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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.

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.

—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.

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

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.

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.

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.

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.

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.

Each neuron possesses a unique firing property, which is largely attributed to heterogeneity in the composition of voltage-gated ion channel complexes. Zebrafish Mauthner (M) cells, which are bilaterally paired giant reticulospinal neurons (RSNs) in the hindbrain and induce rapid escape behavior, generate only a single spike at the onset of depolarization. This single spiking is in contrast with the repetitive firing of the M cell's morphologically homologous RSNs, MiD2cm and MiD3cm, which are also involved in escapes. However, how the unique firing property of M cells is established and the underlying molecular mechanisms remain unclear. In the present study, we first demonstrated that the single-spiking property of M cells was acquired at 4 days postfertilization (dpf), accompanied by an increase in dendrotoxin I (DTX)-sensitive low-threshold K+currents, prior to which the M cell repetitively fires as its homologs. Second, in situ hybridization showed that among DTX-sensitive Kv1 channel α-subunits, zKv1.1a was unexpectedly expressed even in the homologs and the bursting M cells at 2 dpf. In contrast, zKvβ2b, an auxiliary β-subunit of Kv1 channels, was expressed only in the single-spiking M cells. Third, zKv1.1a expressed in Xenopus oocytes functioned as a low-threshold K+channel, and its currents were enhanced by coexpression of zKvβ2b subunits. Finally, knockdown of zKvβ2b expression in zebrafish larvae resulted in repetitive firing of M cells at 4 dpf. Taken together, these results suggest that associative expression of Kvβ2 subunits with Kv1.1 channels is crucial for developmental acquisition of the unique firing properties of the M cells among homologous neurons.

Brain function relies on the ability of neurons to communicate with each other. Interneuronal communication primarily takes place at synapses, where information from one neuron is rapidly conveyed to a second neuron. There are two main modalities of synaptic transmission: chemical and electrical. Far from functioning independently and serving unrelated functions, mounting evidence indicates that these two modalities of synaptic transmission closely interact, both during development and in the adult brain. Rather than conceiving synaptic transmission as either chemical or electrical, this article emphasizes the notion that synaptic transmission is both chemical and electrical, and that interactions between these two forms of interneuronal communication might be required for normal brain development and function.

Immunogold labeling was used to identify serotoninergic synapses of the chemical and mixed types on the ventral dendrite (VD) of Mauthner neurons (MN) in goldfish. These had the characteristic feature that they contained several mitochondria in the bouton and, apart from an active zone (AZ), had an obligatory desmosome-like contact (DlC). The AZ of these synapses generally contacted unlabeled chemical crested synapses, which in turn interacted directly with the VD. These latter synapses had virtually no mitochondria and lacked a DlC, such that they could be identified as inhibitory. This “two-level” organization of excitatory serotoninergic and inhibitory synapses appears to be directly related to the reciprocal mechanism regulating the functional activity of MN via the visual apparatus.

SummaryElectrical synapses are abundant in the vertebrate brain, but their functional and molecular complexities are still poorly understood. We report here that electrical synapses between auditory afferents and goldfish Mauthner cells are constructed by apposition of hemichannels formed by two homologs of mammalian connexin 36 (Cx36) and that, while Cx35 is restricted to presynaptic hemiplaques, Cx34.7 is restricted to postsynaptic hemiplaques, forming heterotypic junctions. This molecular asymmetry is associated with rectification of electrical transmission that may act to promote cooperativity between auditory afferents. Our data suggest that, in similarity to pre- and postsynaptic sites at chemical synapses, one side in electrical synapses should not necessarily be considered the mirror image of the other. While asymmetry based on the presence of two Cx36 homologs is restricted to teleost fish, it might also be based on differences in posttranslational modifications of individual connexins or in the complement of gap junction-associated proteins.

Summary Background During locomotion in vertebrates, reticulospinal neurons in the hindbrain play critical roles in providing descending excitation to the spinal cord locomotor systems. However, despite the fact that many genes that are used to classify the neuronal identities of neurons in the hindbrain have been identified, the molecular identity of the reticulospinal neurons that are critically involved in locomotor drive is not well understood. Chx10-expressing neurons (V2a neurons) are ipsilaterally projecting glutamatergic neurons in the spinal cord and the hindbrain. Many of the V2a neurons in the hindbrain are known to project to the spinal cord in zebrafish, making hindbrain V2a neurons a prime candidate in descending locomotor drive. Results We investigated the roles of hindbrain V2a neurons using optogenetic and electrophysiological approaches. The forced activation of hindbrain V2a neurons using channelrhodopsin efficiently evoked swimming, whereas the forced inactivation of them using Archearhodopsin3 or Halorhodpsin reliably stopped ongoing swimming. Electrophysiological recordings of two populations of hindbrain reticulospinal V2a neurons showed that they were active during swimming. One population of neurons, small V2a neurons in the caudal hindbrain, fired with low rhythmicity, whereas the other population of neurons, large reticulospinal V2a neurons, called MiV1 neurons, fired more rhythmically. Conclusions These results indicated that hindbrain reticulospinal V2a neurons play critical roles in providing excitation to the spinal locomotor circuits during swimming by providing both tonic and phasic inputs to the circuits.

