Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. To analyze somatosensory neuron diversity in larval zebrafish, we identified several enhancers from the zebrafish and pufferfish genomes and used them to create five new reporter transgenes. Sequential deletions of three of these enhancers identified small sequence elements sufficient to drive expression in zebrafish trigeminal and Rohon-Beard (RB) neurons. One of these reporters highlighted a somatosensory neuron subtype that expressed both the p2rx3a and pkca; genes, as well as a previously described trpA1b reporter. To determine whether neurons of this subtype possess characteristic peripheral branching morphologies or central axon projection patterns, we analyzed the morphology of single neurons. Surprisingly, although these analyses revealed diversity in peripheral axon branching and central axon projection, PKCa/p2rx3a/trpA1b-expressing RB cells did not possess characteristic morphological features, suggesting that even within this molecularly defined subtype, individual neurons may possess distinct properties.

Due to their external fertilization, rapid development, and optical clarity, zebrafish larvae are particularly well suited for optogenetic approaches to investigate neural circuit function. Here we demonstrate a procedure for expressing a light-sensitive ion channel in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. Specifically, we created a transgene using a somatosensory neuron enhancer to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicited behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. By introducing a few modifications to previously established methods, this approach was used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). The new transgenes created in this study will be powerful tools for further characterizing the molecular, morphological, and development diversity of larval somatosensory neurons and in combination with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.