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Mauthner cells are the largest neurons in the hindbrain of teleost fish and most amphibians. Each cell has two major dendrites thought to receive segregated streams of sensory input: the lateral dendrite receives mechanosensory input while the ventral dendrite receives visual input. These inputs, which mediate escape responses to sudden stimuli, may be modulated by the availability of sensory information to the animal. To understand the impact of the absence of visual information on the morphologies of Mauthner cells during development and evolutionary time scales, we examined Astyanax mexicanus. This species of tetra is found in two morphs: a seeing surface fish and a blind cavefish. We compared the structure of Mauthner cells in surface fish raised under daily light conditions, surface fish that raised in constant darkness, and two independent lineages of cave populations. The length of ventral dendrites of Mauthner cells in dark-raised surface larvae were longer and more branched, while in both cave morphs the ventral dendrites were smaller or absent. The absence of visual input in surface fish with normal eye development leads to a homeostatic increase in dendrite size, whereas over evolution, the absence of light led to the loss of eyes and a phylogenetic reduction in dendrite size. Consequently, homeostatic mechanisms are under natural selection that provide adaptation to constant darkness.

The central nervous system of the Ciona larva contains only 177 neurons. The precise regulation of neuron subtype-specific morphogenesis and differentiation observed during the formation of this minimal connectome offers a unique opportunity to dissect gene regulatory networks underlying chordate neurodevelopment. Here we compare the transcriptomes of two very distinct neuron types in the hindbrain/spinal cord homolog of Ciona, the Motor Ganglion (MG): the Descending decussating neuron (ddN, proposed homolog of Mauthner Cells in vertebrates) and the MG Interneuron 2 (MGIN2). Both types are invariantly represented by a single bilaterally symmetric left/right pair of cells in every larva. Supernumerary ddNs and MGIN2s were generated in synchronized embryos and isolated by fluorescence-activated cell sorting for transcriptome profiling. Differential gene expression analysis revealed ddN- and MGIN2-specific enrichment of a wide range of genes, including many encoding potential “effectors” of subtype-specific morphological and functional traits. More specifically, we identified the upregulation of centrosome-associated, microtubule-stabilizing/bundling proteins and extracellular guidance cues part of a single intrinsic regulatory program that might underlie the unique polarization of the ddNs, the only descending MG neurons that cross the midline. Consistent with our predictions, CRISPR/Cas9-mediated, tissue-specific elimination of two such candidate effectors, Efcab6-related and Netrin1, impaired ddN polarized axon outgrowth across the midline.

Fast and accurate threat detection is critical for animal survival. Reducing perceptual ambiguity by integrating multiple sources of sensory information can enhance perception and reduce response latency. However, studies addressing the link between behavioral correlates of multisensory integration and its underlying neural basis are rare. Fish that detect an urgent threat escape with an explosive behavior known as C-start. The C-start is driven by an identified neural circuit centered on the Mauthner cell, an identified neuron capable of triggering escapes in response to visual and auditory stimuli. Here we demonstrate that goldfish can integrate visual looms and brief auditory stimuli to increase C-start probability. This multisensory enhancement is inversely correlated to the salience of the stimuli, with weaker auditory cues producing a proportionally stronger multisensory effect. We also show that multisensory stimuli reduced C-start response latency, with most escapes locked to the presentation of the auditory cue. We make a direct link between behavioral data and its underlying neural mechanism by reproducing the behavioral data with an integrate-and-fire computational model of the Mauthner cell. This model of the Mauthner cell circuit suggests that excitatory inputs integrated at the soma are key elements in multisensory decision making during fast C-start escapes. This provides a simple but powerful mechanism to enhance threat detection and survival.

A pair of Mauthner cells (M-cells) can be found in the hindbrain of most teleost fish, as well as amphibians and lamprey. The axons of these reticulospinal neurons cross the midline and synapse on interneurons and motoneurons as they descend the length of the spinal cord. The M-cell initiates fast C-type startle responses (fast C-starts) in goldfish and zebrafish triggered by abrupt acoustic/vibratory stimuli. Starting about 70 days after whole spinal cord crush, less robust startle responses with longer latencies manifest in adult goldfish, Carassius auratus. The morphological and electrophysiological identifiability of the M-cell provides a unique opportunity to study cellular responses to spinal cord injury and the relation of axonal regrowth to a defined behavior. After spinal cord crush at the spinomedullary junction about one-third of the damaged M-axons of adult goldfish send at least one sprout past the wound site between 56 and 85 days postoperatively. These caudally projecting sprouts follow a more lateral trajectory relative to their position in the fasciculus longitudinalis medialis of control fish. Other sprouts, some from the same axon, follow aberrant pathways that include rostral projections, reversal of direction, midline crossings, neuromas, and projection out the first ventral root. Stimulating M-axons in goldfish that had post-injury startle behavior between 198 and 468 days postoperatively resulted in no or minimal EMG activity in trunk and tail musculature as compared to control fish. Although M-cells can survive for at least 468 day (∼1.3 years) after spinal cord crush, maintain regrowth, and elicit putative trunk EMG responses, the cell does not appear to play a substantive role in the emergence of acoustic/vibratory-triggered responses. We speculate that aberrant pathway choice of this neuron may limit its role in the recovery of behavior and discuss structural and functional properties of alternative candidate neurons that may render them more supportive of post-injury startle behavior.

Objectives. To study the structure and formation of Mauthner neuron dendrites in goldfish exposed to neurotoxic β-amyloid fragment 25–35 and prolonged sensory stimulation influencing the afferent inputs to these neurons. Materials and methods. Goldfish Mauthner neurons were studied by light and electron microscopy. Individual dendrites were identified, their volumes were determined, and synapse structure was assessed using virtual 3D images of Mauthner neurons obtained from serial sections of thickness 3 μm. The functional status of Mauthner neurons was evaluated indirectly from the motor lateralization of the fish. Results. Mauthner neurons responded to application of β-amyloid combined with subsequent prolonged sensory stimulation with decreases in the volume of the ventral dendrites, damage to their ultrastructure, and degeneration of a proportion of synapses. Degeneration of more active neurons was more significant than that of less active cells. Newly formed medial dendrites had greater volume and less damaged synapse ultrastructure than ventral dendrites. There were no differences in the sizes of specialized junctions between synapses of the same type on ventral and medial synapses. Conclusions. Medial dendrite formation is a compensatory reaction to dystrophy of the ventral dendrite due to the experimental treatment.

Calcium is an important messenger in the neuronal system, but its specific role in axonal regeneration has not been fully investigated. To clarify it, we constructed a noninvasive in vivo calcium-imaging model of zebrafish Mauthner cells and monitored subcellular calcium dynamics during axonal regeneration. Using the calcium indicator GCamp6f, we observed that the regenerative length correlated with the peak amplitude of the evoked calcium response before axotomy, which suggested that the evoked calcium response might serve as a useful indicator of evoked neuronal activity and axonal regenerative capacity. To investigate this possibility, we overexpressed an inward rectifying potassium channel protein, Kir2.1a, to decrease the Mauthner neuronal activity and found that the inhibition of the calcium response correlated with decreased axonal regeneration. In contrast, treatment of pentylenetetrazol and knockout of the sodium voltage-gated channel α subunit 1 gene increased the calcium response and thus enhanced axonal regeneration. Our results therefore increased the understanding of the correlation between the neural activity and the vertebrate axonal regeneration.-Chen, M., Huang, R.-C., Yang, L.-Q., Ren, D.-L., Hu, B. In vivo imaging of evoked calcium responses indicates the intrinsic axonal regenerative capacity of zebrafish.

Learning is essential for animal survival under changing environments. Even in its simplest form, learning involves interactions between a handful of neuronal circuits, hundreds of neurons and many thousand synapses. In this review I will focus on habituation - a form of non-associative learning during which organisms decrease their response to repetitions of identical sensory stimuli. I will discuss how recent studies of the acoustic startle reflex mediated by the Mauthner cell in the zebrafish larva are helping to understand the neuroplastic processes that underlie habituation. In addition to being a fascinating biological process, habituation is clinically relevant because it is affected in various neuropsychiatric disorders in humans, including autism, schizophrenia, Fragile-X and Tourette's syndromes.