Goldfish are known to exhibit motor asymmetry due to functional asymmetry of their Mauthner neurons that induce the turns to the right or left during free swimming. It has been previously found that if the less active neuron is subjected to prolonged aimed visual stimulation via its ventral dendrite, the motor asymmetry of goldfish is inverted, testifying that this neuron becomes functionally dominant, while the size of the ventral dendrite under these conditions is reduced 2-3 times compared to its counterpart in mirror neuron. Earlier it has been also revealed that training optokinetic stimulation induces adaptation, a substantial resistance of both fish motor asymmetry and morphofunctional state of Mauthner neurons against prolonged optokinetic stimulation. The aim of this work was to study the cellular mechanisms of the effect of an unusual visual afferent input on goldfish motor asymmetry and Mauthner neuron function in norm and under adaptation. It was shown that serotonin applied onto Mauthner neurons greatly reduces their activity whereas its antagonist ondansetron increases it. Against the background of visual stimulation, serotonin strengthens functional asymmetry between neurons whereas ondansetron smoothes it. Taken together these data suggest the involvement of serotonergic excitatory synaptic transmission in the regulation of Mauthner neurons by vision. Ultrastructural study of the ventral dendrites after prolonged optokinetic stimulation has revealed depletions of numeral axo-axonal synapses with specific morphology, identified by means of immunogold label as serotonergic ones. These latter in turn are situated mainly on shaft boutons, which according to specific ultrastructural features are assigned to axo-dendritic inhibitory synapses. Thus, the excitatory serotonergic synapses seem to affect Mauthner neuron indirectly through inhibitory synapses. Further, it was morphometrically established that adaptation is accompanied by the significant decrease of active zones dimensions in both serotonergic and inhibitory synapses. Finally, it was determined in model experiments that the interaction of globular actin with glycine, a main inhibitory neurotransmitter supposedly directly and chronically affecting the ventral dendrite, results in actin filaments formation. It is assumed that glycine-induced cytosolic actin polymerization is a cause of reduction in the ventral dendrite size under stimulation. Thus, it was established that a rather small group of synapses situated on an individual dendrite of the neuron determines the execution of the important form of animal behavior.

Previous work on auditory processing in Opsanus tau has focused on the descending octaval nucleus; however, the magnocellular octaval nucleus receives similar inputs from the otolithic endorgans. The purpose of this study was to assess whether cells in any of the three subdivisions of the magnocellular nucleus respond to auditory frequencies and encode sound source direction. Extracellular recording sites were chosen based on anatomical landmarks, and neurobiotin injections confirmed the location of auditory sites in subdivisions of the magnocellular nucleus. In general, the auditory cells in M2 and M3 responded best to frequencies at or below 100 Hz. Most auditory cells responded well to directional stimuli presented along axes in the horizontal plane. Cells in M3 (not M2) also responded to lateral line stimulation, consistent with otolithic endorgan and lateral line inputs to M3. The convergence of auditory and lateral line inputs in M3, the lack of Mauthner cells in this species, and previous evidence that the magnocellular nucleus does not contribute to ascending auditory pathways suggest to us that the large cells of M3 may play a role in rapid behavioral responses to particle motion stimuli in oyster toadfish.

Summary Inter-individual behavioral variation is thought to increase fitness and aid adaptation to environmental change, but the underlying mechanisms are poorly understood. We find that variation between individuals in neuromodulatory input contributes to individuality in short-term habituation of the zebrafish ( Danio Rerio ) acoustic startle response (ASR). ASR habituation varies greatly between individuals, but differences are stable over days and are heritable. Acoustic stimuli that activate ASR-command Mauthner cells also activate dorsal raphe nucleus (DRN) serotonergic neurons, which project to the vicinity of the Mauthner cells and their inputs. DRN neuron activity decreases during habituation in proportion to habituation and a genetic manipulation that reduces serotonin content in DRN neurons increases habituation, whereas serotonergic agonism or DRN activation with ChR2 reduces habituation. Finally, level of rundown of DRN activity co-segregates with extent of behavioral habituation across generations. Thus, variation between individuals in neuromodulatory input contributes to individuality in a core adaptive behavior. Video Abstract

