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Flexible control of pectoral appendages enables motor behaviors of vastly different strength, speed, and amplitude, as in a human playing the piano or throwing a ball. Such control necessitates a fine-tuned, coordinated activation of motoneurons, which is facilitated by spatially ordered motoneuron pools in mammals. While differently sized neurons are known to contribute to different strengths of pectoral movements, it remains unclear how these pectoral motor pools are organized in less complex pectoral systems as those of teleost fish. We show how pectoral motor control can be extended to increase the speed- and amplitude-range of motor behaviors by investigating anatomical and physiological features of pectoral motoneurons and the motor pools they form in freshwater hatchet fish, well-known for their pectoral aerial escape response. Through the differentiation of one motor pool, the pectoral motor network of hatchet fish acquired additional flexibility to enable specific control of vastly different amplitudes, velocities, and strengths. Similar neuronal organization patterns have been described for controlling fast, intermediate, and slow axial muscles in zebrafish and in tetrapod motor systems controlling pectoral limbs. We show that hatchet fish share organizational principles of their pectoral motor pools with those found in other motor networks in both teleosts and tetrapods. Our data thus suggest that principles of spatial and physiological differentiation of motor pools associated with different pectoral muscles and behaviors might be deeply homologous between actinopterygian and sarcopterygian vertebrates., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Corrales Parada et al.)

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 KV7 2/3 potassium channels. However, although KV7 2/3 exerted a significantly different influence on locomotor and rattle MN physiology, single-cell RNA-seq unexpectedly did not reveal any differences in KV7 2/3 channels' expression. VIDEO ABSTRACT., Competing Interests: Declaration of interests The authors declare no conflict of interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Autism spectrum disorder (ASD) is a persistent neurodevelopmental condition characterized by two core behavioral symptoms: impaired social communication and interaction, as well as stereotypic, repetitive behavior. No distinct cause of ASD is known so far; however, excitatory/inhibitory imbalance and a disturbed serotoninergic transmission have been identified as prominent candidates responsible for ASD etiology., Methods: The GABA B receptor agonist R-Baclofen and the selective agonist for the 5HT 7 serotonin receptor LP-211 have been reported to correct social deficits and repetitive behaviors in mouse models of ASD. To evaluate the efficacy of these compounds in more details, we treated BTBR T + Itpr3 tf /J and B6.129P2- Fmr1 tm 1 Cgr / J mice acutely with R-Baclofen or LP-211 and evaluated the behavior of animals in a series of tests., Results: BTBR mice showed motor deficits, elevated anxiety, and highly repetitive behavior of self-grooming. Fmr1 -KO mice exhibited decreased anxiety and hyperactivity. Additionally, Fmr1 -KO mice's ultrasonic vocalizations were impaired suggesting a reduced social interest and communication of this strain. Acute LP-211 administration did not affect the behavioral abnormalities observed in BTBR mice but improved repetitive behavior in Fmr1 -KO mice and showed a trend to change anxiety of this strain. Acute R-Baclofen treatment improved repetitive behavior only in Fmr1 -KO mice., Conclusion: Our results add value to the current available data on these mouse models and the respective compounds. Yet, additional studies are needed to further test R-Baclofen and LP-211 as potential treatments for ASD therapy., Competing Interests: SS, SF, MD, RR, MP, and BH-P were employed by QPS Austria GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sharghi, Flunkert, Daurer, Rabl, Chagnaud, Leopoldo, Lacivita, Hutter-Paier and Prokesch.)

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 animals 4 , 5 and serves as an aposematic threat and warning display, likely to avoid being preyed upon. 1 , 6 The spectral properties of the rattling sound 1 , 3 and its dependence on the morphology and size of the rattle have been investigated for decades 7-9 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors HZ., (Copyright © 2021 Dunlap, Koukos, Chagnaud, Zakon and Bass.)

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., (© 2020 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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., Competing Interests: BC, JP, PF, AB No competing interests declared, (© 2021, Chagnaud et al.)

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., (© 2020 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2018 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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., (© 2017 S. Karger AG, Basel.)

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.

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., (Copyright © 2016 the authors 0270-6474/16/369097-14$15.00/0.)