—The effects of sensory stimulation on the model of paired Mauthner neurons of monocularly deprived goldfish have been studied by light- and electron microscopy. Three-dimensional reconstruction was performed using serial semi-thin sections in order to determine the volumes of soma and dendrites for the right and left Mauthner neurons. These data suggested that long-term stimulation of some fish caused a decrease in the volume of the lateral dendrites of neurons contralateral with respect to the side of the enucleated eye. As a result, the morphological homeostasis in the dendrites of Mauthner neurons, which was observed in the control fish, was disturbed after the effect of stimulation upon this group of fish. The main mechanism of this effect of stimulation, according to ultrastructural analysis, is the compaction of the cytoskeleton in the lateral dendrite of a neuron and a reduction in the number of synaptic vesicles in the region of afferent synapses. In contrast, stimulation of the remaining fish induced the resistance of the contralateral Mauthner neuron to prolonged stimulation, as well as the hypertrophy of its lateral and shortening of the ventral dendrites. These results suggest that the homeostasis of the dendrites of Mauthner neurons essentially depends on the state of the visual inputs, the load on which increased as a result of the doubled impact.

Social status-dependent modulation of neural circuits has been investigated extensively in vertebrate and invertebrate systems. However, the effects of social status on neuromodulatory systems that drive motor activity are poorly understood. Zebrafish form a stable social relationship that consists of socially dominant and subordinate animals. The locomotor behavior patterns differ according to their social ranks. The sensitivity of the Mauthner startle escape response in subordinates increases compared to dominants while dominants increase their swimming frequency compared to subordinates. Here, we investigated the role of the endocannabinoid system (ECS) in mediating these differences in motor activities. We show that brain gene expression of key ECS protein pathways are socially regulated. Diacylglycerol lipase (DAGL) expression significantly increased in dominants and significantly decreased in subordinates relative to controls. Moreover, brain gene expression of the cannabinoid 1 receptor (CB1R) was significantly increased in subordinates relative to controls. Secondly, increasing ECS activity with JZL184 reversed swimming activity patterns in dominant and subordinate animals. JZL184 did not affect the sensitivity of the startle escape response in dominants while it was significantly reduced in subordinates. Thirdly, blockage of CB1R function with AM-251 had no effect on dominants startle escape response sensitivity, but startle sensitivity was significantly reduced in subordinates. Additionally, AM-251 did not affect swimming activities in either social phenotypes. Fourthly, we demonstrate that the effects of ECS modulation of the startle escape circuit is mediated via the dopaminergic system specifically via the dopamine D1 receptor. Finally, our empirical results complemented with neurocomputational modeling suggest that social status influences the ECS to regulate the balance in synaptic strength between excitatory and inhibitory inputs to control the excitability of motor behaviors. Collectively, this study provides new insights of how social factors impact nervous system function to reconfigure the synergistic interactions of neuromodulatory pathways to optimize motor output.

Animals use a precisely timed motor sequence to escape predators. This requires the nervous system to coordinate several motor behaviors and execute them in a temporal and smooth manner. We here describe a neuronal circuit that faithfully generates a defensive motor sequence in zebrafish larvae. The temporally specific defensive motor sequence consists of an initial escape and a subsequent swim behavior and can be initiated by unilateral stimulation of a single Mauthner cell (M-cell). The smooth transition from escape behavior to swim behavior is achieved by activating a neuronal chain circuit, which permits an M-cell to drive descending neurons in bilateral nucleus of medial longitudinal fascicle (nMLF) via activation of an intermediate excitatory circuit formed by interconnected hindbrain cranial relay neurons. The sequential activation of M-cells and neurons in bilateral nMLF via activation of hindbrain cranial relay neurons ensures the smooth execution of escape and swim behaviors in a timely manner. We propose an existence of a serial model that executes a temporal motor sequence involving three different brain regions that initiates the escape behavior and triggers a subsequent swim. This model has general implications regarding the neural control of complex motor sequences.

SUMMARYIn the spinal cord, cerebrospinal fluid-contacting neurons (CSF-cNs) are GABAergic interoceptive sensory neurons that detect spinal curvature via a functional coupling with the Reissner fiber. This mechanosensory system has recently been found involved in spine morphogenesis and postural control but the underlying mechanisms are not fully understood. In zebrafish, CSF-cNs project an ascending and ipsilateral axon reaching two to six segments away. Rostralmost CSF-cNs send their axons ipsilaterally into the hindbrain, a brain region containing motor nuclei and reticulospinal neurons (RSNs), which send descending motor commands to spinal circuits. Until now, the synaptic connectivity of CSF-cNs has only been investigated in the spinal cord where they synapse onto motor neurons and premotor excitatory interneurons. The identity of CSF-cN targets in the hindbrain and the behavioral relevance of these sensory projections from spinal cord to hindbrain are unknown. Here, we provide anatomical and molecular evidence that rostralmost CSF-cNs synapse onto the axons of large RSNs including the Mauthner cells and early born chx10+ neurons. Functional anatomy and optogenetic-assisted mapping reveal that rostral CSF-cNs also synapse onto the soma and dendrites of cranial motor neurons innervating hypobranchial muscles. During acousto-vestibular evoked escape responses, ablation of rostralmost CSF-cNs results in a weaker escape response with a decreased C-bend amplitude, lower speed and a deficient postural control. Our study demonstrates that spinal sensory feedback enhances speed and stabilizes posture, and reveals a novel spinal gating mechanism acting on the output of descending commands sent from the hindbrain to the spinal cord.eTOCCerebrospinal fluid-contacting neurons are mechanosensory cells that detect spinal curvature. Wu et al. show here that rostralmost CSF-cNs synapse in the hindbrain onto cranial motor neurons and the descending axons of reticulospinal neurons, and enhance speed and power as well as postural control during active locomotion.HighlightsCerebrospinal fluid-contacting neurons (CSF-cNs) in rostral spinal cord form inhibitory synapses onto cranial motor neuronsRostral CSF-cNs synapse onto descending axons of reticulospinal neuronsCSF-cN sensory feedback in the rostral spinal cord enhance speed and power of locomotionRostral CSF-cNs projecting to the hindbrain contribute to postural control

Sensorimotor integration is a pivotal feature of the nervous system for ensuring a coordinated motor response to external stimuli. In essence, such neural circuits can optimize behavioral performance based on the saliency of environmental cues. In zebrafish, habituation of the acoustic startle response (ASR) is a simple behavior integrated into the startle command neurons, called the Mauthner cells. Whereas the essential neuronal components that regulate the startle response have been identified, the principles of how this regulation is integrated at the subcellular regions of the Mauthner cell, which in turn modulate the performance of the behavior, is still not well understood. Here, we reveal mechanistically distinct dynamics of excitatory inputs converging onto the lateral dendrite (LD) and axon initial segment (AIS) of the Mauthner cell byin vivoimaging glutamate release using iGluSnFR, an ultrafast glutamate sensing fluorescent reporter. We find that modulation of glutamate release is dependent on NMDA receptor activity exclusively at the AIS, which is responsible for setting the sensitivity of the startle reflex and inducing a depression of synaptic activity during habituation. In contrast, glutamate-release at the LD is not regulated by NMDA receptors and serves as a baseline component of Mauthner cell activation. Finally, usingin vivocalcium imaging at the feed-forward interneuron population component of the startle circuit, we reveal that these cells indeed play pivotal roles in both setting the startle threshold and habituation by modulating the AIS of the Mauthner cell. These results indicate that a command neuron may have several functionally distinct regions to regulate complex aspects of behavior.

Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins. While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins, it is unknown if channels at electrical synapses require similar scaffold support. Here, we investigated the functional relationship between neuronal Connexins and Zonula Occludens 1 (ZO1), an intracellular scaffolding protein localized to electrical synapses. Using model electrical synapses in zebrafish Mauthner cells, we demonstrated that ZO1 is required for robust synaptic Connexin localization, but Connexins are dispensable for ZO1 localization. Disrupting this hierarchical ZO1/Connexin relationship abolishes electrical transmission and disrupts Mauthner cell-initiated escape responses. We found that ZO1 is asymmetrically localized exclusively postsynaptically at neuronal contacts where it functions to assemble intercellular channels. Thus, forming functional neuronal gap junctions requires a postsynaptic scaffolding protein. The critical function of a scaffolding molecule reveals an unanticipated complexity of molecular and functional organization at electrical synapses., eLife digest Neurons ‘talk’ with each another at junctions called synapses, which can either be chemical or electrical. Communication across a chemical synapse involves a ‘sending’ neuron releasing chemicals that diffuse between the cells and subsequently bind to specialized receptors on the receiving neuron. These complex junctions involve a large number of well-studied molecular actors. Electrical synapses, on the other hand, are believed to be simpler. There, neurons are physically connected via channels formed of ‘connexin’ proteins, which allow electrically charged ions to flow between the cells. However, it is likely that other proteins help to create these structures. In particular, recent evidence shows that without a structurally supporting ‘scaffolding’ protein called ZO1, electrical synapses cannot form in the brain of a tiny freshwater fish known as zebrafish. As their name implies, scaffolding proteins help cells organize their internal structure, for example by anchoring other molecules to the cell membrane. By studying electrical synapses in zebrafish, Lasseigne, Echeverry, Ijaz, Michel et al. now show that these structures are more complex than previously assumed. In particular, the experiments reveal that ZO1 proteins are only present on one side of electrical synapses; despite their deceptively symmetrical anatomical organization, these junctions can be asymmetric, like their chemical cousins. The results also show that ZO1 must be present for connexins to gather at electrical synapses, whereas the converse is not true. This suggests that when a new electrical synapse forms, ZO1 moves into position first: it then recruits or stabilizes connexins to form the channels connecting the two cells. In many animals with a spine, electrical synapses account for about 20% of all neural junctions. Understanding how these structures form and work could help to find new treatments for disorders linked to impaired electrical synapses, such as epilepsy.

Autism spectrum disorders (ASDs) are characterized by abnormal behavioral traits arising from neural circuit dysfunction. While a number of genes have been implicated in ASDs, in most cases, a clear understanding of how mutations in these genes lead to circuit dysfunction and behavioral abnormality is absent. Theautism susceptibility candidate 2(AUTS2) gene is one such gene, associated with ASDs, intellectual disability and a range of other neurodevelopmental conditions. Yet, the function of AUTS2 in neural development and circuit function is not at all known. Here, we undertook functional analysis of Auts2a, the main homolog of AUTS2 in zebrafish, in the context of the escape behavior. Escape behavior in wild type zebrafish is critical for survival and is therefore, reliable, rapid, and has well-defined kinematic properties.Auts2a−/−zebrafish are viable, have normal gross morphology and can generate escape behavior with normal kinematics. However, the behavior is unreliable and delayed, with high trial-to-trial variability in the latency. We demonstrate that this is due to the reduced excitability of Mauthner neurons resulting in unreliable firing with stimuli that normally elicit the escape response. Combined with previous studies that show Auts2-regulation of the transcription of ion channel proteins, our results suggest that Auts2 sets the excitability of neurons by activating a set transcriptional program.Significance statementAUTS2 is one among recently identified autism susceptibility candidate genes, whose function in neuronal circuits is unclear. Using zebrafish as a model organism, we probe the function of Auts2a (homolog of mammalian AUTS2) at the cellular, network and behavioral levels. The escape behavior of Auts2a mutant zebrafish is highly variable with normal short latency escapes, long latency escapes and total failures across trials in the same fish. This occurs because neuronal excitability is inappropriately set in the Mauthner neurons of mutants leading to the large trial-to-trial variability in responses. The behavioral variability is fully explained by variability in firing action potentials in the Mauthner neuron, providing an integrative understanding of how behavioral variability arises from mutations at the genetic level.

Cannabis is one of the most commonly used illicit recreational drugs that is often taken for medicinal purposes. The psychoactive ingredient in cannabis is &Delta, 9-Tetrahydrocannabinol (&Delta, 9-THC, hereafter referred to as THC), which is an agonist at the endocannabinoid receptors CB1R and CB2R. Here, we exposed zebrafish embryos to THC during the gastrulation phase to determine the long-term effects during development. We specifically focused on reticulospinal neurons known as the Mauthner cells (M-cell) that are involved in escape response movements. The M- cells are born during gastrulation, thus allowing us to examine neuronal morphology of neurons born during the time of exposure. After the exposure, embryos were allowed to develop normally and were examined at two days post-fertilization for M-cell morphology and escape responses. THC treated embryos exhibited subtle alterations in M-cell axon diameter and small changes in escape response dynamics to touch. Because escape responses were altered, we also examined muscle fiber development. The fluorescent labelling of red and white muscle fibers showed that while muscles were largely intact, the fibers were slightly disorganized with subtle but significant changes in the pattern of expression of nicotinic acetylcholine receptors. However, there were no overt changes in the expression of nicotinic receptor subunit mRNA ascertained by qPCR. Embryos were allowed to further develop until 5 dpf, when they were examined for overall levels of movement. Animals exposed to THC during gastrulation exhibited reduced activity compared with vehicle controls. Together, these findings indicate that zebrafish exposed to THC during the gastrula phase exhibit small changes in neuronal and muscle morphology that may impact behavior and locomotion.

SUMMARY Sensory experiences dynamically modify whether animals respond to a given stimulus, but it is unclear how innate behavioral thresholds are established. Here, we identify molecular and circuit-level mechanisms underlying the innate threshold of the zebrafish startle response. From a forward genetic screen, we isolated five mutant lines with reduced innate startle thresholds. Using whole-genome sequencing, we identify the causative mutation for one line to be in the fragile X mental retardation protein (FMRP)-interacting protein cyfip2. We show that cyfip2 acts independently of FMRP and that reactivation of cyfip2 restores the baseline threshold after phenotype onset. Finally, we show that cyfip2 regulates the innate startle threshold by reducing neural activity in a small group of excitatory hindbrain interneurons. Thus, we identify a selective set of genes critical to establishing an innate behavioral threshold and uncover a circuit-level role for cyfip2 in this process., Graphical Abstract, In Brief Using forward genetics, electrophysiology, and combined behavior and Ca2+ imaging in zebrafish, Marsden et al. show that cyfip2 regulates the acoustic startle threshold by controlling the activity of excitatory spiral fiber interneurons.

The effects of the Thr–Ser–Lys–Tyr peptide, which was shown to display neuroprotective activity in cell cultures in vitro, were studied in the model of paired Mauthner cells of goldfish. It was found that intracerebral injections provided the peptide to be applied into the zone of the right Mauthner cell under the fourth ventricle of the hindbrain lead to a dose-dependent decrease in the number of spontaneous turns of the goldfish to the left. It was shown that this effect is not eliminated under long-lasting optokinetic stimulation when the fish instinctively follow stimuli with a low spatial frequency that are moving in the nasal-to-temporal direction. We used the method of three-dimensional reconstruction by serial histological sections to study the dendrite morphology of the Mauthner cells in control and experimental goldfish. It was found that optokinetic stimulation of control goldfish evokes the dystrophy of the ventral dendrite of the right Mauthner cell, which is the target of this type of stimulation. Conversely, the peptide stabilize the size of the ventral dendrite of the right Mauthner cell under stimulation. These data could be interpreted as evidence of the neuroprotective effect of the Thr–Ser–Lys–Tyr peptide in vivo.

Zebrafish is an excellent model to study central nervous system (CNS) axonal degeneration and regeneration since we can observe these processes in vivo and in real time in transparent larvae. Previous studies have shown that Mauthner cell (M-cell) axon regenerates poorly after mechanical spinal cord injury. Inconsistent with this result, however, we have found that M-cell possesses a great capacity for axon regeneration after two-photon laser ablation. By using ZEISS LSM 710 two-photon microscope, we performed specific unilateral axotomy of GFP labeled M-cells in the Tol-056 enhancer trap line larvae. Our results showed that distal axons almost degenerated completely at 24h after laser axotomy. After that, the proximal axons initiated a robust regeneration and many of the M-cell axons almost regenerated fully at 4days post axotomy. Furthermore, we also visualized that regenerated axons were remyelinated when we severed fluorescent dye labeled M-cells in the Tg (mbp:EGFP-CAAX) line larvae. Moreover, by single M-cell co-electroporation with Syp:EGFP and DsRed2 plasmids we observed synapses re-establishment in vivo during laser injury-induced axon re-extension which suggested re-innervation of denervated pathways. In addition, we further demonstrated that nocodazole administration could completely abolish this regeneration capacity. These results together suggested that in vivo time-lapse imaging of M-cell axon laser injury may provide a powerful analytical model for understanding the underlying cellular and molecular mechanisms of the CNS axon regeneration.

