Brain aromatase: new lessons from non-mammalian model systems

This review highlights recent studies of the anatomical and functional implications of brain aromatase (estrogen synthase) expression in two vertebrate lineages, teleost fishes and songbirds, that show remarkably high levels of adult brain aromatase activity, protein and gene expression compared to other vertebrate groups. Teleosts and birds have proven to be important neuroethological models for investigating how local estrogen synthesis leads to changes in neural phenotypes that translate into behavior. Region-specific patterns of aromatase expression, and thus estrogen synthesis, include the vocal and auditory circuits that figure prominently into the life history adaptations of vocalizing teleosts and songbirds. Thus, by targeting, for example, vocal motor circuits without inappropriate steroid exposure to other steroid-dependent circuits, such as those involved in either copulatory or spawning behaviors, the neuroendocrine system can achieve temporal and spatial specificity in its modulation of neural circuits that lead to the performance of any one behavior.     

Serotonin distribution in the brain of the plainfin midshipman: Substrates for vocal-acoustic modulation and a reevaluation of the serotonergic system in teleost fishes

Serotonin (5-HT) is a modulator of neural circuitry underlying motor patterning, homeostatic control, and social behavior. While previous studies have described 5-HT distribution in various teleosts, serotonergic raphe subgroups in fish are not well defined and therefore remain problematic for cross-species comparisons. Here we used the plainfin midshipman fish, Porichthys notatus, a well-studied model for investigating the neural and hormonal mechanisms of vertebrate vocal-acoustic communication, to redefine raphe subgroups based on both stringent neuroanatomical landmarks as well as quantitative cell measurements. In addition, we comprehensively characterized 5-HT-immunoreactive (-ir) innervation throughout the brain, including well-delineated vocal and auditory nuclei. We report neuroanatomical heterogeneity in populations of the serotonergic raphe nuclei of the brainstem reticular formation, with three discrete subregions in the superior raphe, an intermediate 5-HT-ir cell cluster, and an extensive inferior raphe population. 5-HT-ir neurons were also observed within the vocal motor nucleus (VMN), forming putative contacts on those cells. In addition, three major 5-HT-ir cell groups were identified in the hypothalamus and one group in the pretectum. Significant 5-HT-ir innervation was found in components of the vocal pattern generator and cranial motor nuclei. All vocal midbrain nuclei showed considerable 5-HT-ir innervation, as did thalamic and hindbrain auditory and lateral line areas and vocal-acoustic integration sites in the preoptic area and ventral telencephalon. This comprehensive atlas offers new insights into the organization of 5-HT nuclei in teleosts and provides neuroanatomical evidence for serotonin as a modulator of vocal-acoustic circuitry and behavior in midshipman fish, consistent with findings in vocal tetrapods.     

Neural correlates of the lombard effect in primate auditory cortex

Speaking is a sensory-motor process that involves constant self-monitoring to ensure accurate vocal production. Self-monitoring of vocal feedback allows rapid adjustment to correct perceived differences between intended and produced vocalizations. One important behavior in vocal feedback control is a compensatory increase in vocal intensity in response to noise masking during vocal production, commonly referred to as the Lombard effect. This behavior requires mechanisms for continuously monitoring auditory feedback during speaking. However, the underlying neural mechanisms are poorly understood. Here we show that when marmoset monkeys vocalize in the presence of masking noise that disrupts vocal feedback, the compensatory increase in vocal intensity is accompanied by a shift in auditory cortex activity toward neural response patterns seen during vocalizations under normal feedback condition. Furthermore, we show that neural activity in auditory cortex during a vocalization phrase predicts vocal intensity compensation in subsequent phrases. These observations demonstrate that the auditory cortex participates in self-monitoring during the Lombard effect, and may play a role in the compensation of noise masking during feedback-mediated vocal control.     

