Prepontine non-giant neurons drive flexible escape behavior in zebrafish.

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.

A Model to Study NMDA Receptors in Early Nervous System Development.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play critical roles in neuronal development and nervous system function. Here, we developed a model to study NMDARs in early development in zebrafish, by generating CRISPR-mediated lesions in the NMDAR genes, grin1a and grin1b, which encode the obligatory GluN1 subunits. While receptors containing grin1a or grin1b show high Ca2+ permeability, like their mammalian counterpart, grin1a is expressed earlier and more broadly in development than grin1b Both grin1a-/- and grin1b-/- zebrafish are viable. Unlike in rodents, where the grin1 knockout is embryonic lethal, grin1 double-mutant fish (grin1a-/-; grin1b-/-), which lack all NMDAR-mediated synaptic transmission, survive until ∼10 d dpf (days post fertilization), providing a unique opportunity to explore NMDAR function during development and in generating behaviors. Many behavioral defects in the grin1 double-mutant larvae, including abnormal evoked responses to light and acoustic stimuli, prey-capture deficits, and a failure to habituate to acoustic stimuli, are replicated by short-term treatment with the NMDAR antagonist MK-801, suggesting that they arise from acute effects of compromised NMDAR-mediated transmission. Other defects, however, such as periods of hyperactivity and alterations in place preference, are not phenocopied by MK-801, suggesting a developmental origin. Together, we have developed a unique model to study NMDARs in the developing vertebrate nervous system.SIGNIFICANCE STATEMENT Rapid communication between cells in the nervous system depends on ion channels that are directly activated by chemical neurotransmitters. One such ligand-gated ion channel, the NMDAR, impacts nearly all forms of nervous system function. It has been challenging, however, to study the prolonged absence of NMDARs in vertebrates, and hence their role in nervous system development, due to experimental limitations. Here, we demonstrate that zebrafish lacking all NMDAR transmission are viable through early development and are capable of a wide range of stereotypic behaviors. As such, this zebrafish model provides a unique opportunity to study the role of NMDAR in the development of the early vertebrate nervous system.

Morphometric analysis and neuroanatomical mapping of the zebrafish brain.

Large-scale genomic studies have recently identified genetic variants causative for major neurodevelopmental disorders, such as intellectual disability and autism. However, determining how underlying developmental processes are affected by these mutations remains a significant challenge in the field. Zebrafish is an established model system in developmental neurogenetics that may be useful in uncovering the mechanisms of these mutations. Here we describe the use of voxel-intensity, deformation field, and volume-based morphometric techniques for the systematic and unbiased analysis of gene knock-down and environmental exposure-induced phenotypes in zebrafish. We first present a computational method for brain segmentation based on transgene expression patterns to create a comprehensive neuroanatomical map. This map allowed us to disclose statistically significant changes in brain microstructure and composition in neurodevelopmental models. We demonstrate the effectiveness of morphometric techniques in measuring changes in the relative size of neuroanatomical subdivisions in atoh7 morphant larvae and in identifying phenotypes in larvae treated with valproic acid, a chemical demonstrated to increase the risk of autism in humans. These tools enable rigorous evaluation of the effects of gene mutations and environmental exposures on neural development, providing an entry point for cellular and molecular analysis of basic developmental processes as well as neurodevelopmental and neurodegenerative disorders.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior.

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior.

Startling responses of zebrafish: an interview with Harold Burgess.

Harold Burgess is a Senior Investigator at the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), part of the National Institutes of Health. Work in his lab combines genetic and imaging techniques to study neural circuits required for sensory guided behavior in zebrafish. In this interview Harold shares his thoughts on the changing field of neural development, pre-publication review, and 'Darwinian experiments' of peer review.

Search strategy is regulated by somatostatin signaling and deep brain photoreceptors in zebrafish.

