A single base pair substitution in zebrafish distinguishes between innate and acute startle behavior regulation.

Behavioral thresholds define the lowest stimulus intensities sufficient to elicit a behavioral response. Establishment of baseline behavioral thresholds during development is critical for proper responses throughout the animal's life. Despite the relevance of such innate thresholds, the molecular mechanisms critical to establishing behavioral thresholds during development are not well understood. The acoustic startle response is a conserved behavior whose threshold is established during development yet is subsequently acutely regulated. We have previously identified a zebrafish mutant line (escapist) that displays a decreased baseline or innate acoustic startle threshold. Here, we identify a single base pair substitution on Chromosome 25 located within the coding sequence of the synaptotagmin 7a (syt7a) gene that is tightly linked to the escapist acoustic hypersensitivity phenotype. By generating animals in which we deleted the syt7a open reading frame, and subsequent complementation testing with the escapist line, we demonstrate that loss of syt7a function is not the cause of the escapist behavioral phenotype. Nonetheless, escapist mutants provide a powerful tool to decipher the overlap between acute and developmental regulation of behavioral thresholds. Extensive behavioral analyses reveal that in escapist mutants the establishment of the innate acoustic startle threshold is impaired, while regulation of its acute threshold remains intact. Moreover, our behavioral analyses reveal a deficit in baseline responses to visual stimuli, but not in the acute regulation of responses to visual stimuli. Together, this work eliminates loss of syt7a as causative for the escapist phenotype and suggests that mechanisms that regulate the establishment of behavioral thresholds in escapist larvae can operate independently from those regulating acute threshold regulation.

Celsr3 drives development and connectivity of the acoustic startle hindbrain circuit.

In the developing brain, groups of neurons organize into functional circuits that direct diverse behaviors. One such behavior is the evolutionarily conserved acoustic startle response, which in zebrafish is mediated by a well-defined hindbrain circuit. While numerous molecular pathways that guide neurons to their synaptic partners have been identified, it is unclear if and to what extent distinct neuron populations in the startle circuit utilize shared molecular pathways to ensure coordinated development. Here, we show that the planar cell polarity (PCP)-associated atypical cadherins Celsr3 and Celsr2, as well as the Celsr binding partner Frizzled 3a/Fzd3a, are critical for axon guidance of two neuron types that form synapses with each other: the command-like neuron Mauthner cells that drive the acoustic startle escape response, and spiral fiber neurons which provide excitatory input to Mauthner cells. We find that Mauthner axon growth towards synaptic targets is vital for Mauthner survival. We also demonstrate that symmetric spiral fiber input to Mauthner cells is critical for escape direction, which is necessary to respond to directional threats. Moreover, we identify distinct roles for Celsr3 and Celsr2, as Celsr3 is required for startle circuit development while Celsr2 is dispensable, though Celsr2 can partially compensate for loss of Celsr3 in Mauthner cells. This contrasts with facial branchiomotor neuron migration in the hindbrain, which requires Celsr2 while we find that Celsr3 is dispensable. Combined, our data uncover critical and distinct roles for individual PCP components during assembly of the acoustic startle hindbrain circuit.

Functional and pharmacological analyses of visual habituation learning in larval zebrafish.

Habituation allows animals to learn to ignore persistent but inconsequential stimuli. Despite being the most basic form of learning, a consensus model on the underlying mechanisms has yet to emerge. To probe relevant mechanisms, we took advantage of a visual habituation paradigm in larval zebrafish, where larvae reduce their reactions to abrupt global dimming (a dark flash). We used Ca2+ imaging during repeated dark flashes and identified 12 functional classes of neurons that differ based on their rate of adaptation, stimulus response shape, and anatomical location. While most classes of neurons depressed their responses to repeated stimuli, we identified populations that did not adapt or that potentiated their response. These neurons were distributed across brain areas, consistent with a distributed learning process. Using a small-molecule screening approach, we confirmed that habituation manifests from multiple distinct molecular mechanisms, and we have implicated molecular pathways in habituation, including melatonin, oestrogen, and GABA signalling. However, by combining anatomical analyses and pharmacological manipulations with Ca2+ imaging, we failed to identify a simple relationship between pharmacology, altered activity patterns, and habituation behaviour. Collectively, our work indicates that habituation occurs via a complex and distributed plasticity processes that cannot be captured by a simple model. Therefore, untangling the mechanisms of habituation will likely require dedicated approaches aimed at sub-component mechanisms underlying this multidimensional learning process.

Mitochondrial proteins encoded by the 22q11.2 neurodevelopmental locus regulate neural stem and progenitor cell proliferation.