Autism spectrum disorders (ASDs) are characterized by abnormal behavioral traits arising from neural circuit dysfunction. While a number of genes have been implicated in ASDs, in most cases, a clear understanding of how mutations in these genes lead to circuit dysfunction and behavioral abnormality is absent. The autism susceptibility candidate 2 (AUTS2) gene is one such gene, associated with ASDs, intellectual disability and a range of other neurodevelopmental conditions. Yet, the role of AUTS2 in neural development and circuit function is not at all known. Here, we undertook functional analysis of Auts2a, the main homolog of AUTS2 in zebrafish, in the context of the escape behavior. Escape behavior in wild type zebrafish is critical for survival and is therefore, reliable, rapid, and has well-defined kinematic properties. Auts2a mutant zebrafish are viable, have normal gross morphology and can generate escape behavior with normal kinematics. However, the behavior is unreliable and delayed, with high trial-to-trial variability in the latency. Using calcium imaging we probed the activity of Mauthner neurons during otic vesicle stimulation and observed lower probability of activation and reduced calcium transients in the mutants. With direct activation of Mauthner by antidromic stimulation, the threshold for activation in mutants was higher than that in wild type, even when inhibition was blocked. Taken together, these results point to reduced excitability of Mauthner neurons in auts2a mutant larvae leading to unreliable escape responses. Our results show a novel role for Auts2a in regulating neural excitability and reliability of behavior.Significance statementAUTS2 is one among recently identified autism susceptibility candidate genes, whose function in neuronal circuits is unclear. Using zebrafish as a model organism, we probe the function of Auts2a (homolog of mammalian AUTS2) at the cellular, network and behavioral levels. The escape behavior of Auts2a mutant zebrafish is highly variable with normal short latency escapes, long latency escapes and total failures across trials in the same fish. This occurs because neuronal excitability is inappropriately set in the Mauthner neurons of mutants leading to large trial-to-trial variability in responses. The behavioral variability is fully explained by variability in firing action potentials in the Mauthner neuron, providing an integrative understanding of how behavioral variability arises from mutations at the genetic level.

Sonic Hedgehog (Shh) signaling plays a major role in vertebrate development, from regulation of proliferation to the patterning of various organs. In amniotes, Shh affects dorsoventral patterning in the inner ear but affects anteroposterior patterning in teleosts. Currently, it remains unknown the function of Shh in inner ear development in terms of how morphogenesis changes in the sarcopterygian/tetrapod lineage coincide with the evolution of limbs and novel auditory organs in the ear. In this study we used the tetrapod, Xenopus laevis, to test how increasing concentrations of the Shh signal pathway antagonist, Vismodegib, affects ear development. Vismodegib treatment dose dependently alters the development of the ear, hypaxial muscle, and indirectly the Mauthner cell through its interaction with the inner ear afferents. Together, these phenotypes have an effect on escape response. The altered Mauthner cell likely contributes to the increased time to respond to a stimulus. In addition, the increased hypaxial muscle in the trunk likely contributes to the subtle change in animal C-start flexion angle. In the ear, Vismodegib treatment results in decreasing segregation between the gravistatic sensory epithelia as the concentration of Vismodegib increases. Furthermore, at higher doses, there is a loss of the horizontal canal but no enantiomorphic transformation, as in bony fish lacking Shh. Like in amniotes, Shh signaling in frogs affects dorsoventral patterning in the ear, suggesting that auditory sensory evolution in sarcopterygians/tetrapods evolved with a shift of Shh axis specification of the ear. This article is protected by copyright. All rights reserved.

The reticulospinal Mauthner cells (M-cells) of the startle circuit have been considered to be dedicated to one basic motor output and the C-type startle response in fish. The neural circuit underlying the C-start, a startle behavior in which the fish forms a "C"-shaped body bend has been described in depth in goldfish and zebrafish [1, 2] and is thought to occur in other species [3, 4]. However, previous research has shown that some species can perform a second type of startle called the S-start [5-7]. This startle response, in which the first movement creates an "S"-shaped body bend achieved with regional muscle activity on left and right sides, cannot be explained by M-cell circuit models. Here we use larval zebrafish to examine the S-start circuit. Since S-starts are elicited through tail stimulation [5-7] and ablating M-cells abolishes short-latency tail-elicited startles [8, 9], we hypothesized that M-cell activity was necessary for S-start generation. Our findings show that the M-cells fire simultaneously to generate the S-start. However, simultaneous M-cell spikes generated through direct current injection were not sufficient to generate S-starts. Through recordings of motoneurons, inhibitory interneurons, and sensory neurons, we uncover a mechanism for generating alternative startle behaviors; local sensory inputs drive inhibitory interneuron activity, which inhibits caudal motoneurons and pre-conditions their excitability prior to the arrival of M-cell spikes in the tail. We suggest that this motoneuron hyperpolarization can bias motor output to left or right sides, determining whether the fish performs a C-start or an S-start behavior.

The ability to detect threatening sensory stimuli and initiate an escape response is essential for survival and under stringent evolutionary pressure. In diverse fish species, acoustic stimuli activate Mauthner neurons, which initiate a stereotypical C-start escape response. This reflexive behavior is highly conserved across aquatic species and provides a model for investigating the neural mechanism underlying the evolution of escape behavior. Here, we define evolved differences in the C-start response between populations of the Mexican cavefish,Astyanax mexicanus. Cave populations ofA. mexicanusinhabit in an environment devoid of light and macroscopic predation, resulting in evolved differences in diverse morphological and behavioral traits. We find that the C-start is present in multiple populations of cavefish and river-dwelling surface fish, but response kinematics and probability differ between populations. The Pachón population of cavefish have an increased response probability, a slower response and reduction of the maximum bend angle, revealing evolved differences between surface and cave populations. In two other independently evolved populations of cavefish, the response probability and the kinematics of the response differ from one another, as well as from surface fish, suggesting the independent evolution of differences in the C-start response. Investigation of surface-cave hybrids reveals a relationship between angular speed and peak angle, suggesting these two kinematic characteristics are related at the genetic or functional levels. Together, these findings provide support for the use ofA. mexicanusas a model to investigate the evolution of escape behavior.

Many species execute ballistic escape reactions to avoid imminent danger. Despite fast reaction times, responses are often highly regulated, reflecting a trade-off between costly motor actions and perceived threat level. However, how sensory cues are integrated within premotor escape circuits remains poorly understood. Here, we show that in zebrafish, less precipitous threats elicit a delayed escape, characterized by flexible trajectories, which are driven by a cluster of 38 prepontine neurons that are completely separate from the fast escape pathway. Whereas neurons that initiate rapid escapes receive direct auditory input and drive motor neurons, input and output pathways for delayed escapes are indirect, facilitating integration of cross-modal sensory information. These results show that rapid decision-making in the escape system is enabled by parallel pathways for ballistic responses and flexible delayed actions and defines a neuronal substrate for hierarchical choice in the vertebrate nervous system., Many species execute ballistic escape reactions to avoid imminent danger, but this is not always the best strategy. This study shows that zebrafish rapidly assess the salience of an auditory threat to select either an immediate ballistic response or a delayed and more flexible escape that uses a distinct and newly discovered neural pathway.

Mauthner neurons in goldfish fry were studied by the methods of light and electron microscopy. The structure and volume of individual dendrites as well as the structure of axodendritic synapses were examined using virtual images of neurons formed from serial 3-μ sections. In short-time (5 h) experiments with application of dopamine, β-amyloid fragment (25-35), and long-term sensory stimulation affecting afferent inputs to Mauthner neurons, the dendrites were larger than the same dendrites under the same conditions without dopamine application. Application of dopamine induced no pathological changes in the structure of axodendritic chemical and electric synapses containing desmosome-like contacts.