BACKGROUND: Animals use sensory cues to efficiently locate resources, but when sensory information is insufficient, they may rely on internally coded search strategies. Despite the importance of search behavior, there is limited understanding of the underlying neural mechanisms in vertebrates. RESULTS: Here, we report that loss of illumination initiates sophisticated light-search behavior in larval zebrafish. Using three-dimensional tracking, we show that at the onset of darkness larvae swim in a helical trajectory that is spatially restricted in the horizontal plane, before gradually transitioning to an outward movement profile. Local and outward swim patterns display characteristic features of area-restricted and roaming search strategies, differentially enhancing phototaxis to nearby and remote sources of light. Retinal signaling is only required to initiate area-restricted search, implying that photoreceptors within the brain drive the transition to the roaming search state. Supporting this, orthopediaA mutant larvae manifest impaired transition to roaming search, a phenotype which is recapitulated by loss of the non-visual opsin opn4a and somatostatin signaling. CONCLUSION: These findings define distinct neuronal pathways for area-restricted and roaming search behaviors and clarify how internal drives promote goal-directed activity.

Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

Motivated state control in larval zebrafish: behavioral paradigms and anatomical substrates.

Over the course of each day, animals prioritize different objectives. Immediate goals may reflect fluctuating internal homeostatic demands, prompting individuals to seek out energy supplies or warmth. At other times, the environment may present temporary challenges or opportunities. Homeostatic demands and environmental signals often elicit persistent changes in an animal's behavior to meet needs and challenges over extended periods of time. These changes reflect the underlying motivational state of the animal. The larval zebrafish has been established as an effective genetically tractable vertebrate system to study neural circuits for sensory-motor reflexes. Fewer studies have exploited zebrafish to study brain circuits that control motivated behavior. In part this is because appropriate conceptual frameworks, anatomical knowledge, and behavioral paradigms are not yet well established. This review sketches a general conceptual framework for studying motivated state control in animal models, how this applies to larval zebrafish, and the current knowledge on neuroanatomical substrates for state control in this model.

Enlightening the brain: linking deep brain photoreception with behavior and physiology.

Vertebrates respond to light with more than just their eyes. In this article, we speculate on the intriguing possibility that a link remains between non-visual opsins and neurohormonal systems that control neuronal circuit formation and activity in mammals. Historically, the retina and pineal gland were considered the only significant light-sensing tissues in vertebrates. However over the last century, evidence has accumulated arguing that extra-ocular tissues in vertebrates influence behavior through non-image-forming photoreception. One such class of extra-ocular light detectors are the long mysterious deep brain photoreceptors. Here, we review recent findings on the cellular identity and the function of deep brain photoreceptors controlling behavior and physiology in zebrafish, and discuss their implications.

Direct activation of the Mauthner cell by electric field pulses drives ultrarapid escape responses.

Rapid escape swims in fish are initiated by the Mauthner cells, giant reticulospinal neurons with unique specializations for swift responses. The Mauthner cells directly activate motoneurons and facilitate predator detection by integrating acoustic, mechanosensory, and visual stimuli. In addition, larval fish show well-coordinated escape responses when exposed to electric field pulses (EFPs). Sensitization of the Mauthner cell by genetic overexpression of the voltage-gated sodium channel SCN5 increased EFP responsiveness, whereas Mauthner ablation with an engineered variant of nitroreductase with increased activity (epNTR) eliminated the response. The reaction time to EFPs is extremely short, with many responses initiated within 2 ms of the EFP. Large neurons, such as Mauthner cells, show heightened sensitivity to extracellular voltage gradients. We therefore tested whether the rapid response to EFPs was due to direct activation of the Mauthner cells, bypassing delays imposed by stimulus detection and transmission by sensory cells. Consistent with this, calcium imaging indicated that EFPs robustly activated the Mauthner cell but only rarely fired other reticulospinal neurons. Further supporting this idea, pharmacological blockade of synaptic transmission in zebrafish did not affect Mauthner cell activity in response to EFPs. Moreover, Mauthner cells transgenically expressing a tetrodotoxin (TTX)-resistant voltage-gated sodium channel retained responses to EFPs despite TTX suppression of action potentials in the rest of the brain. We propose that EFPs directly activate Mauthner cells because of their large size, thereby driving ultrarapid escape responses in fish.

The dorsal raphe modulates sensory responsiveness during arousal in zebrafish.

