Generation, Coordination, and Evolution of Neural Circuits for Vocal Communication.

In many species, vocal communication is essential for coordinating social behaviors including courtship, mating, parenting, rivalry, and alarm signaling. Effective communication requires accurate production, detection, and classification of signals, as well as selection of socially appropriate responses. Understanding how signals are generated and how acoustic signals are perceived is key to understanding the neurobiology of social behaviors. Here we review our long-standing research program focused on Xenopus, a frog genus which has provided valuable insights into the mechanisms and evolution of vertebrate social behaviors. In Xenopus laevis, vocal signals differ between the sexes, through development, and across the genus, reflecting evolutionary divergence in sensory and motor circuits that can be interrogated mechanistically. Using two ex vivo preparations, the isolated brain and vocal organ, we have identified essential components of the vocal production system: the sexually differentiated larynx at the periphery, and the hindbrain vocal central pattern generator (CPG) centrally, that produce sex- and species-characteristic sound pulse frequencies and temporal patterns, respectively. Within the hindbrain, we have described how intrinsic membrane properties of neurons in the vocal CPG generate species-specific vocal patterns, how vocal nuclei are connected to generate vocal patterns, as well as the roles of neurotransmitters and neuromodulators in activating the circuit. For sensorimotor integration, we identified a key forebrain node that links auditory and vocal production circuits to match socially appropriate vocal responses to acoustic features of male and female calls. The availability of a well supported phylogeny as well as reference genomes from several species now support analysis of the genetic architecture and the evolutionary divergence of neural circuits for vocal communication. Xenopus thus provides a vertebrate model in which to study vocal communication at many levels, from physiology, to behavior, and from development to evolution. As one of the most comprehensively studied phylogenetic groups within vertebrate vocal communication systems, Xenopus provides insights that can inform social communication across phyla.

Premotor Neuron Divergence Reflects Vocal Evolution.

To identify mechanisms of behavioral evolution, we investigated the hindbrain circuit that generates distinct vocal patterns in two closely related frog species. Male Xenopus laevis and Xenopus petersii produce courtship calls that include a fast trill: trains of ∼60 Hz sound pulses. Although fast trill rates are similar, X. laevis fast trills have a longer duration and period than those of X. petersii To pinpoint the neural basis of these differences, we used whole-cell patch-clamp recordings in a key premotor hindbrain nucleus (the Xenopus parabrachial area, PBX) in ex vivo brains that produce fictive vocalizations, vocal nerve activity corresponding to advertisement call patterns. We found two populations of PBX neurons with distinct properties: fast trill neurons (FTNs) and early vocal neurons (EVNs). FTNs, but not EVNs, appear to be intrinsically tuned to produce each species' call patterns because: (1) X. laevis FTNs generate longer and slower depolarizations than X. petersii FTNs during their respective fictive vocalizations, (2) current steps in FTNs induce burst durations that are significantly longer in X. laevis than X. petersii, and (3) synaptically isolated FTNs oscillate in response to NMDA in a species-specific manner: longer and slower in X. laevis than in X. petersii Therefore, divergence of premotor neuron membrane properties is a strong candidate for generating vocal differences between species.SIGNIFICANCE STATEMENT The vertebrate hindbrain includes multiple neural circuits that generate rhythmic behaviors including vocalizations. Male African clawed frogs produce courtship calls that are unique to each species and differ in temporal patterns. Here, we identified two functional subtypes of neurons located in the parabrachial nucleus: a hindbrain region implicated in vocal and respiratory control across vertebrates. One of these neuronal subtypes exhibits distinct properties across species that can account for the evolutionary divergence of song patterns. Our results suggest that changes to this group of neurons during evolution may have had a major role in establishing novel behaviors in closely related species.

Probing forebrain to hindbrain circuit functions in Xenopus.