The process by which future behavioral responses are shaped by past experiences is one of the central questions in neuroscience. To gain insight into this process at the molecular and cellular levels, we have applied zebrafish larvae to explore behavioral desensitization to sound. A sudden loud noise often evokes a defensive response known as the acoustic startle response (ASR), which is triggered by firing Mauthner cells in teleosts and amphibians. The probability of evoking ASR by suprathreshold sound is reduced after exposure to repetitive auditory stimuli insufficient in amplitude to evoke the ASR (subthreshold). Although it has been suggested that the potentiation of inhibitory glycinergic inputs into Mauthner cell is involved in this desensitization of the ASR, the molecular basis for the potentiation of glycinergic transmission has been unclear. Through thein vivomonitoring of fluorescently-tagged glycine receptors (GlyRs), we here showed that behavioral desensitization to sound in zebrafish is governed by GlyR clustering in Mauthner cells. We further revealed that CaMKII-dependent phosphorylation of the scaffolding protein gephyrin at serine 325 promoted the synaptic accumulation of GlyR on Mauthner neurons through the enhancement of the gephyrin-GlyR binding, which was indispensable for and could induce desensitization of the ASR. Our study demonstrates an essential molecular and cellular basis of sound-induced receptor dynamics and thus of behavioral desensitization to sound.SIGNIFICANCE STATEMENTBehavioral desensitization in the acoustic startle response of fish is known to involve the potentiation of inhibitory glycinergic input to the Mauthner cell, which is a command neuron for the acoustic startle response. However, the molecular and cellular basis for this potentiation has been unknown. Here we show that an increase in glycine receptor (GlyR) clustering at synaptic sites on zebrafish Mauthner cells is indispensable for and could induce desensitization. Furthermore, we demonstrate that CaMKII-mediated phosphorylation of the scaffolding protein gephyrin promotes GlyR clustering by increasing the binding between the β-loop of GlyRs and gephyrin. Thus, the phosphorylation of gephyrin is a key event which accounts for the potentiation of inhibitory glycinergic inputs observed during sound-evoked behavioral desensitization.

The central nervous system of theCionalarva contains only 177 neurons. The precise regulation of neuron subtype-specific morphogenesis and differentiation observed in during the formation of this minimal connectome offers a unique opportunity to dissect gene regulatory networks underlying chordate neurodevelopment. Here we compare the transcriptomes of two very distinct neuron types in the hindbrain/spinal cord homolog ofCiona, the Motor Ganglion (MG): the Descending decussating neuron (ddN, proposed homolog of Mauthner Cells in vertebrates) and the MG Interneuron 2 (MGIN2). Both types are invariantly represented by a single bilaterally symmetric left/right pair of cells in every larva. Supernumerary ddNs and MGIN2s were generated in synchronized embryos and isolated by fluorescence-activated cell sorting for transcriptome profiling. Differential gene expression analysis revealed ddN- and MGIN2-specific enrichment of a wide range of genes, including many encoding potential “effectors” of subtype-specific morphological and functional traits. More specifically, we identified the upregulation of centrosome-associated, microtubule-stabilizing/bundling proteins and extracellular matrix proteins and axon guidance cues as part of a single intrinsic regulatory program that might underlie the unique polarization of the ddNs, the only descending MG neurons that cross the midline.

During many behaviors in vertebrates, the CNS generates asymmetric activities between the left and right sides to produce asymmetric body movements. For asymmetrical activations of the CNS, reciprocal inhibition between the left and right sides is believed to play a key role. However, the complexity of the CNS makes it difficult to identify the reciprocal inhibition circuits at the level of individual cells and the contribution of each neuron to the asymmetric activity. Using larval zebrafish, we examined this issue by investigating reciprocal inhibition circuits between a pair of Mauthner (M) cells, giant reticulospinal neurons that trigger fast escapes. Previous studies have shown that a class of excitatory neurons, called cranial relay neurons, is involved in the reciprocal inhibition pathway between the M cells. Using transgenic fish, in which two of the cranial relay neurons (Ta1 and Ta2) expressed GFP, we showed that Ta1 and Ta2 constitute major parts of the pathway. In larvae in which Ta1/Ta2 were laser-ablated, the amplitude of the reciprocal IPSPs dropped to less than one-third. Calcium imaging and electrophysiological recording showed that the occurrence probability of bilateral M-cell activation upon sound/vibration stimuli was greatly increased in the Ta1/Ta2-ablated larvae. Behavioral experiments revealed that the Ta1/Ta2 ablation resulted in shallower body bends during sound/vibration-evoked escapes, which is consistent with the observation that increased occurrence of bilateral M-cell activation impaired escape performance. Our study revealed major components of the reciprocal inhibition circuits in the M cell system and the behavioral importance of the circuits.SIGNIFICANCE STATEMENTReciprocal inhibition between the left and right side of the CNS is considered imperative for producing asymmetric movements in animals. It has been difficult, however, to identify the circuits at the individual cell level and their role in behavior. Here, we address this problem by examining the reciprocal inhibition circuits of the hindbrain Mauthner (M) cell system in larval zebrafish. We determined that two paired interneurons play a critical role in the reciprocal inhibition between the paired M cells and that the reciprocal inhibition prevents bilateral firing of the M cells and is thus necessary for the full body bend during M cell-initiated escape. Further, we discussed the cooperation of multiple reciprocal inhibitions working in the hindbrain and spinal cord to ensure high-performance escapes.

Processing of multimodal information is essential for an organism to respond to environmental events. However, how multimodal integration in neurons translates into behavior is far from clear. Here, we investigate integration of biologically relevant visual and auditory information in the goldfish startle escape system in which paired Mauthner-cells (M-cells) initiate the behavior. Sound pips and visual looms as well as multimodal combinations of these stimuli were tested for their effectiveness of evoking the startle response. Results showed that adding a low intensity sound early during a visual loom (low visual effectiveness) produced a supralinear increase in startle responsiveness as compared to an increase expected from a linear summation of the two unimodal stimuli. In contrast, adding a sound pip late during the loom (high visual effectiveness) increased responsiveness consistent with a linear multimodal integration of the two stimuli. Together the results confirm the Inverse Effectiveness Principle (IEP) of multimodal integration proposed in other species. Given the well-established role of the M-cell as a multimodal integrator, these results suggest that IEP is computed in individual neurons that initiate vital behavioral decisions.

Harmful algal blooms (HABs) can produce potent neurotoxins that accumulate in seafood and affect human health. One HAB toxin of concern is domoic acid (DomA), a glutamate analog produced by the marine diatom Pseudo-nitzschia spp. Current regulatory limits are designed to prevent acute neurotoxicity in adult humans. However, research shows that low-level exposure during early life can lead to long-term changes in behavior, neural connectivity, and brain morphology. To determine the underlying mechanisms of developmental toxicity, this dissertation used zebrafish as a tool to: i) Establish the developmental window of susceptibility for DomA toxicity, ii) Characterize the behavioral consequences of exposures, and iii) Identify the cellular targets and processes perturbed by DomA. I found that DomA exposure particularly at 2 days post fertilization (dpf) led to altered startle response behavior, myelination defects, and the downregulation of axonal and myelin structural genes. Using vital dyes and immunolabeling, I assessed DomA-induced alterations in cells required for the startle response. I found no differences in the number of sensory neuromasts or in the sensory cranial ganglia structures that detect the acoustic stimuli. However, the majority of DomA-treated larvae lacked one or both Mauthner cells – hindbrain neurons critical for fast startle responses. DomA-treated larvae also had oligodendrocytes with fewer and shorter myelin sheaths, and appeared to aberrantly myelinate neuronal cell bodies. The loss of the Mauthner neurons and their axons may lead to a cellular environment where oligodendrocytes myelinate neuronal cell bodies in the absence of adequate axonal targets. Indeed, pharmacological treatment that reduced the oligodendrocyte number also led to the reduction in the number of these aberrant, myelinated cell bodies. These results indicate that exposure to DomA at a particular period in neural development targets specific cell types, disrupts myelination in the spinal cord, and leads to prolonged behavioral deficits. These mechanistic insights support hazard assessments of DomA exposures in humans during critical periods in early development.