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.

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., (© 2015. Published by The Company of Biologists Ltd.)

Anesthetics are drugs that reversibly relieve pain, decrease body movements and suppress neuronal activity. Most drugs only cover one of these effects; for instance, analgesics relieve pain but fail to block primary fiber responses to noxious stimuli. Alternately, paralytic drugs block synaptic transmission at neuromuscular junctions, thereby effectively paralyzing skeletal muscles. Thus, both analgesics and paralytics each accomplish one effect, but fail to singularly account for all three. Tricaine methanesulfonate (MS-222) is structurally similar to benzocaine, a typical anesthetic for anamniote vertebrates, but contains a sulfate moiety rendering this drug more hydrophilic. MS-222 is used as anesthetic in poikilothermic animals such as fish and amphibians. However, it is often argued that MS-222 is only a hypnotic drug and its ability to block neural activity has been questioned. This prompted us to evaluate the potency and dynamics of MS-222-induced effects on neuronal firing of sensory and motor nerves alongside a defined motor behavior in semi-intact in vitro preparations of Xenopus laevis tadpoles. Electrophysiological recordings of extraocular motor discharge and both spontaneous and evoked mechanosensory nerve activity were measured before, during and after administration of MS-222, then compared to benzocaine and a known paralytic, pancuronium. Both MS-222 and benzocaine, but not pancuronium caused a dose-dependent, reversible blockade of extraocular motor and sensory nerve activity. These results indicate that MS-222 as benzocaine blocks the activity of both sensory and motor nerves compatible with the mechanistic action of effective anesthetics, indicating that both caine-derivates are effective as single-drug anesthetics for surgical interventions in anamniotes.

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., (© 2014 S. Karger AG, Basel.)

Corollary discharge is essential to an animal's ability to filter self-generated from external stimuli. This includes acoustic communication, although direct demonstration of a corollary discharge that both conveys a vocal motor signal and informs the auditory system about the physical attributes of a self-generated vocalization has remained elusive for vertebrates. Here, we show the underlying synaptic activity of a neuronal vocal corollary discharge pathway in the hindbrain of a highly vocal species of fish. Neurons carrying the vocal corollary discharge are specifically adapted for the transmission of duration information, a predominant acoustic cue. The results reveal that vertebrates, like some insects, have a robust corollary discharge conveying call duration. Along with evidence for the influence of vocal duration on auditory encoding in mammals, these new findings suggest that linking vocal motor and corollary discharge pathways with pattern generating, call duration neurons is a shared network character across the animal kingdom.

In guiding adaptive behavior, efference copy signals or corollary discharge are traditionally considered to serve as predictors of self-generated sensory inputs and by interfering with their central processing are able to counter unwanted consequences of an animal's own actions. Here, in a speculative reflection on this issue, we consider a different functional role for such intrinsic predictive signaling, namely in stabilizing gaze during locomotion where resultant changes in head orientation in space require online compensatory eye movements in order to prevent retinal image slip. The direct activation of extraocular motoneurons by locomotor-related efference copies offers a prospective substrate for assisting self-motion derived sensory feedback, rather than being subtracted from the sensory signal to eliminate unwanted reafferent information. However, implementing such a feed-forward mechanism would be critically dependent on an appropriate phase coupling between rhythmic propulsive movement and resultant head/visual image displacement. We used video analyzes of actual locomotor behavior and basic theoretical modeling to evaluate head motion during stable locomotion in animals as diverse as Xenopus laevis tadpoles, teleost fish and horses in order to assess the potential suitability of spinal efference copies to the stabilization of gaze during locomotion. In all three species, and therefore regardless of aquatic or terrestrial environment, the head displacements that accompanied locomotor action displayed a strong correlative spatio-temporal relationship in correspondence with a potential predictive value for compensatory eye adjustments. Although spinal central pattern generator-derived efference copies offer appropriately timed commands for extraocular motor control during self-generated motion, it is likely that precise image stabilization requires the additional contributions of sensory feedback signals. Nonetheless, the predictability of the visual consequences of stereotyped locomotion renders intrinsic efference copy signaling an appealing mechanism for offsetting these disturbances, thus questioning the exclusive role traditionally ascribed to sensory-motor transformations in stabilizing gaze during vertebrate locomotion.