Microdeletion of a 3Mb region encompassing 45 protein-coding genes at chromosome 22q11.2 (22q11.2DS) predisposes individuals to multiple neurodevelopmental disorders and is one of the greatest genetic risk factors for schizophrenia. Defective mitochondrial function has been hypothesized to contribute to 22q11.2DS pathogenesis; however, which of the six mitochondrial genes contribute to neurodevelopmental phenotypes and their underlying mechanisms remain unresolved. To systematically test 22q11.2DS genes for functional roles in neurodevelopment and behavior, we generated genetic mutants for each of the 37 conserved zebrafish orthologs and performed high throughput behavioral phenotyping using seven behavioral assays. Through this unbiased approach, we identified five single-gene mutants with partially overlapping behavioral phenotypes. Two of these genes, mrpl40 and prodha, encode for mitochondrial proteins and, similar to what we observed in mrpl40 and prodha mutants, pharmacologic inhibition of mitochondrial function during development results in microcephaly. Single mutant analysis shows that both mrpl40 and prodha mutants display aberrant neural stem and progenitor cell proliferation, with each gene regulating distinct cell populations. Finally, double mutants for both mrpl40 and prodha display aggravated behavioral phenotypes and neural stem and progenitor cell analysis reveals a previously unrecognized partially redundant role for mrpl40 and prodha in regulating radial glia-like cell proliferation. Combined, our results demonstrate a critical role for mitochondrial function in neural stem and progenitor cell populations in the developing vertebrate brain and provide compelling evidence that mitochondrial dysfunction during neurodevelopment is linked to brain volume and behavioral phenotypes observed in models of 22q11.2DS.

A single base pair substitution on Chromosome 25 in zebrafish distinguishes between development and acute regulation of behavioral thresholds.

Behavioral thresholds define the lowest stimulus intensities sufficient to elicit a behavioral response. Establishment of baseline behavioral thresholds during development is critical for proper responses throughout the animal's life. Despite the relevance of such innate thresholds, the molecular mechanisms critical to establishing behavioral thresholds during development are not well understood. The acoustic startle response is a conserved behavior whose threshold is established during development yet is subsequently acutely regulated. We have previously identified a zebrafish mutant line ( escapist ) that displays a decreased baseline or innate acoustic startle threshold. Here, we identify a single base pair substitution on Chromosome 25 located within the coding sequence of the synaptotagmin 7a ( syt7a ) gene that is tightly linked to the escapist acoustic hypersensitivity phenotype. By generating animals in which we deleted the syt7a open reading frame, and subsequent complementation testing with the escapist line, we demonstrate that loss of syt7a function is not the cause of the escapist behavioral phenotype. Nonetheless, escapist mutants provide a powerful tool to decipher the overlap between acute and developmental regulation of behavioral thresholds. Extensive behavioral analyses reveal that in escapist mutants the establishment of the innate acoustic startle threshold is impaired, while regulation of its acute threshold remains intact. Moreover, our behavioral analyses reveal a deficit in baseline responses to visual stimuli, but not in the acute regulation of responses to visual stimuli. Together, this work eliminates loss of syt7a as causative for the escapist phenotype and suggests that mechanisms that regulate the establishment of behavioral thresholds in escapist larvae can operate largely independently from those regulating acute threshold regulation.

Target-selective vertebrate motor axon regeneration depends on interaction with glial cells at a peripheral nerve plexus.

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle, and then re-sort into target-specific bundles. Here, we examine this process at a plexus located at the base of the zebrafish pectoral fin, equivalent to tetrapod forelimbs. Using live cell imaging and sparse axon labeling, we find that regenerating motor axons from 3 nerves coalesce into the plexus. There, they intermingle and sort into distinct branches, and then navigate to their original muscle domains with high fidelity that restores functionality. We demonstrate that this regeneration process includes selective retraction of mistargeted axons, suggesting active correction mechanisms. Moreover, we find that Schwann cells are enriched and associate with axons at the plexus, and that Schwann cell ablation during regeneration causes profound axonal mistargeting. Our data provide the first real-time account of regenerating vertebrate motor axons navigating a nerve plexus and reveal a previously unappreciated role for Schwann cells to promote axon sorting at a plexus during regeneration.

Assaying Optic Nerve Regeneration in Larval Zebrafish.