The vertebrate hindbrain includes neural circuits that govern essential functions including breathing, blood pressure and heart rate. Hindbrain circuits also participate in generating rhythmic motor patterns for vocalization. In most tetrapods, sound production is powered by expiration and the circuitry underlying vocalization and respiration must be linked. Perception and arousal are also linked; acoustic features of social communication sounds-for example, a baby's cry-can drive autonomic responses. The close links between autonomic functions that are essential for life and vocal expression have been a major in vivo experimental challenge. Xenopus provides an opportunity to address this challenge using an ex vivo preparation: an isolated brain that generates vocal and breathing patterns. The isolated brain allows identification and manipulation of hindbrain vocal circuits as well as their activation by forebrain circuits that receive sensory input, initiate motor patterns and control arousal. Advances in imaging technologies, coupled to the production of Xenopus lines expressing genetically encoded calcium sensors, provide powerful tools for imaging neuronal patterns in the entire fictively behaving brain, a goal of the BRAIN Initiative. Comparisons of neural circuit activity across species (comparative neuromics) with distinctive vocal patterns can identify conserved features, and thereby reveal essential functional components.

Evolution of vocal patterns: tuning hindbrain circuits during species divergence.

The neural circuits underlying divergent courtship behaviors of closely related species provide a framework for insight into the evolution of motor patterns. In frogs, male advertisement calls serve as unique species identifiers and females prefer conspecific to heterospecific calls. Advertisement calls of three relatively recently (∼8.5 Mya) diverged species - Xenopus laevis, X. petersii and X. victorianus - include rapid trains of sound pulses (fast trills). We show that while fast trills are similar in pulse rate (∼60 pulses s-1) across the three species, they differ in call duration and period (time from the onset of one call to the onset of the following call). Previous studies of call production in X. laevis used an isolated brain preparation in which the laryngeal nerve produces compound action potentials that correspond to the advertisement call pattern (fictive calling). Here, we show that serotonin evokes fictive calling in X. petersii and X. victorianus as it does in X. laevis As in X. laevis, fictive fast trill in X. petersii and X. victorianus is accompanied by an N-methyl-d-aspartate receptor-dependent local field potential wave in a rostral hindbrain nucleus, DTAM. Across the three species, wave duration and period are strongly correlated with species-specific fast trill duration and period, respectively. When DTAM is isolated from the more rostral forebrain and midbrain and/or more caudal laryngeal motor nucleus, the wave persists at species-typical durations and periods. Thus, intrinsic differences within DTAM could be responsible for the evolutionary divergence of call patterns across these related species.

Sex differences and endocrine regulation of auditory-evoked, neural responses in African clawed frogs (Xenopus).

Mating depends on the accurate detection of signals that convey species identity and reproductive state. In African clawed frogs, Xenopus, this information is conveyed by vocal signals that differ in temporal patterns and spectral features between sexes and across species. We characterized spectral sensitivity using auditory-evoked potentials (AEPs), commonly known as the auditory brainstem response, in males and females of four Xenopus species. In female X. amieti, X. petersii, and X. laevis, peripheral auditory sensitivity to their species own dyad-two, species-specific dominant frequencies in the male advertisement call-is enhanced relative to males. Males were most sensitive to lower frequencies including those in the male-directed release calls. Frequency sensitivity was influenced by endocrine state; ovariectomized females had male-like auditory tuning while dihydrotestosterone-treated, ovariectomized females maintained female-like tuning. Thus, adult, female Xenopus demonstrate an endocrine-dependent sensitivity to the spectral features of conspecific male advertisement calls that could facilitate mating. Xenopus AEPs resemble those of other species in stimulus and level dependence, and in sensitivity to anesthetic (MS222). AEPs were correlated with body size and sex within some species. A frequency following response, probably encoded by the amphibian papilla, might facilitate dyad source localization via interaural time differences.

Evolution of Courtship Songs in Xenopus : Vocal Pattern Generation and Sound Production.