During waking behavior, animals adapt their state of arousal in response to environmental pressures. Sensory processing is regulated in aroused states, and several lines of evidence imply that this is mediated at least partly by the serotonergic system. However, there is little information directly showing that serotonergic function is required for state-dependent modulation of sensory processing. Here we find that zebrafish larvae can maintain a short-term state of arousal during which neurons in the dorsal raphe modulate sensory responsiveness to behaviorally relevant visual cues. After a brief exposure to water flow, larvae show elevated activity and heightened sensitivity to perceived motion. Calcium imaging of neuronal activity after flow revealed increased activity in serotonergic neurons of the dorsal raphe. Genetic ablation of these neurons abolished the increase in visual sensitivity during arousal without affecting baseline visual function or locomotor activity. We traced projections from the dorsal raphe to a major visual area, the optic tectum. Laser ablation of the tectum demonstrated that this structure, like the dorsal raphe, is required for improved visual sensitivity during arousal. These findings reveal that serotonergic neurons of the dorsal raphe have a state-dependent role in matching sensory responsiveness to behavioral context.

Mutations in vacuolar H+ -ATPase subunits lead to biliary developmental defects in zebrafish.

We identified three zebrafish mutants with defects in biliary development. One of these mutants, pekin (pn), also demonstrated generalized hypopigmentation and other defects, including disruption of retinal cell layers, lack of zymogen granules in the pancreas, and dilated Golgi in intestinal epithelial cells. Bile duct cells in pn demonstrated an accumulation of electron dense bodies. We determined that the causative defect in pn was a splice site mutation in the atp6ap2 gene that leads to an inframe stop codon. atp6ap2 encodes a subunit of the vacuolar H(+)-ATPase (V-H(+)-ATPase), which modulates pH in intracellular compartments. The Atp6ap2 subunit has also been shown to function as an intracellular renin receptor that stimulates fibrogenesis. Here we show that mutants and morphants involving other V-H(+)-ATPase subunits also demonstrated developmental biliary defects, but did not demonstrate the inhibition of fibrogenic genes observed in pn. The defects in pn are reminiscent of those we and others have observed in class C VPS (vacuolar protein sorting) family mutants and morphants, and we report here that knockdown of atp6ap2 and vps33b had an additive negative effect on biliary development. Our findings suggest that pathways which are important in modulating intracompartmental pH lead to defects in digestive organ development, and support previous studies demonstrating the importance of intracellular sorting pathways in biliary development.

A model of Costeff Syndrome reveals metabolic and protective functions of mitochondrial OPA3.

Costeff Syndrome, which is caused by mutations in the OPTIC ATROPHY 3 (OPA3) gene, is an early-onset syndrome characterized by urinary excretion of 3-methylglutaconic acid (MGC), optic atrophy and movement disorders, including ataxia and extrapyramidal dysfunction. The OPA3 protein is enriched in the inner mitochondrial membrane and has mitochondrial targeting signals, but a requirement for mitochondrial localization has not been demonstrated. We find zebrafish opa3 mRNA to be expressed in the optic nerve and retinal layers, the counterparts of which in humans have high mitochondrial activity. Transcripts of zebrafish opa3 are also expressed in the embryonic brain, inner ear, heart, liver, intestine and swim bladder. We isolated a zebrafish opa3 null allele for which homozygous mutants display increased MGC levels, optic nerve deficits, ataxia and an extrapyramidal movement disorder. This correspondence of metabolic, ophthalmologic and movement abnormalities between humans and zebrafish demonstrates a phylogenetic conservation of OPA3 function. We also find that delivery of exogenous Opa3 can reduce increased MGC levels in opa3 mutants, and this reduction requires the mitochondrial localization signals of Opa3. By manipulating MGC precursor availability, we infer that elevated MGC in opa3 mutants derives from extra-mitochondrial HMG-CoA through a non-canonical pathway. The opa3 mutants have normal mitochondrial oxidative phosphorylation profiles, but are nonetheless sensitive to inhibitors of the electron transport chain, which supports clinical recommendations that individuals with Costeff Syndrome avoid mitochondria-damaging agents. In summary, this paper introduces a faithful Costeff Syndrome model and demonstrates a requirement for mitochondrial OPA3 to limit HMG-CoA-derived MGC and protect the electron transport chain against inhibitory compounds.