The formin family member Fmn2 is a neuronally enriched cytoskeletal remodeling protein conserved across vertebrates. Recent studies have implicated Fmn2 in neurodevelopmental disorders, including sensory processing dysfunction and intellectual disability in humans. Cellular characterization of Fmn2 in primary neuronal cultures has identified its function in the regulation of cell-substrate adhesion and consequently growth cone translocation. However, the role of Fmn2 in the development of neural circuitsin vivo, and its impact on associated behaviors have not been tested. Using automated analysis of behavior and systematic investigation of the associated circuitry, we uncover the role of Fmn2b in zebrafish neural circuit development. As reported in other vertebrates, the zebrafish ortholog of Fmn2 is also enriched in the developing zebrafish nervous system. We find that Fmn2b is required for the development of an excitatory interneuron pathway, the spiral fiber neuron, which is an essential circuit component in the regulation of the Mauthner cell (M-cell)-mediated acoustic startle response. Consistent with the loss of the spiral fiber neurons tracts, high-speed video recording revealed a reduction in the short latency escape events while responsiveness to the stimuli was unaffected. Taken together, this study provides evidence for a circuit-specific requirement of Fmn2b in eliciting an essential behavior in zebrafish. Our findings underscore the importance of Fmn2 in neural development across vertebrate lineages and highlight zebrafish models in understanding neurodevelopmental disorders.

Stimulating neuronal activity increases myelin sheath formation by individual oligodendrocytes, but how myelination is regulated by neuronal activity in vivo is still not fully understood. While in vitro studies have revealed the important role of N-cadherin in myelination, our understanding in vivo remains quite limited. To obtain the role of N-cadherin during activity-dependent regulation of myelinating capacity of individual oligodendrocytes, we successfully built an in vivo dynamic imaging model of the Mauthner cell at the subcellular structure level in the zebrafish central nervous system. Enhanced green fluorescent protein (EGFP)-tagged N-cadherin was used to visualize the stable accumulations and mobile transports of N-cadherin by single-cell electroporation at the single-cell level. We found that pentylenetetrazol (PTZ) significantly enhanced the accumulation of N-cadherin in Mauthner axons, a response that was paralleled by enhanced sheath number per oligodendrocytes. By offsetting this phenotype using oligopeptide (AHAVD) which blocks the function of N-cadherin, we showed that PTZ regulates myelination in an N-cadherin-dependent manner. What is more, we further suggested that PTZ influences N-cadherin and myelination via a cAMP pathway. Consequently, our data indicated that N-cadherin is involved in neuronal activity-dependent regulation of myelinating capacity of zebrafish individual oligodendrocytes in vivo.

The human natural killer cell antigen-1 (HNK-1) is functionally important in development, synaptic activity, and regeneration after injury in the nervous system of several mammalian species. It contains a sulfated glucuronic acid which is carried by neural adhesion molecules and expressed in nonmammalian species, including zebrafish, which, as opposed to mammals, spontaneously regenerate after injury in the adult. To evaluate HNK-1's role in recovery of function after spinal cord injury (SCI) of adult zebrafish, we assessed the effects of the two HNK-1 synthesizing enzymes, glucuronyl transferase and HNK-1 sulfotransferase. Expression of these two enzymes was increased at the messenger RNA (mRNA) level 11 days after injury in the brainstem nuclei that are capable of regrowth of severed axons, namely, the nucleus of medial longitudinal fascicle and intermediate reticular formation, but not at earlier time points after SCI. mRNA levels of glucuronyl transferase and sulfotransferase were increased in neurons, not only of these nuclei but also in the spinal cord caudal to the injury site at 11 days. Mauthner neurons which are not capable of regeneration did not show increased levels of enzyme mRNAs after injury. Reducing protein levels of the enzymes by application of anti-sense morpholinos resulted in reduction of locomotor recovery for glucuronyl transferase, but not for HNK-1 sulfotransferase. The combined results indicate that HNK-1 is upregulated in expression only in those neurons that are intrinsically capable of regeneration and contributes to regeneration after spinal cord injury in adult zebrafish in the absence of its sulfate moiety.

Some neurons, especially in mammalian peripheral nervous system or in lower vertebrate or in vertebrate central nervous system (CNS) regenerate after axotomy, while most mammalian CNS neurons fail to regenerate. There is an emerging consensus that neurons have different intrinsic regenerative capabilities, which theoretically could be manipulated therapeutically to improve regeneration. Population-based comparisons between “good regenerating” and “bad regenerating” neurons in the CNS and peripheral nervous system of most vertebrates yield results that are inconclusive or difficult to interpret. At least in part, this reflects the great diversity of cells in the mammalian CNS. Using mammalian nervous system imposes several methodical limitations. First, the small sizes and large numbers of neurons in the CNS make it very difficult to distinguish regenerating neurons from non-regenerating ones. Second, the lack of identifiable neurons makes it impossible to correlate biochemical changes in a neuron with axonal damage of the same neuron, and therefore, to dissect the molecular mechanisms of regeneration on the level of single neurons. This review will survey the reported responses to axon injury and the determinants of axon regeneration, emphasizing non-mammalian model organisms, which are often under-utilized, but in which the data are especially easy to interpret.

Filtering mechanisms prevent a continuous stream of sensory information from swamping perception, leading to diminished focal attention and cognitive processing. Mechanisms for sensory gating are commonly studied using prepulse inhibition, a paradigm that measures the regulated transmission of auditory information to the startle circuit, however the underlying neuronal pathways are unresolved. Using large-scale calcium imaging, optogenetics and laser ablations, we reveal a cluster of 30 morphologically identified neurons in zebrafish that suppress the transmission of auditory signals during prepulse inhibition. These neurons project to a key sensorimotor interface in the startle circuit — the termination zone of auditory afferents on the dendrite of a startle command neuron. Direct measurement of auditory nerve neurotransmitter release revealed selective presynaptic inhibition of sensory transmission to the startle circuit, sparing signaling to other brain regions. Our results provide the first cellular resolution circuit for prepulse inhibition in a vertebrate, revealing a central role for presynaptic gating of sensory information to a brainstem motor circuit.

Archerfish use two powerful C-starts: One to escape threats, the other to secure prey that they have downed with a shot of water. The two C-starts are kinematically equivalent, are variable in both phases, and the predictive C-starts – used in hunting – are adjusted in the angle of turning and in the final linear speed to where and when their prey will hit the water surface. Presently nothing is known about the circuits that drive the archerfish C-starts. As the starting point for a neuroethological analysis, we first explored the presence and morphology of any paired Mauthner cell, a key cell in the teleost fast-start system. We show that archerfish have a typical Mauthner cell in each medullary hemisphere and that these send by far the largest axons down the spinal cord. Stimulation of the spinal cord caused short-latency all-or-none field potentials that could be detected even at the surface of the medulla and that had the archerfish Mauthner cell as its only source. The archerfish's Mauthner cell is remarkably similar morphologically to that of equally sized goldfish, except that the archerfish's ventral dendrite is slightly longer and its lateral dendrite thinner. Our data provide the necessary starting point for the dissection of the archerfish fast-start system and of any role potentially played by its Mauthner cell in the two C-start manoeuvres. Moreover, they do not support the recently expressed view that Mauthner cells should be reduced in animals with highly variable fast-start manoeuvres.