The extant species of African clawed frogs (Xenopus and Silurana) provide an opportunity to link the evolution of vocal characters to changes in the responsible cellular and molecular mechanisms. In this review, we integrate several robust lines of research: evolutionary trajectories of Xenopus vocalizations, cellular and circuit-level mechanisms of vocalization in selected Xenopus model species, and Xenopus evolutionary history and speciation mechanisms. Integrating recent findings allows us to generate and test specific hypotheses about the evolution of Xenopus vocal circuits. We propose that reduced vocal sex differences in some Xenopus species result from species-specific losses of sexually differentiated neural and neuromuscular features. Modification of sex-hormone-regulated developmental mechanisms is a strong candidate mechanism for reduced vocal sex differences.

Species-specific loss of sexual dimorphism in vocal effectors accompanies vocal simplification in African clawed frogs (Xenopus).

Phylogenetic studies can reveal patterns of evolutionary change, including the gain or loss of elaborate courtship traits in males. Male African clawed frogs generally produce complex and rapid courtship vocalizations, whereas female calls are simple and slow. In a few species, however, male vocalizations are also simple and slow, suggesting loss of male-typical traits. Here, we explore features of the male vocal organ that could contribute to loss in two species with simple, slow male calls. In Xenopus boumbaensis, laryngeal morphology is more robust in males than in females. Larynges are larger, have a more complex cartilaginous morphology and contain more muscle fibers. Laryngeal muscle fibers are exclusively fast-twitch in males but are both fast- and slow-twitch in females. The laryngeal electromyogram, a measure of neuromuscular synaptic strength, shows greater potentiation in males than in females. Male-specific physiological features are shared with X. laevis, as well as with a species of the sister clade, Silurana tropicalis, and thus are likely ancestral. In X. borealis, certain aspects of laryngeal morphology and physiology are sexually monomorphic rather than dimorphic. In both sexes, laryngeal muscle fibers are of mixed-twitch type, which limits the production of muscle contractions at rapid intervals. Muscle activity potentiation and discrete tension transients resemble female rather than male X. boumbaensis. The de-masculinization of these laryngeal features suggests an alteration in sensitivity to the gonadal hormones that are known to control the sexual differentiation of the larynx in other Xenopus and Silurana species.

The Xenopus amygdala mediates socially appropriate vocal communication signals.

Social interaction requires that relevant sensory information is collected, classified, and distributed to the motor areas that initiate an appropriate behavioral response. Vocal exchanges, in particular, depend on linking auditory processing to an appropriate motor expression. Because of its role in integrating sensory information for the purpose of action selection, the amygdala has been implicated in social behavior in many mammalian species. Here, we show that two nuclei of the extended amygdala play essential roles in vocal communication in the African clawed frog, Xenopus laevis. Transport of fluorescent dextran amines identifies the X. laevis central amygdala (CeA) as a target for ascending auditory information from the central thalamic nucleus and as a major afferent to the vocal pattern generator of the hindbrain. In the isolated (ex vivo) brain, electrical stimulation of the CeA, or the neighboring bed nucleus of the stria terminalis (BNST), initiates bouts of fictive calling. In vivo, lesioning the CeA of males disrupts the production of appropriate vocal responses to females and to broadcasts of female calls. Lesioning the BNST in males produces an overall decrease in calling behavior. Together, these results suggest that the anuran CeA evaluates the valence of acoustic cues and initiates socially appropriate vocal responses to communication signals, whereas the BNST plays a role in the initiation of vocalizations.

Courtship behavior: Resurrecting an undead song.

The fly Drosophila yakuba has lost an ancestral component of the male courtship song: this is due to ontogenetic death of effector neurons in the ventral nerve cord, a result of the D. yakuba sex-determining gene dsx producing a male isoform, dsxM, with cell-death-promoting activity similar to that of the female isoform, dsxF, in D. melanogaster.

Distinct neural and neuromuscular strategies underlie independent evolution of simplified advertisement calls.

