent expression of “maleness” and “femaleness” may now include rapid steroid actions on the neurophysiological patterning of behavior.

From social behavior to neural circuitry: Steroid hormones rapidly modulate advertisement calling via a vocal pattern generator

Across vertebrates, androgens are rapidly elevated within minutes in response to aggressive or reproductive stimuli, yet it is unclear what the causal relationship is between fast androgen elevation and the ongoing (minute-by-minute) expression of behavior. This study tested the hypothesis that rapid increases in plasma steroid levels induce similarly rapid increases in both vocal behavior and the neurophysiological output of a central pattern generator that governs vocal behavior. In Gulf toadfish (Opsanus beta), males call to attract females to their nesting sites, and both males and females vocalize in aggressive interactions. Previous field experiments with males showed that simulated territorial challenges produce rapid and concurrent elevations in ongoing calling behavior and circulating levels of the teleost-specific androgen 11-ketotestosterone (11kT), but not the glucocorticoid cortisol. The current field experiments showed that non-invasive (food) delivery of 11kT, but not cortisol, induced an elevation within 10 min in the ongoing calling behavior of males. Electrophysiological experiments revealed that intramuscular injections of either 11kT or cortisol, but neither testosterone nor 17-beta-estradiol, induced increases within 5 min in the output of the vocal pattern generator in males, whereas only cortisol had similarly fast effects in females. The field behavioral results support predictions generated by the challenge hypothesis and also parallel the 11kT-dependent modulation of the vocal pattern generator in males. The cortisol effect on the vocal pattern generator in both sexes predicts that glucocorticoids regulate vocalizations in non-advertisement contexts. Together, these experiments provide strong support for the hypothesis that surges in circulating steroid levels play a causal role in shaping rapid changes in social behavior (vocalizations) through non-genomic-like actions on neural (vocal motor) circuits that directly encode behavioral patterning.

Presynaptic Control of Rapid Estrogen Fluctuations in the Songbird Auditory Forebrain

Within the CNS of vertebrates, estrogens can directly modulate neural circuits that govern a wide range of behaviors, including feeding, spatial navigation, reproduction, and auditory processing. The rapid actions of estrogens in brain (seconds to minutes) have become well established, but it is unclear how estrogens are synthesized and released within restricted temporal and spatial domains in neural circuits. Anatomical localization of the estrogen synthesis enzyme (aromatase) within presynaptic terminals suggests that neuroestrogens can be synthesized directly at the neuronal synapse. A consequent prediction follows that synaptic estrogen production is controlled via classical electrochemical events in neurons. Here, we present evidence that acute fluctuations in local neuroestrogen levels in the forebrain of the zebra finch depend on calcium influx within presynaptic terminals. In vivo experiments using microdialysis linked to a sensitive estrogen ELISA showed that local forebrain neuroestrogens were both suppressed by potassium-evoked excitation and upregulated during 30 min periods of extracellular calcium depletion in a region enriched with presynaptic aromatase. Furthermore, potassium-evoked changes in local neuroestrogens were blocked by targeted delivery of the voltage-gated calcium channel blocker ω-conotoxin GVIA. Together, these experiments indicate that neuroestrogens are controlled by specific, depolarization-sensitive, calcium-dependent events within forebrain presynaptic terminals.

Elevated Aromatase Activity in Forebrain Synaptic Terminals During Song

The enzyme aromatase, which converts androgens into oestrogens, is expressed throughout the brain in zebra finches. Aromatase is enzymatically active in both cell bodies and synaptic terminals of neurones of the songbird brain, particularly within the forebrain motor and auditory networks. Aromatisation within synaptic terminals could thus provide localised and acute modulatory oestrogens within the forebrain during singing and/or audition. In male zebra finches, we tested the hypothesis that forebrain aromatase activity is elevated during singing behaviour and/or hearing male song. The present study reports that aromatase activity is elevated in males that were singing for 30 min compared to nonsinging males, and that this elevation occurs only within the cellular compartment that contains synaptic terminals. In a separate experiment, males that heard acoustic playback of song for 30 min exhibited no differences in aromatase activity or in aromatase mRNA levels, as revealed by quantitative polymerase chain reaction analysis. Therefore, these results indicate that activation of the motor pathway for song production is linked to local elevations in synaptic aromatase activity within the forebrain of male zebra finches. Future experiments could assess whether elevated synaptic aromatase activity during song is dependent on acute regulation of the aromatase protein.

Changing Neuroestrogens Within the Auditory Forebrain Rapidly Transform Stimulus Selectivity in a Downstream Sensorimotor Nucleus

The activity of sensory circuits is shaped by neuromodulators, which can have downstream consequences for both sensorimotor integration and behavioral output. Recent evidence indicates that brain-derived estrogens (“neuroestrogens”) can act as local circuit modulators in the songbird auditory forebrain. Specifically, neuroestrogens fluctuate in the auditory caudomedial nidopallium (NCM) during social interactions and in response to song stimuli. Within minutes of elevation, neuroestrogens also enhance auditory response properties of NCM neurons, and acute blockade of estrogen production in NCM disrupts behavioral song preferences. Here, we test the hypothesis that fluctuating neuroestrogens within NCM influence stimulus selectivity in a downstream sensorimotor nucleus (HVC, used as a proper name) that receives indirect auditory input from NCM. Dual extracellular recordings coupled with retrodialysis delivery show that song selectivity in HVC is rapidly enhanced by increasing neuroestrogens in NCM in adult males. Conversely, inhibiting neuroestrogen production in NCM causes a rapid decline in song selectivity in HVC, demonstrating the endogenous nature of this modulatory network. In contrast, HVC selectivity is unaffected by neuroestrogen delivery to either nearby caudomedial mesopallium or into HVC itself, indicating that neuroestrogen actions are restricted to NCM. In juvenile males, identical neuroestrogen treatment in NCM also does not alter HVC selectivity, consistent with a developmental maturation of the auditory network. Lastly, the rapid actions of estrogens leading to enhanced HVC selectivity appear to be mediated by membrane-bound receptors in NCM. These findings indicate that steroid-dependent modulation of sensory processing is not locally restricted and can be transmitted transynaptically to influence downstream sensorimotor and premotor targets.