Genetic dissection of the adult zebrafish brain by the GAL4-UAS system

○Pradeep Lal1,2, Koichi Kawakami1,2

1) Department of Genetics, Graduate University for Advanced Studies (SOKENDAI)
2) National Institute of Genetics, Mishima, Japan

Zebrafish shows various common vertebrate behavior such as habituation, escape behavior, fear response, operant conditioning, pavlovian conditioning etc. We have developed the pavlovian trace fear conditioning system for zebrafish. In this assay zebrafish shows robust learning in associating green light to electric shock. Although much has been understood about the neural circuit involved in this behavior from other vertebrate model organisms, our understanding of the neural circuits of adult zebrafish brain is limited by the inability to visualize and manipulate different brain regions. Using Tol2 transposon based Gal4-UAS system and enhancer trap, gene trap methods, we have been isolating transgenic fish lines that express Gal4 transactivator in specific tissues. In these lines, the Gal4-expressing cells are visualized by crossing them with the UAS-GFP line. To identify transgenic lines showing Gal4 expression in a subset of neurons in adult zebrafish brain, we observed 351 adult Gal4 gene trap and enhancer trap fish lines and selected 108 lines showing strong GFP expression in the brain. In addition, we observed 115 GFP gene trap and enhancer trap lines and selected 34 lines with GFP in the brain. These selected 142 fish lines were further analyzed by sectioning into 100 μ coronal slices of the brain. The observed GFP fluorescence patterns show unique patterns varying from ubiquitous whole brain patterns to very restricted expression patterns, such as a small region in dorsomedial telencephalon, lateral telencephalon, habenula, restricted region within hypothalamus, cerebellum etc. For most of these regions, anatomical description or marker genes have not been reported. Thus, these lines are useful for describing new regions in the brain and discovering new marker genes. To deduce the role of specific Gal4 labeled neurons in adult zebrafish brain, we need to inhibit their function selectively and look for any resulting change in behavioral abnormality. This can be done by crossing these Gal4 lines with the UAS-neurotoxin line and collecting the double transgenics for Gal4 and UAS-neurotoxin insertion, in which neurotoxin will selectively inhibit the neurotransmitter release in these Gal4 labeled neurons. Currently, I am analyzing the selected Gal4 transgenic lines with UAS-neurotoxin for learning defects in pavlovian trace fear conditioning assay.