The trace amine para-tyramine is structurally and functionally related to the amphetamines and the biogenic amine neurotransmitters. It is currently thought that the biological activities elicited by trace amines such as p-tyramine and the psychostimulant amphetamines are manifestations of their ability to inhibit the clearance of extracellular transmitter and/or stimulate the efflux of transmitter from intracellular stores. Here we report the discovery and pharmacological characterization of a rat G protein-coupled receptor that stimulates the production of cAMP when exposed to the trace amines p-tyramine, beta-phenethylamine, tryptamine, and octopamine. An extensive pharmacological survey revealed that psychostimulant and hallucinogenic amphetamines, numerous ergoline derivatives, adrenergic ligands, and 3-methylated metabolites of the catecholamine neurotransmitters are also good agonists at the rat trace amine receptor 1 (rTAR1). These results suggest that the trace amines and catecholamine metabolites may serve as the endogenous ligands of a novel intercellular signaling system found widely throughout the vertebrate brain and periphery. Furthermore, the discovery that amphetamines, including 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy"), are potent rTAR1 agonists suggests that the effects of these widely used drugs may be mediated in part by this receptor as well as their previously characterized targets, the neurotransmitter transporter proteins.

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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Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system. T HE zebrafish has been an important model through apparent by the 18-19 SS. The first postmitotic neurons of the retina are generated at 28 hr postfertilization which genes necessary for visual system development and function have been identified (reviewed in (hpf) and by 72 hpf the retina is functional (Easter and Nicola 1996; Hu and Easter 1999; Schmitt and Easter and Malicki 2002 and Neuhauss 2003). Zebrafish eyes are large, easily accessible, and structurally Dowling 1999). Retinas of many fish and amphibians also possess a specialized region at their margins, termed similar to the human eye. Eye formation in zebrafish is analogous to that observed in other vertebrate embryos, peripheral or ciliary marginal zones, that perpetually adds cells to the retina during the lifetime of the animal thus providing an excellent model system with which the understanding of vertebrate eye development can ( Johns 1977). Several generations of chemically based forward gebe advanced. Additionally, many disrupted genes and pathways identified as integral to the formation of the netic screens have been undertaken in zebrafish (Driever et al. 1996;Haffter et al. 1996; Matsuda and Mishina zebrafish eye produce phenotypes that resemble disorders of the human visual system. Thus, characterization 2004), some of which have focused on eye development and function (Malicki et al. 1996; Fadool et al. 1997; of the molecular mechanisms of eye development in zebrafish should facilitate a better understanding of these hu- Neuhauss et al. 1999). While these chemically based screens have been instrumental in generating interesting man pathologies (Goldsmith and Harris 2003).Eye development in zebrafish ...

These results demonstrate that the v-ATPase complex plays several critical roles during vertebrate eye development and maintenance, and they suggest that defects in v-ATPase complex function could possibly underlie human ocular disorders that affect the RPE.