Comparing synaptic circuits and networks between brains of different animal groups helps us derive an understanding of how nervous systems might have evolved. The circuits of the startle response pathway in the brains of tailed vertebrates are known from electrophysiological studies on the giant reticulospinal Mauthner cells (M-cells). To identify morphological counterparts in chordate tunicates, a sister group of vertebrates [1, 2], we have compiled a densely reconstructed connectome (defined in [3]) for the CNS in the tadpole larva of Ciona intestinalis (L.), using ssEM [4]. The dorsal, tubular CNS of the ∼1-mm tadpole larva is built on a similar plan to vertebrates, its neurons distributed rostrocaudally in three centers, a brain vesicle, motor ganglion, and caudal nerve cord [5]. A single pair of descending decussating neurons, ddNs, found in the motor ganglion, have similarities to reticulospinal neurons descending from the vertebrate hindbrain to the spinal cord. The pre- and postsynaptic connections and circuits of these ddNs support their homology with decussating vertebrate M-cells. Network analysis reveals that, like M-cells, ddNs receive mechanosensory input from the peripheral nervous system and provide input to motoneurons, premotor interneurons, and ascending commissural inhibitory neurons (ACINs). These circuits uncover a putative homologous startle network in the Ciona tadpole. However, differences in circuits, including a lack of bilateral symmetry in their network, and convergence of inputs from left and right sides, raise questions about the relationship between form and function, and are a possible outcome of the tiny number of neurons in ascidian larvae.

Electrical synapses are an omnipresent feature of nervous systems, from the simple nerve nets of cnidarians to complex brains of mammals. Formed by gap junction channels between neurons, electrical synapses allow direct transmission of voltage signals between coupled cells. The relative simplicity of this arrangement belies the sophistication of these synapses. Coupling via electrical synapses can be regulated by a variety of mechanisms on times scales ranging from milliseconds to days, and active properties of the coupled neurons can impart emergent properties such as signal amplification, phase shifts and frequency-selective transmission. This article reviews the biophysical characteristics of electrical synapses and some of the core mechanisms that control their plasticity in the vertebrate central nervous system.

We used a Tg(glyt2:gfp) transgenic zebrafish expressing the green fluorescent protein (GFP) under control of the glycine transporter 2 (GLYT2) regulatory sequences to study for the first time the glycinergic neurons in the brain of an adult teleost. We also performed in situ hybridization using a GLYT2 probe and glycine immunohistochemistry. This study was combined with biocytin tract tracing from the spinal cord to reveal descending glycinergic pathways. A few groups of GFP(+) /GLYT2(-) cells were observed in the midbrain and forebrain, including numerous pinealocytes. Conversely, a small nucleus of the midbrain tegmentum was GLYT2(+) but GFP(-) . Most of the GFP(+) and GLYT2(+) neurons were observed in the rhombencephalon and spinal cord, and a portion of these cells showed double GLYT2/GFP labeling. In the hindbrain, GFP/GLYT2(+) populations were observed in the medial octavolateral nucleus; the secondary, magnocellular, and descending octaval nuclei; the viscerosensory lobes; and reticular populations distributed from trigeminal to vagal levels. No glycinergic cells were observed in the cerebellum. Tract tracing revealed three conspicuous pairs of GFP/GLYT2(+) reticular neurons projecting to the spinal cord. In the spinal cord, GFP/GLYT2(+) cells were observed in the dorsal and ventral horns. GFP(+) fibers were observed from the olfactory bulbs to the spinal cord, although their density varied among regions. The Mauthner neurons received very rich GFP(+) innervation, mainly around the axon cap. Comparison of the zebrafish glycinergic system with the glycinergic systems of other adult vertebrates reveals shared patterns but also divergent traits in the evolution of this system.

Previous studies have shown that contralateral (to the preferred turning side) optokinetic stimulation and ipsilateral enucleation of the eye lead to significant (by a factor of 2–4) decreases in the volume of the ventral dendrite (VD) in one of the two Mauthner neurons (MN) of the goldfish, which becomes functionally more active. We report here our studies of MN ultrastructure after these unilateral influences from the visual system. In both cases, the whole length of the shrunken VD showed emptying of synapses and compaction of its cytoskeleton as compared with the cytoskeleton of the VD of the contralateral MN and the cytoskeleton of the lateral dendrites and bodies of both neurons. It is suggested that emptied synapses are part of the excitatory visual input and that the control of the functional activity of MN via VD involves both cytoskeletal and synaptic mechanisms.