Independent or convergent evolution can underlie phenotypic similarity of derived behavioural characters. Determining the underlying neural and neuromuscular mechanisms sheds light on how these characters arose. One example of evolutionarily derived characters is a temporally simple advertisement call of male African clawed frogs (Xenopus) that arose at least twice independently from a more complex ancestral pattern. How did simplification occur in the vocal circuit? To distinguish shared from divergent mechanisms, we examined activity from the calling brain and vocal organ (larynx) in two species that independently evolved simplified calls. We find that each species uses distinct neural and neuromuscular strategies to produce the simplified calls. Isolated Xenopus borealis brains produce fictive vocal patterns that match temporal patterns of actual male calls; the larynx converts nerve activity faithfully into muscle contractions and single clicks. In contrast, fictive patterns from isolated Xenopus boumbaensis brains are short bursts of nerve activity; the isolated larynx requires stimulus bursts to produce a single click of sound. Thus, unlike X. borealis, the output of the X. boumbaensis hindbrain vocal pattern generator is an ancestral burst-type pattern, transformed by the larynx into single clicks. Temporally simple advertisement calls in genetically distant species of Xenopus have thus arisen independently via reconfigurations of central and peripheral vocal neuroeffectors.

A neuroendocrine basis for the hierarchical control of frog courtship vocalizations.

Seasonal courtship signals, such as mating calls, are orchestrated by steroid hormones. Sex differences are also sculpted by hormones, typically during brief sensitive periods. The influential organizational-activational hypothesis [50] established the notion of a strong distinction between long-lasting (developmental) and cyclical (adult) effects. While the dichotomy is not always strict [1], experimental paradigms based on this hypothesis have indeed revealed long-lasting hormone actions during development and more transient anatomical, physiological and behavioral effects of hormonal variation in adulthood. Sites of action during both time periods include forebrain and midbrain sensorimotor integration centers, hindbrain and spinal cord motor centers, and muscles. African clawed frog (Xenopus laevis) courtship vocalizations follow the basic organization-activation pattern of hormone-dependence with some exceptions, including expanded steroid-sensitive periods. Two highly-tractable preparations-the isolated larynx and the fictively calling brain-make this model system powerful for dissecting the hierarchical action of hormones. We discuss steroid effects from larynx to forebrain, and introduce new directions of inquiry for which Xenopus vocalizations are especially well-suited.

Convergent and divergent neural circuit architectures that support acoustic communication.

Vocal communication is used across extant vertebrates, is evolutionarily ancient, and been maintained, in many lineages. Here I review the neural circuit architectures that support intraspecific acoustic signaling in representative anuran, mammalian and avian species as well as two invertebrates, fruit flies and Hawaiian crickets. I focus on hindbrain motor control motifs and their ties to respiratory circuits, expression of receptors for gonadal steroids in motor, sensory, and limbic neurons as well as divergent modalities that evoke vocal responses. Hindbrain and limbic participants in acoustic communication are highly conserved, while forebrain participants have diverged between anurans and mammals, as well as songbirds and rodents. I discuss the roles of natural and sexual selection in driving speciation, as well as exaptation of circuit elements with ancestral roles in respiration, for producing sounds and driving rhythmic vocal features. Recent technical advances in whole brain fMRI across species will enable real time imaging of acoustic signaling partners, tying auditory perception to vocal production.

Temporally selective processing of communication signals by auditory midbrain neurons.

Perception of the temporal structure of acoustic signals contributes critically to vocal signaling. In the aquatic clawed frog Xenopus laevis, calls differ primarily in the temporal parameter of click rate, which conveys sexual identity and reproductive state. We show here that an ensemble of auditory neurons in the laminar nucleus of the torus semicircularis (TS) of X. laevis specializes in encoding vocalization click rates. We recorded single TS units while pure tones, natural calls, and synthetic clicks were presented directly to the tympanum via a vibration-stimulation probe. Synthesized click rates ranged from 4 to 50 Hz, the rate at which the clicks begin to overlap. Frequency selectivity and temporal processing were characterized using response-intensity curves, temporal-discharge patterns, and autocorrelations of reduplicated responses to click trains. Characteristic frequencies ranged from 140 to 3,250 Hz, with minimum thresholds of -90 dB re 1 mm/s at 500 Hz and -76 dB at 1,100 Hz near the dominant frequency of female clicks. Unlike units in the auditory nerve and dorsal medullary nucleus, most toral units respond selectively to the behaviorally relevant temporal feature of the rate of clicks in calls. The majority of neurons (85%) were selective for click rates, and this selectivity remained unchanged over sound levels 10 to 20 dB above threshold. Selective neurons give phasic, tonic, or adapting responses to tone bursts and click trains. Some algorithms that could compute temporally selective receptive fields are described.

