Everything in Modulation: Neuromodulators as Keys to Understanding Communication Dynamics.

Across the animal kingdom, the ability to produce communication signals appropriate to social encounters is essential, but how these behaviors are selected and adjusted in a context-dependent manner are poorly understood. This question can be addressed on many levels, including sensory processing by peripheral organs and the central nervous system, sensorimotor integration in decision-making brain regions, and motor circuit activation and modulation. Because neuromodulator systems act at each of these levels, they are a useful lens through which to explore the mechanisms underlying complex patterns of communication. It has been clear for decades that understanding the logic of input-output decision making by the nervous system requires far more than simply identifying the connections linking sensory organs to motor circuits; this is due in part to the fact that neuromodulators can promote distinct and temporally dynamic responses to similar signals. We focus on the vocal circuit dynamics of Xenopus frogs, and describe complementary examples from diverse vertebrate communication systems. While much remains to be discovered about how neuromodulators direct flexibility in communication behaviors, these examples illustrate that several neuromodulators can act upon the same circuit at multiple levels of control, and that the functional consequence of neuromodulation can depend on species-specific factors as well as dynamic organismal characteristics like internal state.

Molecular characterization of frog vocal neurons using constellation pharmacology.

Identification and characterization of neuronal cell classes in motor circuits are essential for understanding the neural basis of behavior. It is a challenging task, especially in a non-genetic-model organism, to identify cell-specific expression of functional macromolecules. Here, we performed constellation pharmacology, calcium imaging of dissociated neurons to pharmacologically identify functional receptors expressed by vocal neurons in adult male and female African clawed frogs, Xenopus laevis. Previously we identified a population of vocal neurons called fast trill neurons (FTNs) in the amphibian parabrachial nucleus (PB) that express N-methyl-d-aspartate (NMDA) receptors and GABA and/or glycine receptors. Using constellation pharmacology, we identified four cell classes of putative fast trill neurons (pFTNs, responsive to both NMDA and GABA/glycine applications). We discovered that some pFTNs responded to the application of substance P (SP), acetylcholine (ACh), or both. Electrophysiological recordings obtained from FTNs using an ex vivo preparation verified that SP and/or ACh depolarize FTNs. Bilateral injection of ACh, SP, or their antagonists into PBs showed that ACh receptors are not sufficient but necessary for vocal production, and SP receptors play a role in shaping the morphology of vocalizations. Additionally, we discovered that the PB of adult female X. laevis also contains all the subclasses of neurons at a similar frequency as in males, despite their sexually distinct vocalizations. These results reveal novel neuromodulators that regulate X. laevis vocal production and demonstrate the power of constellation pharmacology in identifying the neuronal subtypes marked by functional expression of cell-specific receptors in non-genetic-model organisms.NEW & NOTEWORTHY Molecular profiles of neurons are critical for understanding the neuronal functions, but their identification is challenging especially in non-genetic-model organisms. Here, we characterized the functional expression of membrane macromolecules in vocal neurons of African clawed frogs, Xenopus laevis, using a technique called constellation pharmacology. We discovered that receptors for acetylcholine and/or substance P are expressed by some classes of vocal neurons, and their activation plays a role in the production of normal vocalizations.

Inspiring song: The role of respiratory circuitry in the evolution of vertebrate vocal behavior.

Vocalization is a common means of communication across vertebrates, but the evolutionary origins of the neural circuits controlling these behaviors are not clear. Peripheral mechanisms of sound production vary widely: fish produce sounds with a swimbladder or pectoral fins; amphibians, reptiles, and mammalians vocalize using a larynx; birds vocalize with a syrinx. Despite the diversity of vocal effectors across taxa, there are many similarities in the neural circuits underlying the control of these organs. Do similarities in vocal circuit structure and function indicate that vocal behaviors first arose in a single common ancestor, or have similar neural circuits arisen independently multiple times during evolution? In this review, we describe the hindbrain circuits that are involved in vocal production across vertebrates. Given that vocalization depends on respiration in most tetrapods, it is not surprising that vocal and respiratory hindbrain circuits across distantly related species are anatomically intermingled and functionally linked. Such vocal-respiratory circuit integration supports the hypothesis that vocal evolution involved the expansion and functional diversification of breathing circuits. Recent phylogenetic analyses, however, suggest vocal behaviors arose independently in all major tetrapod clades, indicating that similarities in vocal control circuits are the result of repeated co-options of respiratory circuits in each lineage. It is currently unknown whether vocal circuits across taxa are made up of homologous neurons, or whether vocal neurons in each lineage arose from developmentally and evolutionarily distinct progenitors. Integrative comparative studies of vocal neurons across brain regions and taxa will be required to distinguish between these two scenarios.