We experience various sensations through our skin. The sensations are received by distinct sensory channels that are expressed in the trigeminal ganglion (TG) or the dorsal root ganglion (DRG). TG neurons innervate the head skin, whereas DRG neurons innervate the skin of the trunk and limbs. In addition to these neuronal populations, larvae of anamniote vertebrates (lampreys, teleosts, and amphibians) have an additional sensory neuronal population that develops prior to functional maturation of DRG neurons, termed Rohon-Beard (RB) neurons. RB neurons innervate the trunk skin; thus, the TG and RB neurons are responsible for larval somatosensation. After the maturation of DRG neurons, the physiological roles of the RB neurons are replaced progressively by the DRG neurons. Studies of somatosensation in zebrafish have suggested that the transition from RB neurons to DRG neurons is completed within 5 days post fertilization. During this transition, the RB neurons undergo programmed cell death; thus, RB neurons have been considered to be a transient neuronal population. However, recent studies using zebrafish have indicated that some RB neurons survive for at least 2 weeks post-fertilization. These long-lived RBs are distinguished by Protein Kinase C-α (PKCα) expression and comprise

Exposure to repetitive startling stimuli induces habitation, a simple form of learning. Despite its simplicity, the precise cellular mechanisms by which repeated stimulation converts a robust behavioral response to behavioral indifference are unclear. Here, we use head-restrained zebrafish larvae to monitor subcellular Ca2+ dynamics in Mauthner neurons, the startle command neurons, during startle habituation in vivo. Using the Ca2+ reporter GCaMP6s we find that the amplitude of Ca2+ signals in the lateral dendrite of the Mauthner neuron determines startle probability and that depression of this dendritic activity rather than downstream inhibition mediates short-term habituation mediates glycine and N-methyl-D-aspartate (NMDA) receptor dependent short-term habituation. Combined, our results suggest a model for habituation learning in which increased inhibitory drive from feedforward inhibitory neurons combined with decreased excitatory input from auditory afferents decreases dendritic and Mauthner neuron excitability.

Investigation of the Mauthner neurons (MN) morphology in fish Perccottus glehni revealed a significant reduction in their volumes during dormancy, probably stipulated by a need to spare the body energy resources. The initial period of winter dormancy was characterized by a decrease in volumes of both main dendrites with no changes in somatic part of MN. During the terminal period of winter dormancy a total volume of soma and lateral dendrite reduced by 50%, and for the ventral one it did by 80%. It can be assumed that a decrease in integral neuronal volumes is associated with developing hypometabolism, as well as with a significantly decreased inputs of afferent information, entering MN from auditory and visual analyzers. Probl Cryobiol Cryomed 2015; 25(2): 114-121

Summary The Mauthner cell (M-cell) is a command-like neuron in teleost fish whose firing in response to aversive stimuli is correlated with short-latency escapes [1–3]. M-cells have been proposed as evolutionary ancestors of startle response neurons of the mammalian reticular formation [4], and studies of this circuit have uncovered important principles in neurobiology that generalize to more complex vertebrate models [3]. The main excitatory input was thought to originate from multisensory afferents synapsing directly onto the M-cell dendrites [3]. Here, we describe an additional, convergent pathway that is essential for the M-cell-mediated startle behavior in larval zebrafish. It is composed of excitatory interneurons called spiral fiber neurons, which project to the M-cell axon hillock. By in vivo calcium imaging, we found that spiral fiber neurons are active in response to aversive stimuli capable of eliciting escapes. Like M-cell ablations, bilateral ablations of spiral fiber neurons largely eliminate short-latency escapes. Unilateral spiral fiber neuron ablations shift the directionality of escapes and indicate that spiral fiber neurons excite the M-cell in a lateralized manner. Their optogenetic activation increases the probability of short-latency escapes, supporting the notion that spiral fiber neurons help activate M-cell-mediated startle behavior. These results reveal that spiral fiber neurons are essential for the function of the M-cell in response to sensory cues and suggest that convergent excitatory inputs that differ in their input location and timing ensure reliable activation of the M-cell, a feedforward excitatory motif that may extend to other neural circuits.

We examined effects resulting from application of aggregated beta-amyloid on Mauthner neurons (MNs) of ambidextral goldfishes and long-lasting rotatory stimulation on lateral motor behavior of such fishes; these individuals were preliminarily adapted to repetitive rotatory stimulation. Using 3D histological reconstruction of the above cells, we measured the volumes of the right and left MNs and examined the ultrastructure of specialized synaptic compartments in excitatory synapses localized on the surface of the soma, lateral dendrite, and ventral dendrite of these cells. In the course of the experiments, control fishes remain ambidextrals, and the mean volumes of the right and left MNs in these individuals were close to each other. In a fish of this group, dimensions of the active zones (AZs) of chemical synapses localized on the lateral dendrite were considerably greater in the right neuron, while those in synapses on the lateral dendrite were smaller in the left neuron. Experimental fishes subjected to the above-mentioned procedures demonstrated clearly expressed motor lateralization; the mean volumes of their right and left MNs were, however, practically equal to each other. Ultrastructural analysis of peculiarities of MNs of such a fish showed that dimensions of the AZs in chemical synapses on the soma and lateral dendrite of the left MN, which became much less active functionally, significantly exceeded the analogous indices in synapses of the same localization on the right, more active, MN. In mixed synapses, the mean dimensions of gap junctions in control and experimental individuals were similar to each other independently of localization of such synapses. Interrelations between functional asymmetry of MNs manifested in lateralization of motor behavior of the fishes and dimensions of synaptic structures on these cells are discussed.

Second-order sensory neurons are dependent on afferents from the sense organs during a critical period in development for their survival and differentiation. Past research has mostly focused on whole populations of neurons, hampering progress in understanding the mechanisms underlying these critical phases. To move toward a better understanding of the molecular and cellular basis of afferent-dependent neuronal development, we developed a new model to study the effects of ear removal on a single identifiable cell in the hindbrain of a frog, the Mauthner cell. Ear extirpation at various stages of Xenopus laevis development defines a critical period of progressively-reduced dependency of Mauthner cell survival/differentiation on the ear afferents. Furthermore, ear removal results in a progressively decreased reduction in the number of dendritic branches. Conversely, addition of an ear results in an increase in the number of dendritic branches. These results suggest that the duration of innervation and the number of inner ear afferents play a quantitative role in Mauthner cell survival/differentiation, including dendritic development. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1339–1351, 2015

Gap junctions provide for direct intercellular electrical and metabolic coupling. The abundance of gap junctions at "large myelinated club ending (LMCE)" synapses on Mauthner cells (M-cells) of the teleost brain provided a convenient model to correlate anatomical and physiological properties of electrical synapses. There, presynaptic action potentials were found to evoke short-latency electrical "pre-potentials" immediately preceding their accompanying glutamate-induced depolarizations, making these the first unambiguously identified "mixed" (i.e., chemical plus electrical) synapses in the vertebrate CNS. We recently showed that gap junctions at these synapses exhibit asymmetric electrical resistance (i.e., electrical rectification), which we correlated with total molecular asymmetry of connexin composition in their apposing gap junction hemiplaques, with connexin35 (Cx35) restricted to axon terminal hemiplaques and connexin34.7 (Cx34.7) restricted to apposing M-cell plasma membranes. We now show that similarly heterotypic neuronal gap junctions are abundant throughout goldfish brain, with labeling exclusively for Cx35 in presynaptic hemiplaques and exclusively for Cx34.7 in postsynaptic hemiplaques. Moreover, the vast majority of these asymmetric gap junctions occur at glutamatergic axon terminals. The widespread distribution of heterotypic gap junctions at glutamatergic mixed synapses throughout goldfish brain and spinal cord implies that pre- vs. postsynaptic asymmetry at electrical synapses evolved early in the chordate lineage. We propose that the advantages of the molecular and functional asymmetry of connexins at electrical synapses that are so prominently expressed in the teleost CNS are unlikely to have been abandoned in higher vertebrates. However, to create asymmetric coupling in mammals, where most gap junctions are composed of connexin36 (Cx36) on both sides, would require some other mechanism, such as differential phosphorylation of connexins on opposite sides of the same gap junction or on asymmetric differences in the complement of their scaffolding and regulatory proteins.

