Abstract
The central vocal pathway of the African clawed frog, Xenopus laevis, is a powerful vertebrate model to understand mechanisms underlying central pattern generation. However, fast and efficient methods of introducing exogenous genes into the neurons of adult X. laevis are currently not available. Here, we systematically tested methods of transgene delivery into adult X. laevis neurons. Although successfully used for tadpole neurons for over a decade, electroporation was not efficient in transfecting adult neurons. Similarly, adeno-associated virus (AAV) was not reliable, and lentivirus (LV) failed to function as viral vector in adult Xenopus neurons. In contrast, vesicular stomatitis virus (VSV) was a fast and robust vector for adult X. laevis neurons. Although toxic to the host cells, VSV appears to be less virulent to frog neurons than they are to mice neurons. At a single cell level, infected neurons showed normal physiological properties up to 7 days post infection and vocal circuits that included infected neurons generated normal fictive vocalizations up to 9 days post infection. The relatively long time window during which the physiology of VSV-infected neurons can be studied presents an ideal condition for the use of optogenetic tools. We showed that VSV does not gain entry into myelinated axons, but is taken up by both the soma and axon terminal; this is an attractive feature that drives transgene expression in projection neurons. Previous studies showed that VSVs can spread across synapses in anterograde or retrograde directions depending on the types of glycoprotein that are encoded. However, rVSV did not spread across synapses in the Xenopus central nervous system. The successful use of VSV as a transgene vector in amphibian brains not only allows us to exploit the full potential of the genetic tools to answer questions central to understanding central pattern generation, but also opens the door to other research programs that focus on non-genetic model organisms to address unique questions.
Highlights
Understanding the neural mechanisms underlying behavior presents a difficult challenge requiring a well-chosen model system and sophisticated experimental tools
We found that vesicular stomatitis virus (VSV) is a quick, efficient, and robust viral vector that can be used reliably to transduce neurons of adult X. laevis while other methods were either unreliable or entirely ineffective in introducing transgenes in adult X. laevis neurons
Our results suggest that the transduction rate of neurons by VSVs may be higher in the foreand midbrain than in hindbrain of adult X. laevis, quantitative analyses with a larger sample size is necessary in the future to clarify this point
Summary
Understanding the neural mechanisms underlying behavior presents a difficult challenge requiring a well-chosen model system and sophisticated experimental tools. Acute Transgene Delivery in Xenopus Brain neural mechanisms of calling can be studied in vitro because fictive vocalizations can be elicited in the isolated brain of adults (Rhodes et al, 2007), an example only found in select few vertebrate species. The vocalizations of female X. laevis can be rapidly masculinized in an androgen-dependent manner (Potter et al, 2005), providing us with a unique opportunity to explore neural plasticity. Despite these unique advantages, genetic tools that have revolutionized the field of neuroscience in recent years have largely not been available to the central nervous system (CNS) of adult X. laevis. We explored techniques to express exogenous genes in adult neurons of X. laevis
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