Distribution of androgen receptor mRNA expression in vocal,auditory, and neuroendocrine circuits in a teleost fish

Across all major vertebrate groups, androgen receptors (ARs) have been identified in neural circuits that shape reproductive‐related behaviors, including vocalization. The vocal control network of teleost fishes presents an archetypal example of how a vertebrate nervous system produces social, context‐dependent sounds. We cloned a partial cDNA of AR that was used to generate specific probes to localize AR expression throughout the central nervous system of the vocal plainfin midshipman fish (Porichthys notatus). In the forebrain, AR mRNA is abundant in proposed homologs of the mammalian striatum and amygdala, and in anterior and posterior parvocellular and magnocellular nuclei of the preoptic area, nucleus preglomerulosus, and posterior, ventral and anterior tuberal nuclei of the hypothalamus. Many of these nuclei are part of the known vocal and auditory circuitry in midshipman. The midbrain periaqueductal gray, an essential link between forebrain and hindbrain vocal circuitry, and the lateral line recipient nucleus medialis in the rostral hindbrain also express abundant AR mRNA. In the caudal hindbrain‐spinal vocal circuit, high AR mRNA is found in the vocal prepacemaker nucleus and along the dorsal periphery of the vocal motor nucleus congruent with the known pattern of expression of aromatase‐containing glial cells. Additionally, abundant AR mRNA expression is shown for the first time in the inner ear of a vertebrate. The distribution of AR mRNA strongly supports the role of androgens as modulators of behaviorally defined vocal, auditory, and neuroendocrine circuits in teleost fish and vertebrates in general. J. Comp. Neurol. 518:493–512, 2010. © 2009 Wiley‐Liss, Inc.     

Catecholaminergic connectivity to the inner ear,central auditory,and vocal motor circuitry in the plainfin midshipman fish porichthys notatus

Although the neuroanatomical distribution of catecholaminergic (CA) neurons has been well documented across all vertebrate classes, few studies have examined CA connectivity to physiologically and anatomically identified neural circuitry that controls behavior. The goal of this study was to characterize CA distribution in the brain and inner ear of the plainfin midshipman fish (Porichthys notatus) with particular emphasis on their relationship with anatomically labeled circuitry that both produces and encodes social acoustic signals in this species. Neurobiotin labeling of the main auditory end organ, the saccule, combined with tyrosine hydroxylase immunofluorescence (TH‐ir) revealed a strong CA innervation of both the peripheral and central auditory system. Diencephalic TH‐ir neurons in the periventricular posterior tuberculum, known to be dopaminergic, send ascending projections to the ventral telencephalon and prominent descending projections to vocal–acoustic integration sites, notably the hindbrain octavolateralis efferent nucleus, as well as onto the base of hair cells in the saccule via nerve VIII. Neurobiotin backfills of the vocal nerve in combination with TH‐ir revealed CA terminals on all components of the vocal pattern generator, which appears to largely originate from local TH‐ir neurons but may include input from diencephalic projections as well. This study provides strong neuroanatomical evidence that catecholamines are important modulators of both auditory and vocal circuitry and acoustic‐driven social behavior in midshipman fish. This demonstration of TH‐ir terminals in the main end organ of hearing in a nonmammalian vertebrate suggests a conserved and important anatomical and functional role for dopamine in normal audition. J. Comp. Neurol. 522:2887‐2927, 2014. © 2014 Wiley Periodicals, Inc.     

Connectivity and ultrastructure of dopaminergic innervation of the inner ear and auditory efferent system of a vocal fish

Dopamine (DA) is a conserved modulator of vertebrate neural circuitry, yet our knowledge of its role in peripheral auditory processing is limited to mammals. The present study combines immunohistochemistry, neural tract tracing, and electron microscopy to investigate the origin and synaptic characteristics of DA fibers innervating the inner ear and the hindbrain auditory efferent nucleus in the plainfin midshipman, a vocal fish that relies upon the detection of mate calls for reproductive success. We identify a DA cell group in the diencephalon as a common source for innervation of both the hindbrain auditory efferent nucleus and saccule, the main hearing endorgan of the inner ear. We show that DA terminals in the saccule contain vesicles but transmitter release appears paracrine in nature, due to the apparent lack of synaptic contacts. In contrast, in the hindbrain, DA terminals form traditional synaptic contacts with auditory efferent neuronal cell bodies and dendrites, as well as unlabeled axon terminals, which, in turn, form inhibitory‐like synapses on auditory efferent somata. Our results suggest a distinct functional role for brain‐derived DA in the direct and indirect modulation of the peripheral auditory system of a vocal nonmammalian vertebrate.     

