Gekko gecko as a model organism for understanding aspects of laryngeal vocal evolution.

The ability to communicate through vocalization plays a key role in the survival of animals across all vertebrate groups. Although avian reptiles have received much attention relating to their stunning sound repertoire, non-avian reptiles have been wrongfully assumed to have less elaborate vocalization types, and little is known about the biomechanics of sound production and their underlying neural pathways in this group. We investigated alarm calls of Gekko gecko using audio and cineradiographic recordings. Acoustic analysis revealed three distinct call types: a sinusoidal call type (type 1); a train-like call type, characterized by distinct pulse trains (type 3); and an intermediate type, which showed both sinusoidal and pulse train components (type 2). Kinematic analysis of cineradiographic recordings showed that laryngeal movements differ significantly between respiratory and vocal behavior. During respiration, animals repeatedly moved their jaws to partially open their mouths, which was accompanied by small glottal movements. During vocalization, the glottis was pulled back, contrasting with what has previously been reported. In vitro retrograde tracing of the nerve innervating the laryngeal constrictor and dilator muscles revealed round to fusiform motoneurons in the hindbrain-spinal cord transition ipsilateral to the labeled nerve. Taken together, our observations provide insight into the alarm calls generated by G. gecko, the biomechanics of this sound generation and the underlying organization of motoneurons involved in the generation of vocalizations. Our observations suggest that G. gecko may be an excellent non-avian reptile model organism for enhancing our understanding of the evolution of vertebrate vocalization.

The Dorsal Part of the Anterior Tuberal Nucleus Responds to Auditory Stimulation in Zebrafish (Danio rerio).

The zebrafish, a widely used model in neurobiology, relies on hearing in aquatic environments. Unfortunately, its auditory pathways have mainly been studied in larvae. In this study, we examined the involvement of the anterior tuberal nucleus (AT) in auditory processing in adult zebrafish. Our tract-tracing experiments revealed that the dorsal subdivision of AT is strongly bidirectionally connected to the central nucleus of the torus semicircularis (TSc), a major auditory nucleus in fishes. Immunohistochemical visualization of the ribosomal protein S6 (pS6) phosphorylation to map neural activity in response to auditory stimulation substantiated this finding: the dorsal but not the ventral part of AT responded strongly to auditory stimulation. A similar response to auditory stimulation was present in the TSc but not in the nucleus isthmi, a visual region, which we used as a control for testing if the pS6 activation was specific to the auditory stimulation. We also measured the time course of pS6 phosphorylation, which was previously unreported in teleost fish. After auditory stimulation, we found that pS6 phosphorylation peaked between 100 and 130 min and returned to baseline levels after 190 min. This information will be valuable for the design of future pS6 experiments. Our results suggest an anatomical and functional subdivision of AT, where only the dorsal part connects to the auditory network and processes auditory information.

Timing and precision of rattlesnake spinal motoneurons are determined by the KV72/3 potassium channel.

The evolution of novel motor behaviors requires modifications in the central pattern generators (CPGs) controlling muscle activity. How such changes gradually lead to novel behaviors remains enigmatic due to the long time course of evolution. Rattlesnakes provide a unique opportunity to investigate how a locomotor CPG was evolutionarily modified to generate a novel behavior-in this case, acoustic signaling. We show that motoneurons (MNs) in the body and tail spinal cord of rattlesnakes possess fundamentally different physiological characteristics, which allow MNs in the tail to integrate and transmit CPG output for controlling superfast muscles with high temporal precision. Using patch-clamp electrophysiology, we demonstrate that these differences in locomotor and rattle MNs are mainly determined by KV72/3 potassium channels. However, although KV72/3 exerted a significantly different influence on locomotor and rattle MN physiology, single-cell RNA-seq unexpectedly did not reveal any differences in KV72/3 channels' expression. VIDEO ABSTRACT.

Vocal and Electric Fish: Revisiting a Comparison of Two Teleost Models in the Neuroethology of Social Behavior.

