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

Abstract

The acoustic startle response is an evolutionary conserved avoidance behavior. Disruptions in startle behavior, in particular 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. This identified mutants 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 acts independently of supraspinal inputs to regulate locomotion, suggesting its site of action is within spinal circuitry.Moreover, we show that Kv1.1 protein is mis-localized in dolk mutants, suggesting they act in a common genetic pathway to regulate movement magnitude. 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. Author summaryUnderlying all animal behaviors are neural circuits, which are controlled by numerous molecular pathways that direct neuron development and activity. To identify and study these molecular pathways that control behavior, we use a simple vertebrate behavior, the acoustic startle response, in the larval zebrafish. In response to an intense noise, larval zebrafish will quickly turn 3 and swim away to escape. From a genetic screen, we have identified a number of mutants that behave in abnormal ways in response to an acoustic stimulus. We cloned these mutants and identified eight genes that regulate startle behavior. All eight genes are associated with human disorders, and here we focus on two genes, dolk and kcna1a, encoding Dolk, a key regulator of protein glycosylation, and the potassium channel Kv1.1, respectively. We demonstrate that loss of dolk or kcna1a causes larval zebrafish to perform exaggerated swim movements and that Dolk is required for Kv1.1 protein localization to axons of neurons throughout the nervous system, providing strong evidence that dolk and kcna1a act in a common molecular pathway. Combined, our studies provide new insights into the genetic regulation of startle behavior.

The Adaptor Protein 2 (AP2) complex modulates habituation and behavioral selection across multiple pathways and time windows

“…Acoustically-Evoked Behavior: After acclimating to testing conditions on a light box for 45min, healthy fish were placed in individual wells of a custom plexiglass testing arena, either 9mm diameter wells in a 36-well arena or 9mm x 9mm square wells of a 16-well arena, illuminated from below with an IR array (CM-IR200-850, CMVision), attached to a vibrational exciter (4810, Brüel & Kjaer) that administers acoustic stimuli, as previously described 30,97 . Acoustic stimuli were 2ms tones (1000 Hz), varying intensity as needed for each assay.…”

Neurological mechanism of sensory deficits after developmental exposure to non-dioxin-like polychlorinated biphenyls (PCBs)

“…The startle response circuit is of wide relevance and well-established for assessing the influence of specific factors on neuronal cell development, neuromuscular connectivity, and transmission (Burgess and Granato, 2007;Medan and Preuss, 2014;Meserve et al, 2020;Panlilio et al, 2020). We used the startle response circuit to assess the effects of NDL-PCBs on the formation of neuronal populations and neurotransmission and its consequences for mechanosensory disruptions, thus encompassing hearing and touch.…”

“…We used the zebrafish auditory system (Figure 1) as a model to decipher the involvement of these three major targets in disrupting the startle response after developmental exposure to NDL PCB153. Many of the pathways and genes that regulate startle behavior in fish also operate in human neurological disorders (Howe et al 2013;Maximino and Herculano 2010;Meserve et al 2020;Tropepe and Sive 2003;Wolman et al 2015). We demonstrate that environmentally relevant concentrations (10 nM) of NDL PCB153 alter startle response which coincided with axonal swelling and dysregulated dopamine metabolite and GABA levels.…”

“…These signaling processes are widely conserved across vertebrate species, and are important in human neurological diseases. For example, a recent study found that multiple gene mutations causing defective startle response in zebrafish were also associated with disorders of the locomotor system in humans (Meserve et al, 2020).…”

“…In fish, the auditory-evoked startle response plays a critical role in animal survival, and involves cellular functions, sensory-motor pathways, and monoamine neurotransmitters that are conserved among vertebrates (Stewart et al 2014). Many of the pathways and genes that regulate zebrafish startle behavior also operate in human neurological disorders (Howe et al 2013; Maximino and Herculano 2010; Meserve et al 2020; Tropepe and Sive 2003; Wolman et al 2015). In this study, we use the zebrafish startle response to decipher the neuro-disruptive basis of NDL-PCBs on the auditory system during the early stages of development.…”