Axon regeneration, fundamental to nerve repair, and functional recovery, relies on rapid changes in gene expression attributable to microRNA (miRNA) regulation. MiR-133b has been proved to play an important role in different organ regeneration in zebrafish, but its role in regulating axon regeneration in vivo is still controversial. Here, combining single-cell electroporation with a vector-based miRNA-expression system, we have modulated the expression of miR-133b in Mauthner-cells (M-cells) at the single-cell level in zebrafish. Through in vivo imaging, we show that overexpression of miR-133b inhibits axon regeneration, whereas down-regulation of miR-133b, promotes axon outgrowth. We further show that miR-133b regulates axon regeneration by directly targeting a novel regeneration-associated gene, tppp3, which belongs to Tubulin polymerization-promoting protein family. Gain or loss-of-function of tppp3 experiments indicated that tppp3 was a novel gene that could promote axon regeneration. In addition, we observed a reduction of mitochondrial motility, which have been identified to have a positive correlation with axon regeneration, in miR-133b overexpressed M-cells. Taken together, our work provides a novel way to study the role of miRNAs in individual cell and establishes a critical cell autonomous role of miR-133b in zebrafish M-cell axon regeneration. We propose that up-regulation of the newly founded regeneration-associated gene tppp3 may enhance axonal regeneration.

Predator pressure and olfactory cues (alarm substance) have been shown to modulate Mauthner cell (M-cell) initiated startle escape responses (C-starts) in teleost fish. The regulation of such adaptive responses to potential threats is thought to involve the release of steroid hormones such as cortisol. However, the mechanism by which cortisol may regulate M-cell excitability is not known. Here, we used intrasomatic, in vivo recordings to elucidate the acute effects of cortisol on M-cell membrane properties and sound evoked post-synaptic potentials (PSPs). Cortisol tonically decreased threshold current in the M-cell within 10 min before trending towards baseline excitability over an hour later, which may indicate the involvement of non-genomic mechanisms. Consistently, current ramp injection experiments showed that cortisol increased M-cell input resistance in the depolarizing membrane, i.e., by a voltage-dependent postsynaptic mechanism. Cortisol also increases the magnitude of sound-evoked M-cell PSPs by reducing the efficacy of local feedforward inhibition (FFI). Interestingly, another pre-synaptic inhibitory network mediating prepulse inhibition (PPI) remained unaffected. Together, our results suggest that cortisol rapidly increases M-cell excitability via a post-synaptic effector mechanism, likely a chloride conductance, which, in combination with its dampening effect on FFI, will modulate information processing to reach threshold. Given the central role of the M-cell in initiating startle, these results are consistent with a role of cortisol in mediating the expression of a vital behavior.

Key points: The present study examines dendritic integrative processes that occur in many central neurons but have been challenging to study in vivo in the vertebrate brain. The Mauthner cell of goldfish receives auditory and visual information via two separate dendrites, providing a privileged scenario for in vivo examination of dendritic integration. The results show differential attenuation properties in the Mauthner cell dendrites arising at least partly from differences in cable properties and the nonlinear behaviour of the respective dendritic membranes. In addition to distinct modality-dependent membrane specialization in neighbouring dendrites of the Mauthner cell, we report cross-modal dendritic interactions via backpropagating postsynaptic potentials. Broadly, the results of the present study provide an exceptional example for the processing power of single neurons. Abstract: Animals process multimodal information for adaptive behavioural decisions. In fish, evasion of a diving bird that breaks the water surface depends on integrating visual and auditory stimuli with very different characteristics. How do neurons process such differential sensory inputs at the dendritic level? For that, we studied the Mauthner cells (M-cells) in the goldfish startle circuit, which receive visual and auditory inputs via two separate dendrites, both accessible for in vivo recordings. We investigated whether electrophysiological membrane properties and dendrite morphology, studied in vivo, play a role in selective sensory processing in the M-cell. The results obtained show that anatomical and electrophysiological differences between the dendrites combine to produce stronger attenuation of visually evoked postsynaptic potentials (PSPs) than to auditory evoked PSPs. Interestingly, our recordings showed also cross-modal dendritic interaction because auditory evoked PSPs invade the ventral dendrite (VD), as well as the opposite where visual PSPs invade the lateral dendrite (LD). However, these interactions were asymmetrical, with auditory PSPs being more prominent in the VD than visual PSPs in the LD. Modelling experiments imply that this asymmetry is caused by active conductances expressed in the proximal segments of the VD. The results obtained in the present study suggest modality-dependent membrane specialization in M-cell dendrites suited for processing stimuli of different time domains and, more broadly, provide a compelling example of information processing in single neurons. Fil: Medan, Violeta. City University of New York; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Mäki Marttunen, Tuomo. Universidad de Tampere; Finlandia. University of Oslo; Noruega Fil: Sztarker, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Preuss, Thomas. City University of New York; Estados Unidos

Electrical synapses with diverse configurations and functions occur at a variety of interneuronal appositions, thereby significantly expanding the physiological complexity of neuronal circuitry over that provided solely by chemical synapses. Gap junctions between apposed dendritic and somatic plasma membranes form “purely electrical” synapses that allow for electrical communication between coupled neurons. In addition, gap junctions at axon terminals synapsing on dendrites and somata allow for “mixed” (dual chemical + electrical) synaptic transmission. “Dual transmission” was first documented in the autonomic nervous system of birds, followed by its detection in the central nervous systems of fish, amphibia, and reptiles. Subsequently, mixed synapses have been detected in several locations in the mammalian CNS, where their properties and functional roles remain undetermined. Here, we review available evidence for the presence, complex structural composition, and emerging functional properties of mixed synapses in the mammalian CNS.

Escape is among the simplest animal behaviors employed to study the neural mechanisms underlying learning. Teleost fishes exhibit behavioral learning of fast escape initiated with a C-shaped body bend (C-start). C-starts are subdivided into short-latency (SLC) and long-latency (LLC) types in larval zebrafish. Whether these two can be separately modified, and the neural correlates of this modification, however, remains undetermined. We thus performed Ca2+ imaging of Mauthner (M-) cells, a pair of giant hindbrain neurons constituting a core element of SLC circuit, during behavioral learning in larval zebrafish. The Ca2+ response corresponding to a single spiking of the M-cells was coupled with SLCs but not LLCs. Conditioning with a repeated weak sound at subthreshold intensity to elicit C-starts selectively suppressed SLC occurrence for 10min without affecting LLC responsiveness. The short-term desensitization of SLC was associated with the suppression of M-cell activity, suggesting that changes in single neuron responsiveness mediate behavioral learning. The conditioning did not affect the acoustically evoked mechanotransduction of inner ear hair cells, further suggesting plastic change in transmission efficacy within the auditory input circuit between the hair cells and the M-cell.

Mitochondrial transport is essential for neuronal function, whereas the evidence of connections between mitochondrial transport and axonal regeneration in living vertebrate central nervous systems (CNS) remains limited. Here, we developed a novel model which can explore mitochondrial transport in a single Mauthner axon (M axons) of zebrafish by non-invasive in vivo imaging. To ensure the feasibility of this model, we treated labeled zebrafish with nocodazole and demonstrated that nocodazole could disrupt mitochondrial transport in this model. Meanwhile combining with two-photon laser axotomy, we precisely axotomized M axons and recorded M axonal regeneration and their mitochondrial transport in living zebrafish larvae simultaneously. The data proved that the injured axons which have a stronger regenerative capability accompanied with higher mitochondrial motility. Furthermore, in order to stimulate axonal regeneration, treating with dibutyryl cyclic adenosine monophosphate (db-cAMP) could also augment mitochondrial motility. Taken together, our results provided new insights that mitochondrial motility is positively correlated to axonal regeneration in living vertebrate CNS and this promising model we developed will be useful for further studies on the interaction of axonal regeneration with its mitochondrial dynamics through various genetic and pharmacological regulation.

Morphometric methods were used to study the ultrastructure of afferent synapses on the ventral dendrites of Mauthner neurons (MN) during adaptation of goldfish to prolonged, fatiguing, sensory (visual) stimulation inducing increased resistance of MN. The extent of active zones (AZ) in synapses located on the MN ventral dendrite was found to decrease significantly, by 23%, after adaptation. The extent of the AZ of excitatory visual synapses decreased by 29% as compared with controls, with a simultaneous 71% increase in the extent of desmosome-like contacts (DLC) bordering AZ. There was also a 19% reduction in the extent of AZ of inhibitory synapses after adaptation, which is consistent with the important role of inhibitory processes in sensory pathways during memory formation. Considering the actin nature of DLC, it can be suggested that adaptation to visual stimulation is based on a synaptic mechanism of regulation of neurotransmitter secretion by actin.