The communication behaviors of vocal fish and electric fish are among the vertebrate social behaviors best understood at the level of neural circuits. Both forms of signaling rely on midbrain inputs to hindbrain pattern generators that activate peripheral effectors (sonic muscles and electrocytes) to produce pulsatile signals that are modulated by frequency/repetition rate, amplitude and call duration. To generate signals that vary by sex, male phenotype, and social context, these circuits are responsive to a wide range of hormones and neuromodulators acting on different timescales at multiple loci. Bass and Zakon (2005) reviewed the behavioral neuroendocrinology of these two teleost groups, comparing how the regulation of their communication systems have both converged and diverged during their parallel evolution. Here, we revisit this comparison and review the complementary developments over the past 16 years. We (a) summarize recent work that expands our knowledge of the neural circuits underlying these two communication systems, (b) review parallel studies on the action of neuromodulators (e.g., serotonin, AVT, melatonin), brain steroidogenesis (via aromatase), and social stimuli on the output of these circuits, (c) highlight recent transcriptomic studies that illustrate how contemporary molecular methods have elucidated the genetic regulation of social behavior in these fish, and (d) describe recent studies of mochokid catfish, which use both vocal and electric communication, and that use both vocal and electric communication and consider how these two systems are spliced together in the same species. Finally, we offer avenues for future research to further probe how similarities and differences between these two communication systems emerge over ontogeny and evolution.

Frequency modulation of rattlesnake acoustic display affects acoustic distance perception in humans.

The estimation of one's distance to a potential threat is essential for any animal's survival. Rattlesnakes inform about their presence by generating acoustic broadband rattling sounds.1 Rattlesnakes generate their acoustic signals by clashing a series of keratinous segments onto each other, which are located at the tip of their tails.1-3 Each tail shake results in a broadband sound pulse that merges into a continuous acoustic signal with fast-repeating tail shakes. This acoustic display is readily recognized by other animals4,5 and serves as an aposematic threat and warning display, likely to avoid being preyed upon.1,6 The spectral properties of the rattling sound1,3 and its dependence on the morphology and size of the rattle have been investigated for decades7-9 and carry relevant information for different receivers, including ground squirrels that encounter rattlesnakes regularly.10,11 Combining visual looming stimuli with acoustic measurements, we show that rattlesnakes increase their rattling rate (up to about 40 Hz) with decreasing distance of a potential threat, reminiscent of the acoustic signals of sensors while parking a car. Rattlesnakes then abruptly switch to a higher and less variable rate of 60-100 Hz. In a virtual reality experiment, we show that this behavior systematically affects distance judgments by humans: the abrupt switch in rattling rate generates a sudden, strong percept of decreased distance which, together with the low-frequency rattling, acts as a remarkable interspecies communication signal. VIDEO ABSTRACT.

Sound production in piranhas is associated with modifications of the spinal locomotor pattern.

In piranhas, sounds are produced through the vibration of the swim bladder wall caused by the contraction of bilateral sonic muscles. Because they are solely innervated by spinal nerves, these muscles likely evolved from the locomotor hypaxial musculature. The transition from a neuromuscular system initially shaped for slow movements (locomotion) to a system that requires a high contraction rate (sound production) was accompanied with major peripheral structural modifications, yet the associated neural adjustments remain to this date unclear. To close this gap, we investigated the activity of both the locomotor and the sonic musculature using electromyography. The comparison between the activation patterns of both systems highlighted modifications of the neural motor pathway: (1) a transition from a bilateral alternating pattern to a synchronous activation pattern, (2) a switch from a slow- to a high-frequency regime, and (3) an increase in the synchrony of motor neuron activation. Furthermore, our results demonstrate that sound features correspond to the activity of the sonic muscles, as both the variation patterns of periods and amplitudes of sounds highly correspond to those seen in the sonic muscle electromyograms (EMGsonic). Assuming that the premotor network for sound production in piranhas is of spinal origin, our results show that the neural circuit associated with spinal motor neurons transitioned from the slow alternating pattern originally used for locomotion to a much faster simultaneous activation pattern to generate vocal signals.

