Title: Touch Responsiveness in Zebrafish Requires Voltage-gated Calcium Channel 2.1b 1 2 Abbreviated Title: Fakir Is Caused by a Mutation in Cav2.1b 3 4

24 25 The molecular and physiological basis of the touch-unresponsive zebrafish mutant fakir has 26 remained elusive. Here we report that the fakir phenotype is caused by a missense mutation in the gene 27 encoding voltage-gated calcium channel 2.1b (CACNA1Ab). Injection of RNA encoding wild type 28 CaV2.1 restores touch responsiveness in fakir mutants, whereas knockdown of CACNA1Ab via 29 morpholino oligonucleotides recapitulates the fakir mutant phenotype. Fakir mutants display normal 30 current-evoked synaptic communication at the neuromuscular junction, but have attenuated touch31 evoked activation of motor neurons. NMDA-evoked fictive swimming is not affected by the loss of 32 CaV2.1b, suggesting that this channel is not required for motor pattern generation. These results, 33 coupled with the expression of CACNA1Ab by sensory neurons, suggest that CaV2.1b channel activity 34 is necessary for touch-evoked activation of the locomotor network in zebrafish. 35 36 37 38 fakir is caused by a mutation in CaV2.1b Introduction 39 40 Sensory-evoked motor behaviors, common to most animals, are typified by running or flight in 41 terrestrial organisms, and swimming in aquatic organisms. Although these motor behaviors are 42 seemingly different, much of the underlying neural circuitry is similar. Stimuli are first perceived by 43 sensory neurons tuned to particular modalities. These sensory neurons relay inputs to interneurons, 44 which then activate motor neurons and induce muscle contractions resulting in locomotion. While 45 much has been learned about the circuitry underlying sensory-evoked motor behaviors in vertebrates 46 through comparative anatomy and in vivo electrophysiology, proportionally less is known about the 47 contribution of specific genes to the formation and function of these circuits. To overcome this deficit 48 within the context of touch-evoked motor behaviors we and others have turned to the model organism 49 zebrafish. 50 Zebrafish are ideally suited to address these questions as embryos develop externally and 51 possess a relatively simple nervous system amenable to both in vivo electrophysiology (Drapeau et al. 52 1999) and optical techniques (McLean and Fetcho 2011). In addition, recent advances have rendered 53 the zebrafish genome modifiable via forward and reverse genetic techniques (Lawson and Wolfe 54 2011). Finally, zebrafish mature quickly, going from a fertilized egg to an embryo capable of 55 responding to tactile stimuli with highly stereotyped motor behaviors within the first days of life 56 (Saint-Amant and Drapeau 1998). These features have fostered the use of zebrafish in large scale 57 mutagenesis screens aimed at identifying mutations which affect touch-evoked motor behaviors 58 (Granato et al. 1996). 59 Subsequent work with zebrafish mutants isolated from forward genetic screens have identified 60 genes necessary for touch-evoked motor behaviors at the level of skeletal muscle (Hirata et al. 2004; 61