The finding of graded, hyperpolarizing potentials in both receptors and horizontal cells has been somewhat surprising. Svaetichin and his co-workers (24, 30, 37) have by 10.220.33.

Optokinetic and phototactic behaviors of zebrafish larvae were examined for their usefulness in screening for recessive defects in the visual system. The optokinetic response can be reliably and rapidly detected in 5-day larvae, whereas the phototactic response of larvae is variable and not robust enough to be useful for screening. We therefore measured optokinetic responses ofmutagenized larvae as a genetic screen for visual system defects. Third-generation larvae, representing 266 mutagenized genomes, were examined for abnormal optokinetic responses. Eighteen optokinetic-defective mutants were identified and two mutants that did not show obvious morphological defects, no optokinetic response a (noa) and partial optokinetic response a (poa), were studied further. We recorded the electroretinogram (ERG) to determine whether these two mutations affect the retina. The b-wave of noa larvae was grossly abnormal, being delayed in onset and significantly reduced in amplitude. In contrast, the ERG waveform of poa larvae was normal, although the b-wave was reduced in amplitude in bright light. Histologically, the retinas of noa and poa larvae appeared normal. We conclude that noa larvae have a functional defect in the outer retina, whereas the outer retina of poa larvae is likely to be normal. genetic dissection of the zebrafish visual system should be applicable to other vertebrates.Recently, two groups developed chemical mutagenesis procedures and methods for efficiently growing large numbers of zebrafish (9-12). These procedures have made it possible to conduct large-scale genetic screens in which zebrafish larvae from the third generation are analyzed for recessive mutations. Furthermore, a genetic linkage map in zebrafish is now available so mutant genes can be isolated by positional cloning (13).We first characterized two visual behaviors-phototaxis and optokinetic responses-in wild-type zebrafish larvae (3-19 days pf). Preliminary experiments on wild-type larvae (4) suggested that both of these assays would be useful. We then analyzed the optokinetic responses of mutagenized larvae as a primary screen for detecting recessive defects in the visual system. As a secondary screen, we recorded the electroretinogram (ERG) from larvae 5-7 days pf to identify mutations that specifically affect the retina. We describe here the feasibility of this approach for identifying mutations affecting the visual system and describe two mutants isolated on the basis of their abnormal optokinetic response.Benzer (1) was the first to report that mutant Drosophila could be identified by their phototactic behavior. Subsequently, a number of nonphototactic mutants were found to have specific molecular defects in their photoreceptors (2). A phototaxis mutant that failed to respond to UV light, sevenless, lacks UV-sensitive photoreceptor cells (3); analysis of this mutant has defined the role of cell-cell interactions in ommatidial development (for review, see ref. 6).Because there are significant differences between vertebrate and ...

Retinal dystrophies, known in man, dog, mouse, and rat, involve progressive loss of photoreceptor cells with onset during or soon after the developmental period. Functional (electroretinogram), chemical (rhodopsin analyses) and morphological (light and electron microscopy) data obtained in the rat indicated two main processes: (a) overproduction of rhodopsin and an associated abnormal lamellar tissue component, (b) progressive loss of photoreceptor cells. The first abnormality recognized was the appearance of swirling sheets or bundles of extracellular lamellae between normally developing retinal rods and pigment epithelium; membrane thickness and spacing resembled that in normal outer segments. Rhodopsin content reached twice normal values, was present in both rods and extracellular lamellae, and was qualitatively normal, judged by absorption maximum and products of bleaching. Photoreceptors attained virtually adult form and ERG function. Then rod inner segments and nuclei began degenerating; the ERG lost sensitivity and showed selective depression of the a-wave at high luminances. Outer segments and lamellae gradually degenerated and rhodopsin content decreased. No phagocytosis was seen, though pigment cells partially dedifferentiated and many migrated through the outer segment-debris zone toward the retina. Eventually photoreceptor cells and the b-wave of the ERG entirely disappeared. Rats kept in darkness retained electrical activity, rhodopsin content, rod structure, and extracellular lamellae longer than litter mates in light.

Much has been learned about vertebrate development by random mutagenesis followed by phenotypic screening and by targeted gene disruption followed by phenotypic analysis in model organisms. Because the timing of many developmental events is critical, it would be useful to have temporal control over modulation of gene function, a luxury frequently not possible with genetic mutants. Here, we demonstrate that small molecules capable of conditional gene product modulation can be identified through developmental screens in zebrafish. We have identified several small molecules that specifically modulate various aspects of vertebrate ontogeny, including development of the central nervous system, the cardiovascular system, the neural crest, and the ear. Several of the small molecules identified allowed us to dissect the logic of melanocyte and otolith development and to identify critical periods for these events. Small molecules identified in this way offer potential to dissect further these and other developmental processes and to identify novel genes involved in vertebrate development.

Understanding the molecular basis of addiction could be greatly aided by using forward genetic manipulation to lengthen the list of candidate genes involved in this complex process. Here, we report that zebrafish exhibit cocaine-induced conditioned place preference. In a pilot screen of 18 F2 generation families of mutagenized fish, we found three with abnormally low responses to cocaine. This behavior was inherited by the F3 generation in a manner that suggests the abnormalities were because of dominant mutations in single genes. Performance profiles in secondary behavioral screens measuring visual dark-adaptation and learning suggest that the defects were the result of mutations in distinct genes that affect dopaminergic signaling in the retina and brain.A ddiction, the compulsive intake of certain substances despite adverse consequences, continues to be a tremendous public health issue, costing billions of dollars per year (1). To understand addiction better and to design therapeutic strategies, several avenues of investigation have been taken to elucidate the genetic bases of addiction-related behaviors. Selective inbreeding of mouse strains displaying differing degrees of addictionrelated behaviors has been used to correlate the behavior with particular genetic polymorphisms (2). Although this method has great promise, few strong correlations have been made owing to the time required to generate the large numbers of families necessary. Also, the limited number of inbred stains with a given behavioral phenotype prevents characterization of more than a few genes important in addiction-related behaviors. Transgenics have also been used to correlate specific behaviors with the function of known genes (3). However, background effects and compensation by other related genes can complicate analysis of transgenic mouse models. Furthermore, both methods rely heavily on a candidate approach, requiring that the genes of interest be well characterized ahead of time.Methods of forward genetics in which the genome is mutagenized, resulting phenotypes are characterized, and underlying genes are subsequently cloned offer the advantage of not needing to know the genes a priori. Indeed, this approach has been used to determine sensitivity to particular substances such as cocaine or ethanol in Drosophila (4). However, the level of behavioral analysis possible in Drosophila is limited by fundamental differences of their central nervous system relative to vertebrates. Forward genetics on a vertebrate displaying complex, addiction-related behavior would be ideal. By virtue of their large clutch size and relatively low maintenance costs, zebrafish (Danio renio) are currently the vertebrate of choice in forward genetics experimentation (5). The level of behavioral analysis possible in these animals is only now being explored.The role of midbrain dopamine in behaviors related to addiction has been exhaustedly researched (6, 7). Microdialysis, intracerebral injection, lesion, and electrical self-stimulation experiments have all impl...