Gap junction-mediated glycinergic inhibition ensures precise temporal patterning in vocal behavior.

Precise neuronal firing is especially important for behaviors highly dependent on the correct sequencing and timing of muscle activity patterns, such as acoustic signaling. Acoustic signaling is an important communication modality for vertebrates, including many teleost fishes. Toadfishes are well known to exhibit high temporal fidelity in synchronous motoneuron firing within a hindbrain network directly determining the temporal structure of natural calls. Here, we investigated how these motoneurons maintain synchronous activation. We show that pronounced temporal precision in population-level motoneuronal firing depends on gap junction-mediated, glycinergic inhibition that generates a period of reduced probability of motoneuron activation. Super-resolution microscopy confirms glycinergic release sites formed by a subset of adjacent premotoneurons contacting motoneuron somata and dendrites. In aggregate, the evidence supports the hypothesis that gap junction-mediated, glycinergic inhibition provides a timing mechanism for achieving synchrony and temporal precision in the millisecond range for rapid modulation of acoustic waveforms.

Avoiding being stung or bitten - prey capture behaviors of the ant-eating Texas horned lizard (Phrynosoma cornutum).

Horned lizards (Phrynosoma) are specialized predators, including many species that primarily feed on seed harvester ants (Pogonomyrmex). Harvester ants have strong mandibles to husk seeds or defensively bite, and a venomous sting. Texas horned lizards possess a blood plasma factor that neutralizes harvester ant venom and produce copious mucus in the pharynx and esophagus, thus embedding and incapacitating swallowed ants. We used high-speed video recordings to investigate complexities of their lingual prey capture and handling behavior. Lizards primarily strike ants at their mesosoma (thorax plus propodeum of abdomen). They avoid the head and gaster, even if closer to the lizard, and if prey directional movement is reversed. Orientation of captured ants during retraction is with head first (rostral), thus providing initial mucus coating of the mandibles. Prey capture accuracy and precise handling illustrates the specificity of adaptations of horned lizards in avoiding harm, and the challenges lizards face when feeding on dangerous prey.

Morphological diversity of acoustic and electric communication systems of mochokid catfish.

Mochokid catfish offer a distinct opportunity to study a communication system transitioning to a new signaling channel because some produce sounds and others electric discharges. Both signals are generated using an elastic spring system (ESS), which includes a protractor muscle innervated by motoneurons within the protractor nucleus that also has a motoneuron afferent population. Synodontis grandiops and S. nigriventris produce sounds and electric discharges, respectively, and their ESSs show several morphological and physiological differences. The extent to which these differences explain different signal types remains unclear. Here, we compare ESS morphologies and behavioral phenotypes among five mochokids. S. grandiops and S. nigriventris were compared with Synodontis eupterus that is known to produce both signal types, and representative members of two sister genera, Microsynodontis cf. batesii and Mochokiella paynei, for which no data were available. We provide support for the hypothesis that peripheral and central components of the ESS are conserved among mochokids. We also show that the two nonsynodontids are only sonic, consistent with sound production being an ancestral character for mochokids. Even though the three sound producing-only species differ in some ESS characters, several are similar and likely associated with only sound production. We propose that the ability of S. eupterus to generate both electric discharges and sounds may depend on a protractor muscle intermediate in morphology between sound producing-only and electric discharge-only species, and two separate populations of protractor motoneurons. Our results further suggest that an electrogenic ESS in synodontids is an exaptation of a sound producing ESS.

Neuroanatomical and neurophysiological mechanisms of acoustic and weakly electric signaling in synodontid catfish.

