The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

Whereas the zebrafish retina has long been an important model system for developmental and genetic studies, little is known about the responses of the inner retinal neurons. Here we report single-unit ganglion cell recordings from 5-to 6-day-old zebrafish larvae. In wild-type larvae we identify at least five subtypes of ganglion cell responses to full-field illumination, with ON-OFF and ON-type cells predominating. In the nrc mutant retina, in which the photoreceptor terminals develop abnormally, we observe normal OFF responses but abnormal ON-OFF responses and no ON responses. Previously characterized as blind, these mutants lack an optokinetic reflex (OKR), but in another behavioral assay nrc mutant fish have near-normal responses to the offset of light and slow and sluggish responses to the onset of light . Pharmacological block of the ON pathway mimics most of the nrc visual defects. We conclude that the abnormal photoreceptor terminals in nrc mutants predominantly perturb the ON pathway and that the ON pathway is necessary to drive the OKR in larval zebrafish. extracellular recordings ͉ ON and OFF retinal pathway ͉ optokinetic response ͉ retina ͉ retinal ganglion cells

Fragile X syndrome is caused by loss of Fragile X Mental Retardation Protein (FMRP), an RNA binding protein that suppresses protein translation. Here, we identified Down Syndrome Cell Adhesion Molecule (Dscam) RNA, a molecule involved in neural development and implicated in Down syndrome, bound to FMRP. Elevated Dscam protein levels in Drosophila FMRP null animals and in animals with three copies of the Dscam gene both produced specific and similar synaptic targeting errors in a hard-wired neural circuit which impaired the animal’s sensory perception. Reducing Dscam levels in FMRP null animals reduced synaptic targeting errors and rescued behavioral responses. Our results demonstrate that excess Dscam protein may be a common molecular mechanism underlying altered neural wiring in major causes of intellectual disability.

Darkness serves as a stimulus for vertebrate photoreceptors; they are actively depolarized in the dark and hyperpolarize in the light. Here, we show that larval zebrafish essentially turn off their visual system at night when they are not active. Electroretinograms recorded from larval zebrafish show large differences between day and night; the responses are normal in amplitude throughout the day but are almost absent after several hours of darkness at night. Behavioral testing also shows that larval zebrafish become unresponsive to visual stimuli at night. This phenomenon is largely circadian driven as fish show similar dramatic changes in visual responsiveness when maintained in continuous darkness, although light exposure at night partially restores the responses. Visual responsiveness is decreased at night by at least two mechanisms: photoreceptor outer segment activity decreases and synaptic ribbons in cone pedicles disassemble.photoreceptors | circadian rhythm | synaptic plasticity

Accurate measurement of the amount of specific protein a cell produces is important for investigating basic molecular processes. We have developed a technique that allows for quantitation of protein levels in single cells in vivo. This protein quantitation ratioing (PQR) technique uses a genetic tag that produces a stoichiometric ratio of a fluorescent protein reporter and the protein of interest during protein translation. The fluorescence intensity is proportional to the number of molecules produced of the protein of interest and is used to determine the relative amount of protein within the cell. We use PQR to quantify protein expression of different genes using quantitative imaging, electrophysiology, and phenotype. We use genome editing to insert Protein Quantitation Reporters into endogenous genomic loci in three different genomes for quantitation of endogenous protein levels. The PQR technique will allow for a wide range of quantitative experiments examining gene-to-phenotype relationships with greater accuracy.