Acoustic signaling behaviors are widespread among bony vertebrates, which include the majority of living fishes and tetrapods. Developmental studies in sound-producing fishes and tetrapods indicate that central pattern generating networks dedicated to vocalization originate from the same caudal hindbrain rhombomere (rh) 8-spinal compartment. Together, the evidence suggests that vocalization and its morphophysiological basis, including mechanisms of vocal-respiratory coupling that are widespread among tetrapods, are ancestral characters for bony vertebrates. Premotor-motor circuitry for pectoral appendages that function in locomotion and acoustic signaling develops in the same rh8-spinal compartment. Hence, vocal and pectoral phenotypes in fishes share both developmental origins and roles in acoustic communication. These findings lead to the proposal that the coupling of more highly derived vocal and pectoral mechanisms among tetrapods, including those adapted for nonvocal acoustic and gestural signaling, originated in fishes. Comparative studies further show that rh8 premotor populations have distinct neurophysiological properties coding for equally distinct behavioral attributes such as call duration. We conclude that neural network innovations in the spatiotemporal patterning of vocal and pectoral mechanisms of social communication, including forelimb gestural signaling, have their evolutionary origins in the caudal hindbrain of fishes.

Rapid temporal modulation of acoustic signals among several vertebrate lineages has recently been shown to depend on the actions of superfast muscles. We hypothesized that such fast events, known to require synchronous activation of muscle fibers, would rely on motoneuronal properties adapted to generating a highly synchronous output to sonic muscles. Using intracellular in vivo recordings, we identified a suite of premotor network inputs and intrinsic motoneuronal properties synchronizing the oscillatory-like, simultaneous activation of superfast muscles at high gamma frequencies in fish. Motoneurons lacked spontaneous activity, firing synchronously only at the frequency of premotor excitatory input. Population-level motoneuronal output generated a spike-like, vocal nerve volley that directly determines muscle contraction rate and, in turn, natural call frequency. In the absence of vocal output, motoneurons showed low excitability and a weak afterhyperpolarization, leading to rapid accommodation in firing rate. By contrast, vocal activity was accompanied by a prominent afterhyperpolarization, indicating a dependency on network activity. Local injection of a GABA(A) receptor antagonist demonstrated the necessity of electrophysiologically and immunohistochemically confirmed inhibitory GABAergic input for motoneuronal synchrony and vocalization. Numerous transneuronally labeled motoneurons following single-cell neurobiotin injection together with electrophysiological collision experiments confirmed gap junctional coupling, known to contribute to synchronous activity in other neural networks. Motoneuronal synchrony at the premotor input frequency was maintained during differential recruitment of variably sized motoneurons. Differential motoneuron recruitment led, however, to amplitude modulation (AM) of vocal output and, hence, natural call AM. In summary, motoneuronal intrinsic properties, in particular low excitability, predisposed vocal motoneurons to the synchronizing influences of premotor inputs to translate a temporal input code into a coincident and extremely synchronous, but variable-amplitude, output code. We propose an analogous suite of neuronal properties as a key innovation underlying similarly rapid acoustic events observed among amphibians, reptiles, birds, and mammals.

Fish and aquatic frogs detect minute water motion by means of a specialized mechanosensory system, the lateral line. Ubiquitous in fish, the lateral-line system is characterized by hair-cell based sensory structures across the fish's surface called neuromasts. These neuromasts occur free-standing on the skin as superficial neuromasts (SN) or are recessed into canals as canal neuromasts. SNs respond to rapid changes of water velocity in a small layer of fluid around the fish, including the so-called boundary layer. Although omnipresent, the boundary layer's impact on the SN response is still a matter of debate. For the first time using an information-theoretic approach to this sensory system, we have investigated the SN afferents encoding capabilities. Combining covariance analysis, phase analysis, and modeling of recorded neuronal responses of primary lateral line afferents, we show that encoding by the SNs is adequately described as a linear, velocity-responsive mechanism. Afferent responses display a bimodal distribution of opposite Wiener kernels that likely reflected the two hair-cell populations within a given neuromast. Using frozen noise stimuli, we further demonstrate that SN afferents respond in an extremely precise manner and with high reproducibility across a broad frequency band (10-150 Hz), revealing that an optimal decoder would need to rely extensively on a temporal code. This was further substantiated by means of signal reconstruction of spike trains that were time shifted with respect to their original. On average, a time shift of 3.5 ms was enough to diminish the encoding capabilities of primary afferents by 70%. Our results further demonstrate that the SNs' encoding capability is linearly related to the stimulus outside the boundary layer, and that the boundary layer can, therefore, be neglected while interpreting lateral line response of SN afferents to hydrodynamic stimuli.