Sexual dimorphism: Neural circuit switches in the Drosophila brain.

The sex-determining genes Double Sex and Fruitless are expressed in sexually differentiated neurons of the Drosophila brain. A tiny cluster of neurons, aDN cells, serves as a key circuit switch with sexually dimorphic properties: those of female flies respond to visual signals in males, while those of male flies respond to smell and humidity in females, supporting effective courtship and communal egg laying behaviors, respectively.

Regulation of respiratory and vocal motor pools in the isolated brain of Xenopus laevis.

The aquatic frog Xenopus laevis uses a complex vocal repertoire during mating and male-male interactions. Calls are produced without breathing, allowing the frogs to vocalize for long periods underwater. The Xenopus vocal organ, the larynx, is innervated by neurons in cranial motor nucleus (n.) IX-X, which contains both vocal (laryngeal) and respiratory (glottal) motor neurons. The primary descending input to n.IX-X comes from the pretrigeminal nucleus of the dorsal tegmental area of the medulla (DTAM), located in the rostral hindbrain. We wanted to characterize premotor inputs to respiratory and vocal motor neurons and to determine what mechanisms might be involved in regulating two temporally distinct rhythmic behaviors: breathing and calling. Using isolated brain and larynx preparations, we recorded extracellular activity from the laryngeal nerve and muscles and intracellular activity in laryngeal and glottal motor neurons. Spontaneous nerve activities mimicking respiratory and vocal patterns were observed. DTAM projection neurons (DTAM(IX-X) neurons) provide direct input to glottal and laryngeal motor neurons. Electrical stimulation produced short-latency coordinated activity in the laryngeal nerve. DTAM(IX-X) neurons provide excitatory monosynaptic inputs to laryngeal motor neurons and mixed excitatory and inhibitory inputs to glottal motor neurons. DTAM stimulation also produced a delayed burst of glottal motor neuron activity. Together, our data suggest that neurons in DTAM produce vocal motor output by directly activating laryngeal motor neurons and that DTAM may coordinate vocal and respiratory motor activity.

The return to water in ancestral Xenopus was accompanied by a novel mechanism for producing and shaping vocal signals.

Listeners locate potential mates using species-specific vocal signals. As tetrapods transitioned from water to land, lungs replaced gills, allowing expiration to drive sound production. Some frogs then returned to water. Here we explore how air-driven sound production changed upon re-entry to preserve essential acoustic information on species identity in the secondarily aquatic frog genus Xenopus. We filmed movements of cartilage and muscles during evoked sound production in isolated larynges. Results refute the current theory for Xenopus vocalization, cavitation, and favor instead sound production by mechanical excitation of laryngeal resonance modes following rapid separation of laryngeal arytenoid discs. Resulting frequency resonance modes (dyads) are intrinsic to the larynx rather than due to neuromuscular control. Dyads are a distinctive acoustic signature. While their component frequencies overlap across species, their ratio is shared within each Xenopus clade providing information on species identity that could facilitate both conspecific localization and ancient species divergence. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Breathing and calling: neuronal networks in the Xenopus laevis hindbrain.