Zebrafish have a remarkable capacity for spontaneously regenerating their central nervous system. Larval zebrafish are optically transparent and therefore are widely used to dynamically visualize cellular processes in vivo, such as nerve regeneration. Regeneration of retinal ganglion cell (RGC) axons within the optic nerve has been previously studied in adult zebrafish. In contrast, assays of optic nerve regeneration have previously not been established in larval zebrafish. In order to take advantage of the imaging capabilities in the larval zebrafish model, we recently developed an assay to physically transect RGC axons and monitor optic nerve regeneration in larval zebrafish. We found that RGC axons rapidly and robustly regrow to the optic tectum. Here, we describe the methods for performing the optic nerve transections, as well as methods for visualizing RGC regeneration in larval zebrafish.

Integration of cooperative and opposing molecular programs drives learning-associated behavioral plasticity.

Habituation is a foundational learning process critical for animals to adapt their behavior to changes in their sensory environment. Although habituation is considered a simple form of learning, the identification of a multitude of molecular pathways including several neurotransmitter systems that regulate this process suggests an unexpected level of complexity. How the vertebrate brain integrates these various pathways to accomplish habituation learning, whether they act independently or intersect with one another, and whether they act via divergent or overlapping neural circuits has remained unclear. To address these questions, we combined pharmacogenetic pathway analysis with unbiased whole-brain activity mapping using the larval zebrafish. Based on our findings, we propose five distinct molecular modules for the regulation of habituation learning and identify a set of molecularly defined brain regions associated with four of the five modules. Moreover, we find that in module 1 the palmitoyltransferase Hip14 cooperates with dopamine and NMDA signaling to drive habituation, while in module 3 the adaptor protein complex subunit Ap2s1 drives habituation by antagonizing dopamine signaling, revealing two distinct and opposing roles for dopaminergic neuromodulation in the regulation of behavioral plasticity. Combined, our results define a core set of distinct modules that we propose act in concert to regulate habituation-associated plasticity, and provide compelling evidence that even seemingly simple learning behaviors in a compact vertebrate brain are regulated by a complex and overlapping set of molecular mechanisms.

Multivariate analysis of variegated expression in Neurons: A strategy for unbiased localization of gene function to candidate brain regions in larval zebrafish.

Behavioral screens in model organisms have greatly facilitated the identification of genes and genetic pathways that regulate defined behaviors. Identifying the neural circuitry via which specific genes function to modify behavior remains a significant challenge in the field. Tissue- and cell type-specific knockout, knockdown, and rescue experiments serve this purpose, yet in zebrafish screening through dozens of candidate cell-type-specific and brain-region specific driver lines for their ability to rescue a mutant phenotype remains a bottleneck. Here we report on an alternative strategy that takes advantage of the variegation often present in Gal4-driven UAS lines to express a rescue construct in a neuronal tissue-specific and variegated manner. We developed and validated a computational pipeline that identifies specific brain regions where expression levels of the variegated rescue construct correlate with rescue of a mutant phenotype, indicating that gene expression levels in these regions may causally influence behavior. We termed this unbiased correlative approach Multivariate Analysis of Variegated Expression in Neurons (MAVEN). The MAVEN strategy advances the user's capacity to quickly identify candidate brain regions where gene function may be relevant to a behavioral phenotype. This allows the user to skip or greatly reduce screening for rescue and proceed to experimental validation of candidate brain regions via genetically targeted approaches. MAVEN thus facilitates identification of brain regions in which specific genes function to regulate larval zebrafish behavior.

Mitochondrial genes in the 22q11.2 deleted region regulate neural stem and progenitor cell proliferation.

Microdeletion of a 3Mbp region encompassing 45 protein-coding genes at chromosome 22q11.2 (22q11.2DS) predisposes to multiple neurodevelopmental disorders and is one of the greatest genetic risk factors for schizophrenia. Defective mitochondrial function has been hypothesized to contribute to 22q11.2DS pathogenesis; however, which of the six mitochondrial genes contribute to neurodevelopmental phenotypes and their underlying mechanisms remain unresolved. To systematically test 22q11.2DS genes for functional roles in neurodevelopment and behavior, we generated genetic mutants for each of the 37 conserved zebrafish orthologs and performed high throughput behavioral phenotyping using seven behavioral assays. Through this unbiased approach, we identified five single-gene mutants with partially overlapping behavioral phenotypes. Two of these genes, mrpl40 and prodha , encode for mitochondrial proteins and, similar to what we observed in mrpl40 and prodha mutants, pharmacologic inhibition of mitochondrial function during development results in microcephaly. Finally, we show that both mrpl40 and prodha mutants display neural stem and progenitor cell phenotypes, with each gene regulating different neural stem cell populations. Combined, our results demonstrate a critical role for mitochondrial function in neural stem and progenitor cell populations in the developing vertebrate brain and provide compelling evidence that mitochondrial dysfunction during neurodevelopment is linked to brain volume and behavioral phenotypes observed in models of 22q11.2DS.