Temporal patterning is an essential feature of neural networks producing precisely timed behaviours such as vocalizations that are widely used in vertebrate social communication. Here we show that intrinsic and network properties of separate hindbrain neuronal populations encode the natural call attributes of frequency and duration in vocal fish. Intracellular structure/function analyses indicate that call duration is encoded by a sustained membrane depolarization in vocal prepacemaker neurons that innervate downstream pacemaker neurons. Pacemaker neurons, in turn, encode call frequency by rhythmic, ultrafast oscillations in their membrane potential. Pharmacological manipulations show prepacemaker activity to be independent of pacemaker function, thus accounting for natural variation in duration which is the predominant feature distinguishing call types. Prepacemaker neurons also innervate key hindbrain auditory nuclei thereby effectively serving as a call-duration corollary discharge. We propose that premotor compartmentalization of neurons coding distinct acoustic attributes is a fundamental trait of hindbrain vocal pattern generators among vertebrates.

In many fishes, prey capture is guided primarily by vision. In the paddlefish, the electrosense can completely substitute for the visual system to detect tiny daphnia, their primary prey. Electroreceptors are distributed over the entire rostrum, head, and gill covers, and there are no accessory structures like a lens to form an image. To accurately locate planktonic prey in three-dimensional space, the poor spatial resolving power of peripheral receptors has to be improved by another mechanism. We have investigated information processing in the electrosensory system of the paddlefish at hind- and midbrain levels by recording single cells extracellularly. We stimulated with a linear array of electrodes that simulated a moving dipole field. In addition, global electric fields were applied to simulate the temporal component of a moving dipole only. Some stimulation were done with sinusoidal fields. The fire rate of cells in the hindbrain followed the first derivative of the stimulus wave form. In contrast, the response of tectal cells were similar to the third derivative. This improves spatial resolution and receptive fields of tectal units are much smaller than the ones of hind brain units. The principle is similar to a Laplacian of Gaussian filter that is commonly used in digital image processing. However, instead of working in the space domain, the paddlefish edge detection filter works in the time domain, thus eliminating the need for extensive interconnections in an array of topographically organized neurons.

Paddlefish use their electrosense to locate small water fleas (daphnia), their primary prey, in three-dimensional space. High sensitivity and a representation of object location are essential for this task. High sensitivity can be achieved by convergence of information from a large number of receptors and object location is usually represented in the nervous system by topographic maps. However the first electrosensory center in the brain, the dorsal octavolateral nucleus in the hindbrain, is neither topographically organized nor does it show a higher sensitivity than primary afferent fibers. Here, we investigated the response properties of electrosensory neurons in the dorsal octavolateral nucleus (DON), the lateral mesencephalic nucleus (LMN) and the tectum mesencephali (TM). LMN units are characterized by large receptive fields, which suggest a high degree of convergence. TM units have small receptive fields and are topographically arranged, at least in the rostro-caudal axis, the only dimension we could test. Well-defined receptive fields, however, could only be detected in the TM with a moving DC stimulus. The receptive fields of TM units, as determined by slowly scanning the rostrum and head with a 5 Hz stimulus, were very large and frequently two or more receptive fields were present. The receptive fields for LMN units were located in the anterior half of the rostrum whereas TM units had receptive fields predominantly on the head and at the base of the rostrum. A detailed analysis of the prey catching behavior revealed that it consists of two phases that coincide with the location of the receptive fields in LMN and TM, respectively. This suggests that LMN units are responsible for the initial orienting response that occurs when the prey is alongside the anterior first half of the rostrum. TM units, in contrast, had receptive fields at locations where the prey is located when the fish opens its mouth and attempts the final strike.