To what extent do modifications in the nervous system and peripheral effectors contribute to novel behaviors? Using a combination of morphometric analysis, neuroanatomical tract-tracing, and intracellular neuronal recording, we address this question in a sound-producing and a weakly electric species of synodontid catfish, Synodontis grandiops, and Synodontis nigriventris, respectively. The same peripheral mechanism, a bilateral pair of protractor muscles associated with vertebral processes (elastic spring mechanism), is involved in both signaling systems. Although there were dramatic species differences in several morphometric measures, electromyograms provided strong evidence that simultaneous activation of paired protractor muscles accounts for an individual sound and electric discharge pulse. While the general architecture of the neural network and the intrinsic properties of the motoneuron population driving each target was largely similar, differences could contribute to species-specific patterns in electromyograms and the associated pulse repetition rate of sounds and electric discharges. Together, the results suggest that adaptive changes in both peripheral and central characters underlie the transition from an ancestral sound to a derived electric discharge producing system, and thus the evolution of a novel communication channel among synodontid catfish. Similarities with characters in other sonic and weakly electric teleost fish provide a striking example of convergent evolution in functional adaptations underlying the evolution of the two signaling systems among distantly related taxa.

Serotonin systems in three socially communicating teleost species, the grunting toadfish (Allenbatrachus grunniens), a South American marine catfish (Ariopsis seemanni), and the upside-down catfish (Synodontis nigriventris).

We investigated immunohistochemically the distribution of serotonergic cell populations in three teleost species (one toadfish, Allenbatrachus grunniens, and two catfishes, Synodontis nigriventris and Ariopsis seemanni). All three species exhibited large populations of 5-HT positive neurons in the paraventricular organ (PVO) and the dorsal (Hd) and caudal (Hc) periventricular hypothalamic zones, plus a smaller one in the periventricular pretectum, a few cells in the pineal stalk, and - only in catfishes - in the preoptic region. Furthermore, the rhombencephalic superior and inferior raphe always contained ample serotonergic cells. In each species, a neuronal mass extended into the hypothalamic lateral recess. Only in the toadfish, did this intraventricular structure contain serotonergic cells and arise from Hd, whereas in the catfishes it emerged from medially and represents the dorsal tuberal nucleus seen in other catfishes as well. Serotonergic cells in PVO, Hd and Hc were liquor-contacting. Those of the PVO extended into the midline area of the periventricular posterior tubercular nucleus in both catfishes. Dopaminergic, liquor-contacting neurons were additionally investigated using an antibody against tyrosine hydroxylase (TH) in S. nigriventris showing that TH was never co-localized with serotonin. Because TH antibodies are known to reveal mostly or only the TH1 enzyme, we hypothesize that th1-expressing dopamine cells (unlike th2-expressing ones) do not co-localize with serotonin. Since the three investigated species engage in social communication using swim bladder associated musculature, we investigated the serotonergic innervation of the hindbrain vocal or electromotor nuclei initiating the social signal. We found in all three species serotonergic fibers seemingly originating from close-by serotonergic neurons of inferior raphe or anterior spinal cord. Minor differences appear to be rather species-specific than dependent on the type of social communication.

A New Perspective on Predictive Motor Signaling.

Adaptive behavior relies on complex neural processing in multiple interacting networks of both motor and sensory systems. One such interaction employs intrinsic neuronal signals, so-called 'corollary discharge' or 'efference copy', that may be used to predict the sensory consequences of a specific behavioral action, thereby enabling self-generated (reafferent) sensory information and extrinsic (exafferent) sensory inflow to be dissociated. Here, by using well-established examples, we seek to identify the distinguishing features of corollary discharge and efference copy within the framework of predictive motor-to-sensory system coordination. We then extend the more general concept of predictive signaling by showing how neural replicas of a particular motor command not only inform sensory pathways in order to gate reafferent stimulation, but can also be used to directly coordinate distinct and otherwise independent behaviors to the original motor task. Moreover, this motor-to-motor pairing may additionally extend to a gating of sensory input to either or both of the coupled systems. The employment of predictive internal signaling in such motor systems coupling and remote sensory input control thus adds to our understanding of how an organism's central nervous system is able to coordinate the activity of multiple and generally disparate motor and sensory circuits in the production of effective behavior.

