Viral vectors to deliver trasngenes into neurons of adult African clawed frogs, Xenopus laevis

Journal ArticleThe African clawed frog, Xenopus laevis, produce sexually distinct vocalizations to coordinate reproduction. Xenopus vocal behavior is an ideal model to study the neural basis of behavior for a variety of reasons. Unlike most other behaviors exhibited by vertebrate species, the neural pathways for vocal production is simple because the sound is produced by a single pair of muscles. Fictive vocalizations can be readily elicited from the isolated brains of adult male X. laevis, allowing detailed experimental analyses of the vocal circuitry. Furthermore, female vocalizations can be masculinized in response to androgen in a relatively short amount of time, providing us with an opportunity to understand androgen-induced modification of a neural circuits. Additionally, an abundance of species within the Xenopus genus allows comparative analyses of the vocal neural circuitry across species. The ultimate goal of our research program is to understand how sex-specific and species-specific motor programs are generated, and how neural output can be modified under selective pressure. Application of optogenetic tools to the fictive preparation will greatly enhance our ability to test the causal relations between neuronal activity and vocal motor programs. However, a major obstacle to achieving this level of understanding is the current lack of tools to manipulate gene expression in adult X. laevis. Transgenic lines have been generated in X. laevis, but progress is slowed by long generation times. Electroporation can be used for the acute delivery of genes into cells, but suffers from low efficacy. Viral vectors, in contrast, are efficient, stable, and thus are a great alternative. Although most viral vectors used in the field of neuroscience target mammals as host, we have found that recombinant vesicular stomatitis viral (VSV) vector can be readily used to transduce neurons of adult Xenopus laevis in vivo. Although the vector is toxic to the host neurons in mammals, the transduced frog neurons remained healthy for a significantly longer period of time (up to 9 days) than mouse neurons, and the central vocal pathways including vocal neurons transduced by VSVs can generate fictive vocalizations. Recently, we found that dissociated adult frog neurons transduced with VSV encoding the Guillardia theta anion channel rhodopsin 2 (GtACR2, created by Connie Cepko's laboratory) can be readily inhibited by illumination, holding promise of the vector as an optogenetic inhibition tool in Xenopus. Given their broad tropism, VSV vectors are likely to serve as a powerful tool to deliver transgenes into other non-genetic model organisms