A paradox in the evolution of primate vocal learning

The importance of auditory feedback in the development of spoken language in humans is striking. Paradoxically, although auditory-feedback-dependent vocal plasticity has been shown in a variety of taxonomic groups, there is little evidence that our nearest relatives--non-human primates--require auditory feedback for the development of species-typical vocal signals. Because of the apparent lack of developmental plasticity in the vocal production system, neuroscientists have largely ignored the neural mechanisms of non-human primate vocal production and perception. Recently, the absence of evidence for vocal plasticity from developmental studies has been contrasted with evidence for vocal plasticity in adults. We argue that this new evidence makes non-human primate vocal behavior an attractive model system for neurobiological analysis.     

Estrogenic modulation of auditory processing: A vertebrate comparison

Sex-steroid hormones are well-known regulators of vocal motor behavior in several organisms. A large body of evidence now indicates that these same hormones modulate processing at multiple levels of the ascending auditory pathway. The goal of this review is to provide a comparative analysis of the role of estrogens in vertebrate auditory function. Four major conclusions can be drawn from the literature: First, estrogens may influence the development of the mammalian auditory system. Second, estrogenic signaling protects the mammalian auditory system from noise- and age-related damage. Third, estrogens optimize auditory processing during periods of reproductive readiness in multiple vertebrate lineages. Finally, brain-derived estrogens can act locally to enhance auditory response properties in at least one avian species. This comparative examination may lead to a better appreciation of the role of estrogens in the processing of natural vocalizations and mayprovide useful insights toward alleviating auditory dysfunctions emanating from hormonal imbalances.     

Sensory feedback control of mammalian vocalizations

Somatosensory and auditory feedback mechanisms are dynamic components of the vocal motor pattern generator in mammals. This review explores how sensory cues arising from central auditory and somatosensory pathways actively guide the production of both simple sounds and complex phrases in mammals. While human speech is a uniquely sophisticated example of mammalian vocal behavior, other mammals can serve as examples of how sensory feedback guides complex vocal patterns. Echolocating bats in particular are unique in their absolute dependence on voice control for survival: these animals must constantly adjust the acoustic and temporal patterns of their orientation sounds to efficiently navigate and forage for insects at high speeds under the cover of darkness. Many species of bats also utter a broad repertoire of communication sounds. The functional neuroanatomy of the bat vocal motor pathway is basically identical to other mammals, but the acute significance of sensory feedback in echolocation has made this a profitable model system for studying general principles of sensorimotor integration with regard to vocalizing. Bats and humans are similar in that they both maintain precise control of many different voice parameters, both exhibit a similar suite of responses to altered auditory feedback, and for both the efficacy of sensory feedback depends upon behavioral context. By comparing similarities and differences in the ways sensory feedback influences voice in humans and bats, we may shed light on the basic architecture of the mammalian vocal motor system and perhaps be able to better distinguish those features of human vocal control that evolved uniquely in support of speech and language.     

Wireless multi-channel single unit recording in freely moving and vocalizing primates

The ability to record well-isolated action potentials from individual neurons in naturally behaving animals is crucial for understanding neural mechanisms underlying natural behaviors. Traditional neurophysiology techniques, however, require the animal to be restrained which often restricts natural behavior. An example is the common marmoset (Callithrix jacchus), a highly vocal New World primate species, used in our laboratory to study the neural correlates of vocal production and sensory feedback. When restrained by traditional neurophysiological techniques marmoset vocal behavior is severely inhibited. Tethered recording systems, while proven effective in rodents pose limitations in arboreal animals such as the marmoset that typically roam in a three-dimensional environment. To overcome these obstacles, we have developed a wireless neural recording technique that is capable of collecting single-unit data from chronically implanted multi-electrodes in freely moving marmosets. A lightweight, low power and low noise wireless transmitter (headstage) is attached to a multi-electrode array placed in the premotor cortex of the marmoset. The wireless headstage is capable of transmitting 15 channels of neural data with signal-to-noise ratio (SNR) comparable to a tethered system. To minimize radio-frequency (RF) and electro-magnetic interference (EMI), the experiments were conducted within a custom designed RF/EMI and acoustically shielded chamber. The individual electrodes of the multi-electrode array were periodically advanced to densely sample the cortical layers. We recorded single-unit data over a period of several months from the frontal cortex of two marmosets. These recordings demonstrate the feasibility of using our wireless recording method to study single neuron activity in freely roaming primates.     