If exposed to bulk water flow, fish lateral line afferents respond only to flow fluctuations (AC) and not to the steady (DC) component of the flow. Consequently, a single lateral line afferent can encode neither bulk flow direction nor velocity. It is possible, however, for a fish to obtain bulk flow information using multiple afferents that respond only to flow fluctuations. We show by means of particle image velocimetry that, if a flow contains fluctuations, these fluctuations propagate with the flow. A cross-correlation of water motion measured at an upstream point with that at a downstream point can then provide information about flow velocity and flow direction. In this study, we recorded from pairs of primary lateral line afferents while a fish was exposed to either bulk water flow, or to the water motion caused by a moving object. We confirm that lateral line afferents responded to the flow fluctuations and not to the DC component of the flow, and that responses of many fiber pairs were highly correlated, if they were time-shifted to correct for gross flow velocity and gross flow direction. To prove that a cross-correlation mechanism can be used to retrieve the information about gross flow velocity and direction, we measured the flow-induced bending motions of two flexible micropillars separated in a downstream direction. A cross-correlation of the bending motions of these micropillars did indeed produce an accurate estimate of the velocity vector along the direction of the micropillars.

Many fishes and amphibians are able to sense weak electric fields from prey animals or other sources. The response properties of primary afferent fibers innervating the electroreceptors and information processing at the level of the hindbrain is well investigated in a number of taxa. However, there are only a few studies in higher brain areas. We recorded from electrosensory neurons in the lateral mesencephalic nucleus (LMN) and from neurons in the dorsal octavolateral nucleus (DON) of the paddlefish. We stimulated with sine wave stimuli of different amplitudes and frequencies and with moving DC stimuli. During sinusoidal stimulation, DON units increased their firing rate during the negative cycle of the sine wave and decreased their firing rate to the positive cycle. Lateral mesencephalic nucleus units increased their rate for both half cycles of the sine wave. Lateral mesencephalic nucleus units are more sensitive than DON units, especially to small moving dipoles. Dorsal octavolateral nucleus units respond to a moving DC dipole with an increase followed by a decrease in spike rate or vice versa, depending on movement direction and dipole orientation. Lateral mesencephalic nucleus units, in contrast, increased their discharge rate for all stimuli. Any change in discharge rate of DON units is converted in the LMN to a discharge rate increase. Lateral mesencephalic nucleus units therefore appear to code the presence of a stimulus regardless of orientation and motion direction.

Paddlefish use their peculiar rostrum to detect minute electric fields from their main prey, small water fleas. Electroreceptors over the rostrum and head sense these fields and send the information into a single hindbrain area, the dorsal octavolateral nucleus (DON). From there, information is sent to various midbrain structures, including the tectum. The response properties of primary afferent fibers and DON units has been well investigated, but nothing is known about electrosensory units in the midbrain. Here we recorded the responses of single units in the midbrain tectum and DON to uniform electric fields. Tectal units exhibited little spontaneous activity and responded to sine waves with a few, well phase-locked spikes. Phase locking was still significant at amplitudes one order of magnitude lower than in the DON. If stimulated with sinusoidal electric fields of different frequencies, phase locking in DON units decreased proportionally with frequency whereas the response of tectal units depended little on frequency. This is in agreement with behavioral studies showing that relevant frequencies range from DC to ca 20 Hz.

Fish acquire information about their aquatic environment by means of their mechanosensory lateral-line system. This system consists of superficial and canal neuromasts that sense perturbations in the water surrounding them. Based on a hydrodynamic model presented here, we propose a mechanism through which fish can localize the source of these perturbations. In doing so we include the curvature of the fish body, a realistic lateral line canal inter-pore distance for the lateral-line canals, and the surface boundary layer. Using our model to explore receptor behavior based on experimental data of responses to dipole stimuli we suggest that superficial and canal neuromasts employ the same mechanism, hence provide the same type of input to the central nervous system. The analytical predictions agree well with spiking responses recorded experimentally from primary lateral-line nerve fibers. From this, and taking into account the central organization of the lateral-line system, we present a simple biophysical model for determining the distance to a source.