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.

Feedback to the future: motor neuron contributions to central pattern generator function.

Motor behaviors depend on neural signals in the brain. Regardless of where in the brain behavior patterns arise, the central nervous system sends projections to motor neurons, which in turn project to and control temporally appropriate muscle contractions; thus, motor neurons are traditionally considered the last relay from the central nervous system to muscles. However, in an array of species and motor systems, an accumulating body of evidence supports a more complex role of motor neurons in pattern generation. These studies suggest that motor neurons not only relay motor patterns to the periphery, but directly contribute to pattern generation by providing feedback to upstream circuitry. In spinal and hindbrain circuits in a variety of animals - including flies, worms, leeches, crustaceans, rodents, birds, fish, amphibians and mammals - studies have indicated a crucial role for motor neuron feedback in maintaining normal behavior patterns dictated by the activity of a central pattern generator. Hence, in this Review, we discuss literature examining the role of motor neuron feedback across many taxa and behaviors, and set out to determine the prevalence of motor neuron participation in motor circuits.

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.

Motor Neurons Tune Premotor Activity in a Vertebrate Central Pattern Generator.

Central patterns generators (CPGs) are neural circuits that drive rhythmic motor output without sensory feedback. Vertebrate CPGs are generally believed to operate in a top-down manner in which premotor interneurons activate motor neurons that in turn drive muscles. In contrast, the frog (Xenopus laevis) vocal CPG contains a functionally unexplored neuronal projection from the motor nucleus to the premotor nucleus, indicating a recurrent pathway that may contribute to rhythm generation. In this study, we characterized the function of this bottom-up connection. The X. laevis vocal CPG produces a 50-60 Hz "fast trill" song used by males during courtship. We recorded "fictive vocalizations" in the in vitro CPG from the laryngeal nerve while simultaneously recording premotor activity at the population and single-cell level. We show that transecting the motor-to-premotor projection eliminated the characteristic firing rate of premotor neurons. Silencing motor neurons with the intracellular sodium channel blocker QX-314 also disrupted premotor rhythms, as did blockade of nicotinic synapses in the motor nucleus (the putative location of motor neuron-to-interneuron connections). Electrically stimulating the laryngeal nerve elicited primarily IPSPs in premotor neurons that could be blocked by a nicotinic receptor antagonist. Our results indicate that an inhibitory signal, activated by motor neurons, is required for proper CPG function. To our knowledge, these findings represent the first example of a CPG in which precise premotor rhythms are tuned by motor neuron activity.SIGNIFICANCE STATEMENT Central pattern generators (CPGs) are neural circuits that produce rhythmic behaviors. In vertebrates, motor neurons are not commonly known to contribute to CPG function, with the exception of a few spinal circuits where the functional significance of motor neuron feedback is still poorly understood. The frog hindbrain vocal circuit contains a previously unexplored connection from the motor to premotor region. Our results indicate that motor neurons activate this bottom-up connection, and blocking this signal eliminates normal premotor activity. These findings may promote increased awareness of potential involvement of motor neurons in a wider range of CPGs, perhaps clarifying our understanding of network principles underlying motor behaviors in numerous organisms, including humans.

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.

Coding rate and duration of vocalizations of the frog, Xenopus laevis.

Vocalizations involve complex rhythmic motor patterns, but the underlying temporal coding mechanisms in the nervous system are poorly understood. Using a recently developed whole-brain preparation from which "fictive" vocalizations are readily elicited in vitro, we investigated the cellular basis of temporal complexity of African clawed frogs (Xenopus laevis). Male advertisement calls contain two alternating components--fast trills (∼300 ms) and slow trills (∼700 ms) that contain clicks repeated at ∼60 and ∼30 Hz, respectively. We found that males can alter the duration of fast trills without changing click rates. This finding led us to hypothesize that call rate and duration are regulated by independent mechanisms. We tested this by obtaining whole-cell patch-clamp recordings in the "fictively" calling isolated brain. We discovered a single type of premotor neuron with activity patterns correlated with both the rate and duration of fast trills. These "fast-trill neurons" (FTNs) exhibited long-lasting depolarizations (LLDs) correlated with each fast trill and action potentials that were phase-locked with motor output-neural correlates of call duration and rate, respectively. When depolarized without central pattern generator activation, FTNs produced subthreshold oscillations and action potentials at fast-trill rates, indicating FTN resonance properties are tuned to, and may dictate, the fast-trill rhythm. NMDA receptor (NMDAR) blockade eliminated LLDs in FTNs, and NMDAR activation in synaptically isolated FTNs induced repetitive LLDs. These results suggest FTNs contain an NMDAR-dependent mechanism that may regulate fast-trill duration. We conclude that a single premotor neuron population employs distinct mechanisms to regulate call rate and duration.