Reassessing the mechanisms and origins of vocal learning in birds.

The most widely accepted hypothesis of vocal imitation in birds pre-dates many recent studies on the behavior, anatomy, physiology and cell biology of this phenomenon. It states that vocal learning involves two steps: (1) an auditory memory is laid down, and then (2) vocal output is modified until the auditory feedback it generates matches the model. This black-box model of vocal imitation disregards circuitry. We now know that the brain pathways for vocal learning in birds include a series of well-defined nuclei and projections. Some of these nuclei and projections develop late in ontogeny, at the time when auditory models are first acquired and imitated. We also know that the pathways involved in song production respond to sound, an observation that blurs the demarcation between what is an auditory and what is a motor circuit. These and other recent discoveries call for a reassessment of the mechanisms and origins of vocal learning in birds and mammals.     

Effects of voice harmonic complexity on ERP responses to pitch-shifted auditory feedback

Objective

The present study investigated the neural mechanisms of voice pitch control for different levels of harmonic complexity in the auditory feedback.

Methods

Event-related potentials (ERPs) were recorded in response to +200 cents pitch perturbations in the auditory feedback of self-produced natural human vocalizations, complex and pure tone stimuli during active vocalization and passive listening conditions.

Results

During active vocal production, ERP amplitudes were largest in response to pitch shifts in the natural voice, moderately large for non-voice complex stimuli and smallest for the pure tones. However, during passive listening, neural responses were equally large for pitch shifts in voice and non-voice complex stimuli but still larger than that for pure tones.

Conclusions

These findings suggest that pitch change detection is facilitated for spectrally rich sounds such as natural human voice and non-voice complex stimuli compared with pure tones. Vocalization-induced increase in neural responses for voice feedback suggests that sensory processing of naturally-produced complex sounds such as human voice is enhanced by means of motor-driven mechanisms (e.g. efference copies) during vocal production.

Significance

This enhancement may enable the audio-vocal system to more effectively detect and correct for vocal errors in the feedback of natural human vocalizations to maintain an intended vocal output for speaking.     

Vocal-acoustic circuitry and descending vocal pathways in teleost fish: convergence with terrestrial vertebrates reveals conserved traits

Vocal behavior is multifaceted and requires that vocal-motor patterning be integrated at multiple brain levels with auditory, neuroendocrine, and other social behavior processes (e.g., courtship and aggression). We now provide anatomical evidence for an extensive vocal network in teleost fishes (Batrachoididae: Porichthys notatus; Opsanus beta) that is strongly integrated with neuroendocrine and auditory pathways and that exhibits striking similarities to the vocal-acoustic circuitry known for mammals. Biotin compound injections into neurophysiologically identified vocal regions of the forebrain (preoptic area and anterior hypothalamus) and of the midbrain (periaqueductal gray and paralemniscal tegmentum) reveal extensive connectivity within and between these regions, as well as reciprocal relationships with the auditory thalamus and/or auditory midbrain (torus semicircularis). Thus, specific components of the basal forebrain and midbrain are here designated as the forebrain vocal-acoustic complex (fVAC) and midbrain vocal-acoustic complex (mVAC), respectively. Biotin injections into the mVAC and a previously identified hindbrain vocal pattern generator likewise provide anatomical evidence for a distributed network of descending projections to the vocal pacemaker-motoneuron circuitry. Together, the present experiments establish a vocal-auditory-neuroendocrine network in teleost fish that links the forebrain and midbrain to the hindbrain vocal pattern generator (i.e., fVAC --> mVAC --> pattern generator) and provides an anatomical framework for the previously identified neuropeptide modulation of vocal activity elicited from the forebrain and midbrain, which contributes to the expression of sex- and male morph-specific behavior. We conclude with a broad comparison of these findings with those for other vertebrate taxa and suggest that the present findings provide novel insights into the structure of conserved behavioral regulatory circuits that have led to evolutionary convergence in vocal-acoustic systems.     