The discharges of anterior and posterior lateral line nerve afferents were recorded while stimulating goldfish, Carassius auratus, with bulk water flow. With increasing flow velocity lateral line afferents increased their discharge rates. However, an increased response to flow rates occurred even if flow direction was reversed. Thus, individual lateral line afferents did not encode the direction of running water. Frequency spectra of the water motions quantified with particle image velocimetry revealed flow fluctuations that increased with increasing flow velocity. Maximal spectral amplitudes of the flow fluctuations were below 5 Hz (bulk flow velocity 4-15 cms(-1)). The frequency spectra of the firing rates of lateral line afferents also showed an increase in amplitude when fish were exposed to running water. The maximal spectral amplitudes of the recorded data were in the frequency range 3-8 Hz. This suggests that the lateral line afferents mainly responded to the higher frequency fluctuations that developed under flow conditions, but not to the direct current flow or the lower frequency fluctuations. Although individual lateral line afferents encoded neither flow velocity nor flow direction we suggest that higher order lateral line neurons can do so by monitoring flow fluctuations as they move across the surface of the fish.

Fish use the lateral line system for prey detection, predator avoidance, schooling behavior, intraspecific communication and spatial orientation. In addition the lateral line may be important for station holding and for the detection of the hydrodynamic trails (vortex streets) generated by swimming fish. We investigated the responses of anterior lateral line nerve fibers of goldfish, Carassius auratus, to unidirectional water flow (10 cm s(-1)) and to running water that contained a Kármán vortex street. Compared to still water conditions, both unidirectional water flow and Kármán vortex streets caused a similar increase in the discharge rate of anterior lateral line nerve fibers. If exposed to a Kármán vortex street, the amplitude of spike train frequency spectra increased at the vortex shedding frequency. This increase was especially pronounced if the fish intercepted the edge of a Kármán vortex street. Our data show that the vortex shedding frequency can be retrieved from the responses of anterior lateral line nerve fibers.

We investigated how fibres in the anterior lateral line nerve of goldfish, Carassius auratus, respond to sinusoidal water motions in a background of still or running water. Two types of fibres were distinguished: type I fibres, which most likely innervate superficial neuromasts, were stimulated by running water (10 cm s(-1)) while type II fibres, which most likely innervate canal neuromasts, were not stimulated by running water. The responses of type I fibres to sinusoidal water motions were masked in running water whereas responses of type II fibres were not masked. These findings are in agreement with previous data obtained from the posterior lateral line nerve of goldfish. Furthermore, we demonstrate here that for type I fibres the degree of response masking increased with increasing flow velocity. Finally, the ratio between responses that were masked in running water (type I) and those that were not masked (type II) increases with increasing flow velocity. Flow fluctuations that were generated by a cylinder in front of the fish did not affect ongoing activity in the flow, nor the dipole-evoked responses. The findings are discussed with respect to particle image velocimetry data of the water motions generated in the experiments.

The lateral line system of fish is sensitive to weak water motions. We recorded from posterior lateral line nerve afferents while stimulating goldfish, Carassius auratus, with unidirectional water flow and with a vortex ring. Posterior lateral line afferents of goldfish were either flow sensitive or flow insensitive. Both types of afferents responded to a vortex ring that passed the fish laterally with one to three reproducible patterns of neural activity, followed by activity patterns that were less reproducible. Using particle image velocimetry, we visualized and quantified the water motions in the vertical plane next to the surface of the fish while recording from lateral line afferents. Early response components correlated with the direction of water motions that occurred at the position of the neuromast recorded from. By contrast, neural activity that occurred after the vortex had passed the fish barely predicted the direction of water motions. These results are in agreement with the known directional sensitivity of hair cells and indicate that fish might be able to extract sensory information from complex stimuli like vortices by comparing the activity of a whole array of neuromasts. The stimulus used in this study is novel to lateral line research and resembles some of the hydrodynamic stimuli that fish might encounter in their natural environments. We expect that by combining naturalistic hydrodynamic stimuli and central nervous recordings, we will learn if and how hydrodynamic feature detection is accomplished by the lateral line system.