A Critical Review of Zebrafish Neurological Disease Models-2. Application: Functional and Neuroanatomical Phenotyping Strategies and Chemical Screens.

Extensive phylogenetic conservation of molecular pathways and neuroanatomical structures, associated with efficient methods for genetic modification, have been exploited increasingly to generate zebrafish models of human disease. A range of powerful approaches can be deployed to analyze these models with the ultimate goal of elucidating pathogenic mechanisms and accelerating efforts to find effective treatments. Unbiased neurobehavioral assays can provide readouts that parallel clinical abnormalities found in patients, although some of the most useful assays quantify responses that are not routinely evaluated clinically, and differences between zebrafish and human brains preclude expression of the full range of neurobehavioral abnormalities seen in disease. Imaging approaches that use fluorescent reporters and standardized brain atlases coupled with quantitative measurements of brain structure offer an unbiased means to link experimental manipulations to changes in neural architecture. Together, quantitative structural and functional analyses allow dissection of the cellular and physiological basis underlying neurological phenotypes. These approaches can be used as outputs in chemical modifier screens, which provide a major opportunity to exploit zebrafish models to identify small molecule modulators of pathophysiology that may be informative for understanding disease mechanisms and possible therapeutic approaches.

A Critical Review of Zebrafish Neurological Disease Models-1. The Premise: Neuroanatomical, Cellular and Genetic Homology and Experimental Tractability.

The last decade has seen a dramatic rise in the number of genes linked to neurological disorders, necessitating new models to explore underlying mechanisms and to test potential therapies. Over a similar period, many laboratories adopted zebrafish as a tractable model for studying brain development, defining neural circuits and performing chemical screens. Here we discuss strengths and limitations of using the zebrafish system to model neurological disorders. The underlying premise for many disease models is the high degree of homology between human and zebrafish genes, coupled with the conserved vertebrate Bauplan and repertoire of neurochemical signaling molecules. Yet, we caution that important evolutionary divergences often limit the extent to which human symptoms can be modeled meaningfully in zebrafish. We outline advances in genetic technologies that allow human mutations to be reproduced faithfully in zebrafish. Together with methods that visualize the development and function of neuronal pathways at the single cell level, there is now an unprecedented opportunity to understand how disease-associated genetic changes disrupt neural circuits, a level of analysis that is ideally suited to uncovering pathogenic changes in human brain disorders.

Tbx2b is required for ultraviolet photoreceptor cell specification during zebrafish retinal development.

The vertebrate rod and cone photoreceptors are highly specialized sensory neurons that transduce light into the chemical and electrical signals of the nervous system. Although the physiological properties of cones and rods are well known, only a handful of genes have been identified that regulate the specification of photoreceptor subtypes. Taking advantage of the mosaic organization of photoreceptors in zebrafish, we report the isolation of a mutation resulting in a unique change in photoreceptor cell fate. Mutation of the lots-of-rods (lor) locus results in a near one-for-one transformation of UV-cone precursors into rods. The transformed cells exhibit morphological characteristics and a gene-expression pattern typical of rods, but differentiate in a temporal and spatial pattern consistent with UV-cone development. In mutant larvae and adults, the highly ordered photoreceptor mosaic is maintained and degeneration is not observed, suggesting that lor functions after the specification of the other photoreceptor subtypes. In genetic chimeras, lor functions cell-autonomously in the specification of photoreceptor cell fate. Linkage analysis and genetic-complementation testing indicate that lor is an allele of tbx2b/fby (from beyond). fby was identified by a pineal complex phenotype, and carries a nonsense mutation in the T-box domain of the tbx2b transcription factor. Homozygous fby mutant larvae and lor/fby transheterozygotes also display the lots-of-rods phenotype. Based upon these data, we propose a previously undescribed function for tbx2b in photoreceptor cell precursors, to promote the UV cone fate by repressing the rod differentiation pathway.

A cerebellar-prepontine circuit for tonic immobility triggered by an inescapable threat.

