Behavioral screening for cocaine sensitivity in mutagenized zebrafish

Abstract

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...

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Cerebellar-Dependent Learning in Larval Zebrafish

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Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish

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