CNS Hypomyelination Disrupts Axonal Conduction and Behavior in Larval Zebrafish

Myelination is essential for central nervous system (CNS) formation, health and function. As a model organism, larval zebrafish have been extensively employed to investigate the molecular and cellular basis of CNS myelination, because of their genetic tractability and suitability for non-invasive live cell imaging. However, it has not been assessed to what extent CNS myelination affects neural circuit function in zebrafish larvae, prohibiting the integration of molecular and cellular analyses of myelination with concomitant network maturation. To test whether larval zebrafish might serve as a suitable platform with which to study the effects of CNS myelination and its dysregulation on circuit function, we generated zebrafish myelin regulatory factor (myrf) mutants with CNS-specific hypomyelination and investigated how this affected their axonal conduction properties and behavior. We found that myrf mutant larvae exhibited increased latency to perform startle responses following defined acoustic stimuli. Furthermore, we found that hypomyelinated animals often selected an impaired response to acoustic stimuli, exhibiting a bias toward reorientation behavior instead of the stimulus-appropriate startle response. To begin to study how myelination affected the underlying circuitry, we established electrophysiological protocols to assess various conduction properties along single axons. We found that the hypomyelinated myrf mutants exhibited reduced action potential conduction velocity and an impaired ability to sustain high-frequency action potential firing. This study indicates that larval zebrafish can be used to bridge molecular and cellular investigation of CNS myelination with multiscale assessment of neural circuit function. SIGNIFICANCE STATEMENT Myelination of CNS axons is essential for their health and function, and it is now clear that myelination is a dynamic life-long process subject to modulation by neuronal activity. However, it remains unclear precisely how changes to myelination affects animal behavior and underlying action potential conduction along axons in intact neural circuits. In recent years, zebrafish have been employed to study cellular and molecular mechanisms of myelination, because of their relatively simple, optically transparent, experimentally tractable vertebrate nervous system. Here we find that changes to myelination alter the behavior of young zebrafish and action potential conduction along individual axons, providing a platform to integrate molecular, cellular, and circuit level analyses of myelination using this model

Melanopsin Carboxy-terminus phosphorylation plasticity and bulk negative charge, not strict site specificity, achieves phototransduction deactivation.

Melanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsin's Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and β-arrestin binding are centered around these critical phosphorylation sites (S388-S395). The degree of functional versatility described in this study, along with ipRGC biophysical heterogeneity and the possible use of multiple signal transduction cascades, might contribute to the diverse ipRGC light responses for use in non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4

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

BackgroundThe 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.MethodsTo 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.ResultsWhole-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.ConclusionsTaken 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