A glia cell dependent mechanism at a peripheral nerve plexus critical for target-selective axon regeneration.

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle, and then re-sort into target-specific bundles. Here, we examine this process at a plexus located at the base of the zebrafish pectoral fin, equivalent to tetrapod forelimbs. Using live cell imaging and sparse axon labeling, we find that regenerating motor axons from three nerves coalesce into the plexus. There, they intermingle and sort into distinct branches, and then navigate to their original muscle domains with high fidelity that restores functionality. We demonstrate that this regeneration process includes selective retraction of mistargeted axons, suggesting active correction mechanisms. Moreover, we find that Schwann cells are enriched and associate with axons at the plexus, and that Schwann cell ablation during regeneration causes profound axonal mistargeting. Our data provide the first real time account of regenerating vertebrate motor axons navigating a nerve plexus and reveal a previously unappreciated role for Schwann cells to promote axon sorting at a plexus during regeneration.

The calcium-sensing receptor (CaSR) regulates zebrafish sensorimotor decision making via a genetically defined cluster of hindbrain neurons.

Decision making is a fundamental nervous system function that ranges widely in complexity and speed of execution. We previously established larval zebrafish as a model for sensorimotor decision making and identified the G-protein-coupled calcium-sensing receptor (CaSR) to be critical for this process. Here, we report that CaSR functions in neurons to dynamically regulate the bias between two behavioral outcomes: escapes and reorientations. By employing a computational guided transgenic strategy, we identify a genetically defined neuronal cluster in the hindbrain as a key candidate site for CaSR function. Finally, we demonstrate that transgenic CaSR expression targeting this cluster consisting of a few hundred neurons shifts behavioral bias in wild-type animals and restores decision making deficits in CaSR mutants. Combined, our data provide a rare example of a G-protein-coupled receptor that biases vertebrate sensorimotor decision making via a defined neuronal cluster.

cacna2d3, a voltage-gated calcium channel subunit, functions in vertebrate habituation learning and the startle sensitivity threshold.

BACKGROUND: The ability to filter sensory information into relevant versus irrelevant stimuli is a fundamental, conserved property of the central nervous system and is accomplished in part through habituation learning. Synaptic plasticity that underlies habituation learning has been described at the cellular level, yet the genetic regulators of this plasticity remain poorly understood, as do circuits that mediate sensory filtering. METHODS: To identify genes critical for plasticity, a forward genetic screen for zebrafish genes that mediate habituation learning was performed, which identified a mutant allele, doryp177, that caused reduced habituation of the acoustic startle response. In this study, we combine whole-genome sequencing with behavioral analyses to characterize and identify the gene affected in doryp177 mutants. RESULTS: Whole-genome sequencing identified the calcium voltage-gated channel auxiliary subunit alpha-2/delta-3 (cacna2d3) as a candidate gene affected in doryp177 mutants. Behavioral characterization of larvae homozygous for two additional, independently derived mutant alleles of cacna2d3, together with failure of these alleles to complement doryp177, confirmed a critical role for cacna2d3 in habituation learning. Notably, detailed analyses of the acoustic response in mutant larvae also revealed increased startle sensitivity to acoustic stimuli, suggesting a broader role for cacna2d3 in controlling innate response thresholds to acoustic stimuli. CONCLUSIONS: Taken together, our data demonstrate a critical role for cacna2d3 in sensory filtering, a process that is disrupted in human CNS disorders, e.g. ADHD, schizophrenia, and autism.

Zebrafish behavior as a gateway to nervous system assembly and plasticity.

Nervous system assembly relies on a diversity of cellular processes ranging from dramatic tissue reorganization to local, subcellular changes all driven by precise molecular programs. Combined, these processes culminate in an animal's ability to plan and execute behaviors. Animal behavior can, therefore, serve as a functional readout of nervous system development. Benefitting from an expansive and growing set of molecular and imaging tools paired with an ever-growing number of assays of diverse behaviors, the zebrafish system has emerged as an outstanding platform at the intersection of nervous system assembly, plasticity and behavior. Here, we summarize recent advancements in the field, including how developing neural circuits are refined to shape complex behaviors and plasticity.

Robo2 Drives Target-Selective Peripheral Nerve Regeneration in Response to Glia-Derived Signals.