Inhibitory and modulatory inputs to the vocal central pattern generator of a teleost fish.

Vocalization is a behavioral feature that is shared among multiple vertebrate lineages, including fish. The temporal patterning of vocal communication signals is set, in part, by central pattern generators (CPGs). Toadfishes are well-established models for CPG coding of vocalization at the hindbrain level. The vocal CPG comprises three topographically separate nuclei: pre-pacemaker, pacemaker, motor. While the connectivity between these nuclei is well understood, their neurochemical profile remains largely unexplored. The highly vocal Gulf toadfish, Opsanus beta, has been the subject of previous behavioral, neuroanatomical and neurophysiological studies. Combining transneuronal neurobiotin-labeling with immunohistochemistry, we map the distribution of inhibitory neurotransmitters and neuromodulators along with gap junctions in the vocal CPG of this species. Dense GABAergic and glycinergic label is found throughout the CPG, with labeled somata immediately adjacent to or within CPG nuclei, including a distinct subset of pacemaker neurons co-labeled with neurobiotin and glycine. Neurobiotin-labeled motor and pacemaker neurons are densely co-labeled with the gap junction protein connexin 35/36, supporting the hypothesis that transneuronal neurobiotin-labeling occurs, at least in part, via gap junction coupling. Serotonergic and catecholaminergic label is also robust within the entire vocal CPG, with additional cholinergic label in pacemaker and prepacemaker nuclei. Likely sources of these putative modulatory inputs are neurons within or immediately adjacent to vocal CPG neurons. Together with prior neurophysiological investigations, the results reveal potential mechanisms for generating multiple classes of social context-dependent vocalizations with widely divergent temporal and spectral properties.

Sensing External and Self-Motion with Hair Cells: A Comparison of the Lateral Line and Vestibular Systems from a Developmental and Evolutionary Perspective.

Detection of motion is a feature essential to any living animal. In vertebrates, mechanosensory hair cells organized into the lateral line and vestibular systems are used to detect external water or head/body motion, respectively. While the neuronal components to detect these physical attributes are similar between the two sensory systems, the organizational pattern of the receptors in the periphery and the distribution of hindbrain afferent and efferent projections are adapted to the specific functions of the respective system. Here we provide a concise review comparing the functional organization of the vestibular and lateral line systems from the development of the organs to the wiring from the periphery and the first processing stages. The goal of this review is to highlight the similarities and differences to demonstrate how evolution caused a common neuronal substrate to adapt to different functions, one for the detection of external water stimuli and the generation of sensory maps and the other for the detection of self-motion and the generation of motor commands for immediate behavioral reactions.

Moving or being moved: that makes a difference.

During head/body movements, gaze stability is ensured by transformation of motion-related sensory signals into respective motor commands. Passively induced motion in all vertebrates including amphibians evokes a robust vestibulo-ocular reflex, suggesting an equally important role of this motor reaction during actively induced motion. However, during self-induced movements including locomotion, motor efference copies offer a convenient additional substrate for counteracting retinal image displacements. During such locomotor activity in Xenopus laevis tadpoles, spinal central pattern generator-derived efference copies elicit spatio-temporally specific eye movements, which are functionally appropriate to offset swimming-related retinal image displacements. In addition, passively induced horizontal semicircular canal signals are suppressed, making intrinsic spino-extraocular motor coupling the dominating mechanism for gaze stabilization during locomotion. The presence of functionally appropriate efference copy-driven eye movements in adult frogs with limb-based locomotion suggests that this mechanism might play a role for gaze stability during rhythmic locomotion also in other vertebrates.

Ontogenetic Development of Vestibulo-Ocular Reflexes in Amphibians.

