HIGASHIJIMA, S.; MANDEL, G.; FETCHO, J. R.; SUNY Stony Brook; SUNY Stony Brook; SUNY Stony Brook: Optical and genetic studies of neuronal networks for movement II

Genetic tools available for zebrafish offer many advantages for the study of neuronal circuitry. By injecting a DNA construct coding for green fluorescent protein (GFP) into one-cell stage embryos, we can label all types of neurons by using a pan-neuronal promoter, or specific types by using a cell type specific promoter. This labeling technique is very powerful for morphological studies. Furthermore, if a genetically encoded calcium indicator such as cameleon is used instead of GFP, it is possible to perform calcium imaging in the labeled neurons. This compliments calcium imaging with chemical indicators because it allows the labeling of neurons that are difficult to fill with other approaches, such as backfilling. To develop cameleon in zebrafish, we asked whether cameleon could detect neuronal activity during escape behaviors. We found that this was the case in neurons that we knew to be activated (motoneurons), showing the feasibility of using cameleon to monitor activity of neurons in vivo. To express cameleon in specific types of spinal interneurons, we tested regulatory regions of several genes that are expressed in a subset of neurons in the spinal cord. These include the glycine transporter2 (GlyT2; expressed in glycinergic neurons), the vesicular glutamate transporters (expressed in glutamatergic neurons), and some transcription factors such as En1 and Evx, which are thought to define subsets of spinal interneurons. We have so far successfully driven expression with regulatory regions of GlyT2 and En1. Injection of a DNA construct of GlyT2-driven cameleon labeled many commissural neurons, while injection of an En1-driven construct labeled many ipsilateral ascending neurons. We are now performing calcium imaging in those neurons to explore their contributions to motor behaviors.