Peripheral nerves are divided into multiple branches leading to divergent synaptic targets. This poses a remarkable challenge for regenerating axons as they select their original trajectory at nerve branch-points. Despite implications for functional regeneration, the molecular mechanisms underlying target selectivity are not well characterized. Danio Rerio (zebrafish) motor nerves are composed of a ventral and a dorsal branch that diverge at a choice-point, and we have previously shown that regenerating axons faithfully select their original branch and targets. Here we identify robo2 as a key regulator of target-selective regeneration (sex of experimental subjects unknown). We demonstrate that robo2 function in regenerating axons is required and sufficient to drive target-selective regeneration, and that robo2 acts in response to glia located precisely where regenerating axons select the branch-specific trajectory to prevent and correct axonal errors. Combined, our results reveal a glia-derived mechanism that acts locally via axonal robo2 to promote target-selective regeneration.SIGNIFICANCE STATEMENT Despite its relevance for functional recovery, the molecular mechanisms that direct regenerating peripheral nerve axons toward their original targets are not well defined. Zebrafish spinal motor nerves are composed of a dorsal and a ventral branch that diverge at a stereotyped nerve branch-point, providing a unique opportunity to decipher the molecular mechanisms critical for target-selective regeneration. Using a combination of live cell imaging and molecular-genetic manipulations, we demonstrate that the robo2 guidance receptor is necessary and sufficient to promote target-selective regeneration. Moreover, we demonstrate that robo2 is part of a genetic pathway that generates transient, spatially restricted, and tightly coordinated signaling events that direct axons of the dorsal nerve branch toward their original, pre-injury targets.

Agrin/Lrp4 signal constrains MuSK-dependent neuromuscular synapse development in appendicular muscle.

The receptor tyrosine kinase MuSK, its co-receptor Lrp4 and the Agrin ligand constitute a signaling pathway that is crucial in axial muscle for neuromuscular synapse development, yet whether this pathway functions similarly in appendicular muscle is unclear. Here, using the larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, we show that, similar to axial muscle, developing appendicular muscles form aneural acetylcholine receptor (AChR) clusters prior to innervation. As motor axons arrive, neural AChR clusters form, eventually leading to functional synapses in a MuSK-dependent manner. We find that loss of Agrin or Lrp4 function, which abolishes synaptic AChR clusters in axial muscle, results in enlarged presynaptic nerve regions and progressively expanding appendicular AChR clusters, mimicking the consequences of motoneuron ablation. Moreover, musk depletion in lrp4 mutants partially restores synaptic AChR patterning. Combined, our results provide compelling evidence that, in addition to the canonical pathway in which Agrin/Lrp4 stimulates MuSK activity, Agrin/Lrp4 signaling in appendicular muscle constrains MuSK-dependent neuromuscular synapse organization. Thus, we reveal a previously unappreciated role for Agrin/Lrp4 signaling, thereby highlighting distinct differences between axial and appendicular synapse development.

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1.

The acoustic startle response is an evolutionarily conserved avoidance behavior. Disruptions in startle behavior, particularly startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. We uncovered mutations in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 and Dolk play critical roles in the spinal cord to regulate movement magnitude during the startle response and spontaneous swim movements. Moreover, we show that Kv1.1 protein is mislocalized in dolk mutants, suggesting they act in a common genetic pathway. Combined, our results identify a diverse set of eight genes, all associated with human disorders, that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway.

Acute Regulation of Habituation Learning via Posttranslational Palmitoylation.

Habituation is an adaptive learning process that enables animals to adjust innate behaviors to changes in their environment. Despite its well-documented implications for a wide diversity of behaviors, the molecular and cellular basis of habituation learning is not well understood. Using whole-genome sequencing of zebrafish mutants isolated in an unbiased genetic screen, we identified the palmitoyltransferase Huntingtin interacting protein 14 (Hip14) as a critical regulator of habituation learning. We demonstrate that Hip14 regulates depression of sensory inputs onto an identified hindbrain neuron and provide evidence that Hip14 palmitoylates the Shaker-like K+ voltage-gated channel subunit (Kv1.1), thereby regulating Kv1.1 subcellular localization. Furthermore, we show that, like for Hip14, loss of Kv1.1 leads to habituation deficits and that Hip14 is dispensable in development and instead acts acutely to promote habituation. Combined, these results uncover a previously unappreciated role for acute posttranslational palmitoylation at defined circuit components to regulate learning.

Vagus Topographic Map: Wandering through a gRAdient.

Topographic map development requires precise coordination between navigating axons and their targets in a dynamic environment. In this issue of Developmental Cell, Isabella et al. describe a mechanism in which a changing gradient of the morphogen retinoic acid regulates the expression of guidance factors to shape topographic axon targeting.