Xenopus laevis is an aquatic anuran with a complex vocal repertoire. Unlike terrestrial frogs, vocalizations are independent of respiration, and a single muscle group--the laryngeal dilators--produces underwater calls. We sought to identify the premotor neural network that underlies vocal behaviors. Vocal patterns generated by premotor networks control laryngeal motor neurons in cranial nucleus (n.) IX-X. Glottal motor neurons, active during respiration, are also present in n.IX-X. We used horseradish peroxidase (HRP), Lucifer yellow, and fluorescently conjugated dextrans to characterize the organization of n.IX-X and to trace premotor neuron projections. Premotor nuclei include the inferior reticular formation (Ri) adjacent to n.IX-X and the pretrigeminal nucleus of the dorsal tegmental area of the medulla (DTAM), the primary descending input to n.IX-X. Intramuscular HRP injections revealed a spatially segregated pattern, with glottal motor neurons in anterior n.IX-X and laryngeal motor neurons in the caudal portion of the nucleus. Dextran injections identified commissural n.IX-X neurons that project to the contralateral motor nucleus and DTAM-projecting n.IX-X neurons. Both neuronal types are clustered in anteromedial n.IX-X, closely associated with glottal motor neurons. Ri neurons project to ipsilateral and contralateral DTAM. Projections from DTAM target n.IX-X bilaterally, and all four identified subtypes receive DTAM input. In contrast, Ri neurons receive little input from DTAM. We hypothesize that connectivity between neurons in n.IX-X, Ri and DTAM may provide mechanisms to generate laryngeal and glottal activity patterns and that DTAM may coordinate vocal and respiratory motor pools, perhaps acting to switch between these two mutually exclusive behaviors.

Vocal communication in frogs.

The robust nature of vocal communication in frogs has long attracted the attention of natural philosophers and their biologically inclined successors. Each frog species produces distinctive calls that facilitate pre-mating reproductive isolation and thus speciation. In many terrestrial species, a chorus of simultaneously calling males attracts females to breeding sites; reproductive females then choose and locate one male, using distinctive acoustic cues. Males compete with each other vocally and sometimes physically as well. Anuran acoustic signaling systems are thus subject to the strong pressures of sexual selection. We are beginning to understand the ways in which vocal signals are produced and decoded by the nervous system and the roles of neurally active hormones in both processes.

Testing the evolutionary conservation of vocal motoneurons in vertebrates.

Medullary motoneurons drive vocalization in many vertebrate lineages including fish, amphibians, birds, and mammals. The developmental history of vocal motoneuron populations in each of these lineages remains largely unknown. The highly conserved transcription factor Paired-like Homeobox 2b (Phox2b) is presumed to be expressed in all vertebrate hindbrain branchial motoneurons, including laryngeal motoneurons essential for vocalization in humans. We used immunohistochemistry and in situ hybridization to examine Phox2b protein and mRNA expression in caudal hindbrain and rostral spinal cord motoneuron populations in seven species across five chordate classes. Phox2b was present in motoneurons dedicated to sound production in mice and frogs (bullfrog, African clawed frog), but not those in bird (zebra finch) or bony fish (midshipman, channel catfish). Overall, the pattern of caudal medullary motoneuron Phox2b expression was conserved across vertebrates and similar to expression in sea lamprey. These observations suggest that motoneurons dedicated to sound production in vertebrates are not derived from a single developmentally or evolutionarily conserved progenitor pool.

Functional specialization of male and female vocal motoneurons.

Vocal behaviors of African clawed frogs (Xenopus laevis) are produced by a single pair of muscles. This simplification, relative to other vertebrates, allows us to more easily determine how CNS motor pathways function to produce sex-specific songs. We describe here certain sexually differentiated properties of vocal motoneurons that are matched to male and female vocal demands. Both active and passive membrane properties differ between the sexes. Male motoneurons have lower input resistances and larger membrane capacitances than female motoneurons. Two distinct firing patterns are found, in different proportions, in males and females. The strongly adapting neurons that predominate in males initiate spikes at short, reliable latencies, whereas the weakly adapting motoneurons characteristic of females translate graded levels of depolarization into different firing rates. Low-threshold potassium currents (IKL) predominate in males. Hyperpolarization-activated cationic currents (IH) are found almost exclusively in males. Modeling results indicate that sex-typical active and passive properties can account for the occurrence of strongly and weakly adapting spike trains in the sexes. In particular, IKL seem to play an important role in determining the firing patterns of neurons. We suggest that these physiological differences facilitate transformation of synaptic inputs into male- and female-specific outputs that generate sexually distinct songs in vivo.