Sudden changes in the environment are frequently perceived as threats and provoke defensive behavioral states. One such state is tonic immobility, a conserved defensive strategy characterized by powerful suppression of movement and motor reflexes. Tonic immobility has been associated with multiple brainstem regions, but the underlying circuit is unknown. Here, we demonstrate that a strong vibratory stimulus evokes tonic immobility in larval zebrafish defined by suppressed locomotion and sensorimotor responses. Using a circuit-breaking screen and targeted neuron ablations, we show that cerebellar granule cells and a cluster of glutamatergic ventral prepontine neurons (vPPNs) that express key stress-associated neuropeptides are critical components of the circuit that suppresses movement. The complete sensorimotor circuit transmits information from sensory ganglia through the cerebellum to vPPNs to regulate reticulospinal premotor neurons. These results show that cerebellar regulation of a neuropeptide-rich prepontine structure governs a conserved and ancestral defensive behavior that is triggered by an inescapable threat.

Disruption of grin2B, an ASD-associated gene, produces social deficits in zebrafish.

BACKGROUND: Autism spectrum disorder (ASD), like many neurodevelopmental disorders, has complex and varied etiologies. Advances in genome sequencing have identified multiple candidate genes associated with ASD, including dozens of missense and nonsense mutations in the NMDAR subunit GluN2B, encoded by GRIN2B. NMDARs are glutamate-gated ion channels with key synaptic functions in excitatory neurotransmission. How alterations in these proteins impact neurodevelopment is poorly understood, in part because knockouts of GluN2B in rodents are lethal. METHODS: Here, we use CRISPR-Cas9 to generate zebrafish lacking GluN2B (grin2B-/-). Using these fish, we run an array of behavioral tests and perform whole-brain larval imaging to assay developmental roles and functions of GluN2B. RESULTS: We demonstrate that zebrafish GluN2B displays similar structural and functional properties to human GluN2B. Zebrafish lacking GluN2B (grin2B-/-) surprisingly survive into adulthood. Given the prevalence of social deficits in ASD, we assayed social preference in the grin2B-/- fish. Wild-type fish develop a strong social preference by 3 weeks post fertilization. In contrast, grin2B-/- fish at this age exhibit significantly reduced social preference. Notably, the lack of GluN2B does not result in a broad disruption of neurodevelopment, as grin2B-/- larvae do not show alterations in spontaneous or photic-evoked movements, are capable of prey capture, and exhibit learning. Whole-brain imaging of grin2B-/- larvae revealed reduction of an inhibitory neuron marker in the subpallium, a region linked to ASD in humans, but showed that overall brain size and E/I balance in grin2B-/- is comparable to wild type. LIMITATIONS: Zebrafish lacking GluN2B, while useful in studying developmental roles of GluN2B, are unlikely to model nuanced functional alterations of human missense mutations that are not complete loss of function. Additionally, detailed mammalian homologies for larval zebrafish brain subdivisions at the age of whole-brain imaging are not fully resolved. CONCLUSIONS: We demonstrate that zebrafish completely lacking the GluN2B subunit of the NMDAR, unlike rodent models, are viable into adulthood. Notably, they exhibit a highly specific deficit in social behavior. As such, this zebrafish model affords a unique opportunity to study the roles of GluN2B in ASD etiologies and establish a disease-relevant in vivo model for future studies.

The X-linked acrogigantism-associated gene gpr101 is a regulator of early embryonic development and growth in zebrafish.

We recently described X-linked acrogigantism (X-LAG), a condition of early childhood-onset pituitary gigantism associated with microduplications of the GPR101 receptor. The expression of GPR101 in hyperplastic pituitary regions and tumors in X-LAG patients, and GPR101's normally transient pituitary expression during fetal development, suggest a role in the regulation of growth. Nevertheless, little is still known about GPR101's physiological functions, especially during development. By using zebrafish models, we investigated the role of gpr101 during embryonic development and somatic growth. Transient ectopic gpr101 expression perturbed the embryonic body plan but did not affect growth. Loss of gpr101 led to a significant reduction in body size that was even more pronounced in the absence of maternal transcripts, as well as subfertility. These changes were accompanied by gastrulation and hypothalamic defects. In conclusion, both gpr101 loss- and gain-of-function affect, in different ways, fertility, embryonic patterning, growth and brain development.