Intraventricular infusion of arginine vasotocin induces singing in a female songbird

Arginine vasotocin (AVT) has been implicated in the activation of courtship and aggressive behaviors in many vertebrate taxa. Here, we tested its effect on singing and other vocal behavior in a songbird. Female white-crowned sparrows (Z. l. gambelii) were implanted with chronic cannulae aimed at the third ventricle. Infusions of AVT dramatically increased the number of songs and other vocalizations during a 40 min period following infusion. Half of the subjects sang full song following AVT treatment. No bird sang after treatment with saline; any type of vocalization after saline treatment was rare. Female white-crowned sparrows are known to sing in both spring and winter in the wild; this behavior is thought to be aggressive, functioning in dominance interactions and territoriality. Central infusion of AVT induced singing and other vocalizations in estrogen-primed, photostimulated subjects as well as in non-reproductive subjects housed on short photoperiods. Thus, the effects of AVT on vocal behavior may not require breeding levels of gonadal steroids and are probably not seasonal. We hypothesize that both in the breeding and non-breeding seasons, AVT increases motivation to sing.     

The pallial basal ganglia pathway modulates the behaviorally driven gene expression of the motor pathway

The discrete neural network for songbird vocal communication provides an effective system to study neural mechanisms of learned motor behaviors in vertebrates. This system consists of two pathways--a vocal motor pathway used to produce learned vocalizations and a vocal pallial basal ganglia loop used to learn and modify the vocalizations. However, it is not clear how the loop exerts control over the motor pathway. To study the mechanism, we used expression of the neural activity-induced gene ZENK (or egr-1), which shows singing-regulated expression in a social context-dependent manner: high levels in both pathways when singing undirected and low levels in the lateral part of the loop and in the robust nucleus of the arcopallium (RA) of the motor pathway when singing directed to another animal. Here, we show that there are two parallel interactive parts within the pallial basal ganglia loop, lateral and medial, which modulate singing-driven ZENK expression of the motor pathway nuclei RA and HVC, respectively. Within the loop, the striatal and pallial nuclei appear to have opposing roles; the striatal vocal nucleus lateral AreaX is required for high ZENK expression in its downstream nuclei, particularly during undirected singing, while the pallial vocal lateral magnocellular nucleus of the anterior nidopallium is required for lower expression, particularly during directed singing. These results suggest a dynamic molecular interaction between the basal ganglia pathway and the motor pathway during production of a learned motor behavior.     

Storing maternal memories: Hypothesizing an interaction of experience and estrogen on sensory cortical plasticity to learn infant cues

Much of the literature on maternal behavior has focused on the role of infant experience and hormones in a canonical subcortical circuit for maternal motivation and maternal memory. Although early studies demonstrated that the cerebral cortex also plays a significant role in maternal behaviors, little has been done to explore what that role may be. Recent work though has provided evidence that the cortex, particularly sensory cortices, contains correlates of sensory memories of infant cues, consistent with classical studies of experience-dependent sensory cortical plasticity in non-maternal paradigms. By reviewing the literature from both the maternal behavior and sensory cortical plasticity fields, focusing on the auditory modality, we hypothesize that maternal hormones (predominantly estrogen) may act to prime auditory cortical neurons for a longer-lasting neural trace of infant vocal cues, thereby facilitating recognition and discrimination. This couldthen more efficiently activate the subcortical circuit to elicit and sustain maternal behavior.     

