Neuronal Circuits Controlling Behavior: Genetic Analysis in Zebrafish

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

This project has three main goals. 1. Understanding central mechanisms that control sensory processing. In zebrafish, as in mammals, auditory startle responses are inhibited when an intense acoustic stimulus is preceded by a weak prepulse. This form of startle modulation, termed prepulse inhibition, is diminished in neurological conditions including schizophrenia. Previously, we showed that, in zebrafish, as in mammals, several distinct cellular mechanisms mediate prepulse inhibition, depending on the time interval between the prepulse and the startle stimulus, with NMDA–receptor signaling playing a key role for intervals greater than 100 ms. Accordingly, zebrafish with gene mutations in specific NMDA receptor subunits linked to schizophrenia show diminished prepulse inhibition. We have now also identified neurons that mediate prepulse inhibition at intervals of less than 100 ms and confirmed previous reports that a GABAergic mechanism is involved. In order to understand how neurons in this pathway transmit information we developed a new strategy that allows selective ablation of targeted output synapses from a neuron of interest (1). To achieve this, we fuse a photosensitizer to a presynaptic protein and use a digital micromirror device to illuminate output synapses of neurons of interest only onto specific post-synaptic neurons. This method has broad utility in neural circuit studies. 2. Functional mapping of neuronal architecture mediating short-term behavioral states. Over the course of the day, goals change in response to both internal and external cues. At any given moment, an individual’s behavioral state strongly influences decisions on how to interact with the environment. We aim to identify the neural systems that maintain short-term behavioral states and to determine how they interact with central mechanisms for sensory processing. We have developed several paradigms in which the behavioral state of zebrafish is temporarily altered. Our strategy is to allow larvae to remain in the arena until their motor behavior achieves a stable state, then introduce a temporary perturbation to the environment and examine changes in behavior that persist on a time-scale of several minutes after the perturbation is removed. One robust paradigm that we recently used to study behavioral state control is flow-induced arousal. After exposure to a brief water flow stimulus, zebrafish larvae show elevated motor activity and sensory responsiveness that persists for several minutes after termination of water movement. We previously showed that the serotonergic raphe nucleus regulates sensory responsiveness during this state, but the neural basis for hyperactivity was not known. Using a genetically encoded reporter of calcium activity (CaMPARI2), we showed that flow-induced arousal is accompanied by a distributed increase in brain activity. To identify neurons that mediate this flow-induced swimming, we performed an ablation screen of a library of Gal4 transgenic lines and recovered a Gal4 line that labels neurons required for this behavior. By comparing the pattern of expression in this line to CaMPARI2 labeling, we have pinpointed specific neurons that mediate flow-induced swimming, and found molecular markers for these neurons. 3. Analysis of zebrafish models of neurodevelopmental disorders. We collaborate widely with clinicians to generate and characterize zebrafish models for mutations discovered in humans (often through exome-sequencing) that are likely to have a neurodevelopmental origin. We use the CRISPR/Cas9 system to generate lesions in zebrafish genes that are homologous to those disrupted in the human disorders. We then apply behavioral analysis, transcriptomics, and voxel-based morphometry as part of a broad phenotyping strategy. A unique feature of brain imaging in zebrafish is the ability to visualize the total architecture of the brain while simultaneously recording the position and morphology of every constituent labeled neuron. High precision alignment then permits statistically robust whole-brain analysis of neuronal composition and morphology in zebrafish mutant models, pinpointing brain regions with changes that are difficult to detect visually. The technique can be applied to almost any zebrafish neurodevelopmental model, thereby enabling robust and quantitative detection of subtle changes in brain structure or composition. Through this work, we aim to provide insight into the fundamental molecular and cellular processes associated with each disorder. Using voxelwise morphometry, we discovered that mutations in xrcc1, a human disease gene, leads to a highly localized reduction in tissue volume in the larval cerebellum. Xrcc1 is a scaffold protein that negatively regulates Parp1, a protein involved in single-strand break repair (2). We showed that cerebellar defects in xrcc1 mutants can be rescued by simultaneously knocking down parp1, suggesting that parp1 inhibitors might be useful therapeutic candidates for xrcc1 related disorders. We also studied srrm4, which earlier studies had suggested was a key regulator of alternatively spliced microexons during neural development that were implicated in the etiology of autism. We found that srrm4 is indeed expressed during neural development in zebrafish, enriched in granule populations in the cerebellum and torus longitudinalis. However, we could not reproduce previously described morpholino phenotypes in mutants, and found that loss of srrm4 had little effect on neural development or microexon splicing in zebrafish (3). In a third project on neurodevelopmental disorders, we studied a new zebrafish model for Timothy syndrome (4). Timothy syndrome (TS) is a rare pediatric disorder characterized by cardiac arrhythmia and neurodevelopmental impairments (seizures and intellectual disability). In TS, a de novo G406 missense mutation in cacna1c, the pore-forming subunit of the voltage-gated calcium channel Cav1.2 leads to a sustained depolarization of electrically excitable cells in the brain and heart, due to an impairment in voltage-dependent inactivation. To assess the effects of the TS mutation on neural development, we characterized a new zebrafish model, generated using CRISPR-Cas9 to knock-in the the patient mutation into the homologous residue of cacna1c. Similar to clinical manifestations in patients, homozygous mutant zebrafish showed cardiac arrhythmia (bradycardia and intermittent 2:1 atrioventricular block), elevated neural activity and susceptibility to seizure-like behavior, providing evidence for model validity. As in mouse and organoid models, we saw abnormalities in forebrain inhibitory neurons; the greatest change, however, was a reduction in the en1b labeled midbrain-hindbrain boundary region, consistent with a reduction in cerebellar and optic tectum volume. We used the TS fish to assess a previously proposed therapeutic strategy: because TS mutations affect one of the two alternatively spliced variants of exon 8, antisense splice-site oligonucleotides might suppress incorporation of the mutant exon and thereby benefit patients. Supporting this idea, we found that a splice-blocking morpholino against the mutant exon shifted isoform use to the intact exon 8 variant and restored normal cardiac contraction frequency in mutants. These results validated the use of zebrafish as system in which to analyze the etiology of, and therapeutic strategies for TS. However we were puzzled to find that heterozygous fish were adult viable and fertile because the TS mutation is dominant in patients. Based on a report that at temperatures above 37°C, Cav1.2 channels open spontaneously, we speculated that heterozygotes may be spared due to the low temperature at which zebrafish are raised and tested (28°C). Supporting this, heterozygous larvae showed tachycardia and seizures when tested at 37°C, thus implicating fever as a modifiable risk in TS. The zebrafish model therefore offers a valuable new platform for dissecting TS pathophysiology and identifying strategies to mitigate fever-related crises.