Vestibulo-ocular reflexes (VOR) ensure gaze stability during locomotion and passively induced head/body movements. In precocial vertebrates such as amphibians, vestibular reflexes are required very early at the onset of locomotor activity. While the formation of inner ears and the assembly of sensory-motor pathways is largely completed soon after hatching, angular and translational/tilt VOR display differential functional onsets and mature with different time courses. Otolith-derived eye movements appear immediately after hatching, whereas the appearance and progressive amelioration of semicircular canal-evoked eye movements is delayed and dependent on the acquisition of sufficiently large semicircular canal diameters. Moreover, semicircular canal functionality is also required to tune the initially omnidirectional otolith-derived VOR. The tuning is due to a reinforcement of those vestibulo-ocular connections that are co-activated by semicircular canal and otolith inputs during natural head/body motion. This suggests that molecular mechanisms initially guide the basic ontogenetic wiring, whereas semicircular canal-dependent activity is required to establish the spatio-temporal specificity of the reflex. While a robust VOR is activated during passive head/body movements, locomotor efference copies provide the major source for compensatory eye movements during tail- and limb-based swimming of larval and adult frogs. The integration of active/passive motion-related signals for gaze stabilization occurs in central vestibular neurons that are arranged as segmentally iterated functional groups along rhombomere 1-8. However, at variance with the topographic maps of most other sensory systems, the sensory-motor transformation of motion-related signals occurs in segmentally specific neuronal groups defined by the extraocular motor output targets.

Galvanic Vestibular Stimulation: Cellular Substrates and Response Patterns of Neurons in the Vestibulo-Ocular Network.

UNLABELLED: Galvanic vestibular stimulation (GVS) uses modulated currents to evoke neuronal activity in vestibular endorgans in the absence of head motion. GVS is typically used for a characterization of vestibular pathologies; for studies on the vestibular influence of gaze, posture, and locomotion; and for deciphering the sensory-motor transformation underlying these behaviors. At variance with the widespread use of this method, basic aspects such as the activated cellular substrate at the sensory periphery or the comparability to motion-induced neuronal activity patterns are still disputed. Using semi-intact preparations of Xenopus laevis tadpoles, we determined the cellular substrate and the spatiotemporal specificity of GVS-evoked responses and compared sinusoidal GVS-induced activity patterns with motion-induced responses in all neuronal elements along the vestibulo-ocular pathway. As main result, we found that, despite the pharmacological block of glutamatergic hair cell transmission by combined bath-application of NMDA (7-chloro-kynurenic acid) and AMPA (CNQX) receptor blockers, GVS-induced afferent spike activity persisted. However, the amplitude modulation was reduced by ∼30%, suggesting that both hair cells and vestibular afferent fibers are normally recruited by GVS. Systematic alterations of electrode placement with respect to bilateral semicircular canal pairs or alterations of the bipolar stimulus phase timing yielded unique activity patterns in extraocular motor nerves, compatible with a spatially and temporally specific activation of vestibulo-ocular reflexes in distinct planes. Despite the different GVS electrode placement in semi-intact X. laevis preparations and humans and the more global activation of vestibular endorgans by the latter approach, this method is suitable to imitate head/body motion in both circumstances. SIGNIFICANCE STATEMENT: Galvanic vestibular stimulation is used frequently in clinical practice to test the functionality of the sense of balance. The outcome of the test that relies on the activation of eye movements by electrical stimulation of vestibular organs in the inner ear helps to dissociate vestibular impairments that cause vertigo and imbalance in patients. This study uses an amphibian model to investigate at the cellular level the underlying mechanism on which this method depends. The outcome of this translational research unequivocally revealed the cellular substrate at the vestibular sensory periphery that is activated by electrical currents, as well as the spatiotemporal specificity of the evoked eye movements, thus facilitating the interpretation of clinical test results.

Spinal corollary discharge modulates motion sensing during vertebrate locomotion.