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.

Vocal pathway degradation in gonadectomized Xenopus laevis adults.

Reproductive behaviors of many vertebrate species are activated in adult males by elevated androgen levels and abolished by castration. Neural and muscular components controlling these behaviors contain numerous hormone-sensitive sites including motor initiation centers (such as the basal ganglia), central pattern generators (CPGs), and muscles; therefore it is difficult to confirm the role of each hormone-activated target using behavioral assays alone. Our goal was to address this issue by determining the site of androgen-induced vocal activation using male Xenopus laevis, a species in which androgen dependence of vocal activation has been previously determined. We compared in vivo calling patterns and functionality of two in vitro preparations-the isolated larynx and an isolated brain from which fictive courtship vocalizations can be evoked--in castrated and control males. The isolated larynx allowed us to test whether castrated males were capable of transducing male-typical nerve signals into vocalizations and the fictively vocalizing brain preparation allowed us to directly examine vocal CPG function separate from the issue of vocal initiation. The results indicate that all three components--vocal initiation, CPG, and larynx--require intact gonads. Vocal production decreased dramatically in castrates and laryngeal contractile properties of castrated males were demasculinized, whereas no changes were observed in control animals. In addition, fictive calls of castrates were degraded compared with those of controls. To our knowledge, this finding represents the first demonstration of gonad-dependent maintenance of a CPG for courtship behavior in adulthood. Because previous studies showed that androgen-replacement can prevent castration-induced vocal impairments, we conclude that degradation of vocal initiation centers, larynx, and CPG function are most likely due to steroid hormone deprivation.

NMDAR-dependent control of call duration in Xenopus laevis.

Many rhythmic behaviors, such as locomotion and vocalization, involve temporally dynamic patterns. How does the brain generate temporal complexity? Here, we use the vocal central pattern generator (CPG) of Xenopus laevis to address this question. Isolated brains can elicit fictive vocalizations, allowing us to study the CPG in vitro. The X. laevis advertisement call is temporally modulated; calls consist of rhythmic click trills that alternate between fast (approximately 60 Hz) and slow (approximately 30 Hz) rates. We investigated the role of two CPG nuclei--the laryngeal motor nucleus (n.IX-X) and the dorsal tegmental area of the medulla (DTAM)--in setting rhythm frequency and call durations. We discovered a local field potential wave in DTAM that coincides with fictive fast trills and phasic activity that coincides with fictive clicks. After disrupting n.IX-X connections, the wave persists, whereas phasic activity disappears. Wave duration was temperature dependent and correlated with fictive fast trills. This correlation persisted when wave duration was modified by temperature manipulations. Selectively cooling DTAM, but not n.IX-X, lengthened fictive call and fast trill durations, whereas cooling either nucleus decelerated the fictive click rate. The N-methyl-d-aspartate receptor (NMDAR) antagonist dAPV blocked waves and fictive fast trills, suggesting that the wave controls fast trill activation and, consequently, call duration. We conclude that two functionally distinct CPG circuits exist: 1) a pattern generator in DTAM that determines call duration and 2) a rhythm generator (spanning DTAM and n.IX-X) that determines click rates. The newly identified DTAM pattern generator provides an excellent model for understanding NDMAR-dependent rhythmic circuits.

Sexually differentiated central pattern generators in Xenopus laevis.

Understanding the neural mechanisms that underlie the function of central pattern generators (CPGs) presents a formidable challenge requiring sophisticated tools and well-chosen model systems. In this article, we describe recent work on vocalizations of the African clawed frog Xenopus laevis. These behaviors are driven by sexually differentiated CPGs and are exceptionally well suited to this objective. In particular, a simplified mechanism of vocal production (independent of respiratory musculature) allows straightforward interpretations of nerve activity with respect to behavior. Furthermore, the development of a fictively vocalizing isolated brain, together with the finding of rapid androgen-induced masculinization of female vocalizations, provides an invaluable tool for determining how new behaviors arise from existing circuits.

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.

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.