Network architectures and circuit function: testing alternative hypotheses in multifunctional networks

Understanding how species-typical movement patterns are organized in the nervous system is a central question in neurobiology. The current explanations involve 'alphabet' models in which an individual neuron may participate in the circuit for several behaviors but each behavior is specified by a specific neural circuit. However, not all of the well-studied model systems fit the 'alphabet' model. The 'equation' model provides an alternative possibility, whereby a system of parallel motor neurons, each with a unique (but overlapping) field of innervation, can account for the production of stereotyped behavior patterns by variable circuits. That is, it is possible for such patterns to arise as emergent properties of a generalized neural network in the absence of feedback, a simple version of a 'self-organizing' behavioral system. Comparison of systems of identified neurons suggest that the 'alphabet' model may account for most observations where CPGs act to organize motor patterns. Other well-known model systems, involving architectures corresponding to feed-forward neural networks with a hidden layer, may organize patterned behavior in a manner consistent with the 'equation' model. Such architectures are found in the Mauthner and reticulospinal circuits, 'escape' locomotion in cockroaches, CNS control of Aplysia gill, and may also be important in the coordination of sensory information and motor systems in insect mushroom bodies and the vertebrate hippocampus. The hidden layer of such networks may serve as an 'internal representation' of the behavioral state and/or body position of the animal, allowing the animal to fine-tune oriented, or particularly context-sensitive, movements to the prevalent conditions. Experiments designed to distinguish between the two models in cases where they make mutually exclusive predictions provide an opportunity to elucidate the neural mechanisms by which behavior is organized in vivo and in vitro.     

The oscine song system considered in the context of the avian brain: lessons learned from comparative neurobiology.

The oscine song system has emerged as one of the leading model systems for studying motor learning in vertebrates, combining an easily recorded behavior with a discrete neural substrate. That neural substrate seems to be distinct from other structures in the avian brain and thus is often studied in isolation. However, the song system is unlikely to have evolved ex nihilo, and should share some features with the parts of the avian brain from which it evolved. Identification of its evolutionary precursors should help us apply what we know about the song system to other vertebrate motor systems, and vice versa. Here, I review the homologies between parts of the avian and mammalian telencephala and explain where the song system nuclei reside in this context. The organization of the song system is then compared to other parts of the avian brain and the brains of nonoscine birds. Study of the nonoscine brain has revealed a 'general motor pathway' from caudolateral neostriatum (NCL) to intermediate archistriatum (Ai) that resembles the song system motor pathway in its anatomical organization. No part of this motor pathway projects directly to brainstem vocal or respiratory centers in nonoscines, but it does innervate a wide variety of motor and premotor neuron populations that mediate other behaviors. This general motor pathway may be accompanied by an 'anterior forebrain pathway', suggesting that the song system is simply a specialization of a part of this preexisting circuit. This hypothesis has implications for how accessory structures of the song system (e.g. HVc shelf, LMAN shell) are regarded, can help explain how the forebrain vocal control systems of three avian taxa (parrots, hummingbirds, and songbirds) could have evolved independently yet be so similar in organization, and makes testable predictions concerning the anatomy of the song system and the nonoscine brain.     

Distribution and Dynamics in the Expression of Androgen and Estrogen Receptors in Vocal Control Systems of Songbirds

Developmental and seasonal changes in the production of androgens and estrogens seem to control sex-specific differentiation and seasonal changes in sexual behaviors such as singing of songbirds. These steroids affect the brain by binding to intracellular located receptors. Here we analyze whether the expression of androgen receptors (AR) and estrogen receptors (ER) is a limiting factor for differentiation of the vocal pattern and the vocal control system of zebra finches and canaries. AR and ER are localised in the brain using in situ hybridizations with cRNA probes of the AR and ER of the zebra finch. AR are widely expressed in the vocal control system and allow androgen-dependent alterations of the development and function of most vocal control areas. The expression of AR in some vocal control areas such as NIF, DLM, and AVT differs between individuals. This individual variability suggests genetic differences or transient steroid-independent expression of AR. ER are found only in the HVC and thus restrict estrogen-dependent developmental and functional changes of the singing to the HVC area. AR- and ER-mRNA expression per cell in the HVC of adult canaries undergoes seasonal changes so that ER are higher expressed from fall to the early breeding season. During ontogeny, ER start to occur in the zebra finch HVC at posthatching day 15 and in the canary HVC at posthatching day 30. As the HVC is already sexual dimorphic in size at these times, HVC-based estrogen-ER–dependent mechanisms seem not to be important for the initial sexual dimorphic development of the HVC.