During active movements, neural replicas of the underlying motor commands may assist in adapting motion-detecting sensory systems to an animal's own behaviour. The transmission of such motor efference copies to the mechanosensory periphery offers a potential predictive substrate for diminishing sensory responsiveness to self-motion during vertebrate locomotion. Here, using semi-isolated in vitro preparations of larval Xenopus, we demonstrate that shared efferent neural pathways to hair cells of vestibular endorgans and lateral line neuromasts express cyclic impulse bursts during swimming that are directly driven by spinal locomotor circuitry. Despite common efferent innervation and discharge patterns, afferent signal encoding at the two mechanosensory peripheries is influenced differentially by efference copy signals, reflecting the different organization of body/water motion-detecting processes in the vestibular and lateral line systems. The resultant overall gain reduction in sensory signal encoding in both cases, which likely prevents overstimulation, constitutes an adjustment to increased stimulus magnitudes during locomotion.

Locomotor corollary activation of trigeminal motoneurons: coupling of discrete motor behaviors.

During motor behavior, corollary discharges of the underlying motor commands inform sensory-motor systems about impending or ongoing movements. These signals generally limit the impact of self-generated sensory stimuli but also induce motor reactions that stabilize sensory perception. Here, we demonstrate in isolated preparations of Xenopus laevis tadpoles that locomotor corollary discharge provokes a retraction of the mechanoreceptive tentacles during fictive swimming. In the absence of sensory feedback, these signals activate a cluster of trigeminal motoneurons that cause a contraction of the tentacle muscle. This corollary discharge encodes duration and strength of locomotor activity, thereby ensuring a reliable coupling between locomotion and tentacle motion. The strict phase coupling between the trigeminal and spinal motor activity, present in many cases, suggests that the respective corollary discharge is causally related to the ongoing locomotor output and derives at least in part from the spinal central pattern generator; however, additional contributions from midbrain and/or hindbrain locomotor centers are likely. The swimming-related retraction might protect the touch-receptive Merkel cells on the tentacle from sensory over-stimulation and damage and/or reduce the hydrodynamic drag. The intrinsic nature of the coupling of tentacle retraction to locomotion is an excellent example of a context-dependent, direct link between otherwise discrete motor behaviors.

Vocal behavior and vocal central pattern generator organization diverge among toadfishes.

Among fishes, acoustic communication is best studied in toadfishes, a single order and family that includes species commonly known as toadfish and midshipman. However, there is a lack of comparative anatomical and physiological studies, making it difficult to identify both shared and derived mechanisms of vocalization among toadfishes. Here, vocal nerve labeling and intracellular in vivo recording and staining delineated the hindbrain vocal network of the Gulf toadfish Opsanus beta. Dextran-biotin labeling of the vocal nerve or intracellular neurobiotin fills of motoneurons delineated a midline vocal motor nucleus (VMN). Motoneurons showed bilaterally extensive dendritic arbors both within and lateral to the paired motor nuclei. The motoneuron activity matched that of the spike-like vocal nerve motor volley that determines the natural call duration and frequency. Ipsilateral vocal nerve labeling with biocytin or neurobiotin yielded dense bilateral transneuronal filling of motoneurons and coextensive columns of premotor neurons. These premotor neurons generated pacemaker-like action potentials matched 1:1 with vocal nerve and motoneuron firing. Transneuronal transport further revealed connectivity within and between the pacemaker-motor circuit and a rostral prepacemaker nucleus. Unlike the pacemaker-motor circuit, prepacemaker firing did not match the frequency of vocal nerve activity but instead was predictive of the duration of the vocal nerve volley that codes for call duration. Transneuronally labeled terminal-like boutons also occurred in auditory-recipient hindbrain nuclei, including neurons innervating the inner ear and lateral line organs. Together with studies of midshipman, we propose that separate premotor populations coding vocal frequency and duration with direct premotor coupling to auditory-lateral line nuclei are plesiomorphic characters for toadfishes. Unlike in midshipman, transneuronal labeling in toadfishes reveals an expansive column of pacemaker neurons that is weakly coupled to prepacemaker neurons, a character that likely depends on the extent of gap junction coupling. We propose that these and other anatomical characters contribute to neurophysiological properties that, in turn, sculpt the species-typical patterning of frequency and amplitude-modulated vocalizations.