Development of an Acute Method to Deliver Transgenes Into the Brains of Adult Xenopus laevis

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.

Application of Recombinant Rabies Virus toXenopusTadpole Brain

“…Virus-mediated gene expression in non-mammalian vertebrates has had mixed results. AAV and lentivirus, which have been used with great success in mammals ( Nassi et al, 2015 ; Parr-Brownlie et al, 2015 ; Haery et al, 2019 ), infect Xenopus and zebrafish either inconsistently or not at all ( Zhu et al, 2009 ; Yamaguchi et al, 2018 ). Xenopus express two homologues of the Cosackie and adenovirus receptor (CAR) rendering them susceptible to infection by adenovirus and adenovirus-mediated expression of EGFP lasts for at least 10 d in non-neuronal tissue ( Kawakami et al, 2006 ; Dutton et al, 2009 ).…”

“…However, there was very little expression detected by immunohistochemistry in tectal neurons following electroporation in vivo. Yamaguchi et al, (2018) demonstrated that B19G incorporated into the VSV genome was also insufficient to produce transneuronal transfer, suggesting that the difficulty of expressing B19G in vivo in Xenopus is not specific to electroporation.…”

“…Neither adeno-associated virus (AAV) nor lentivirus transduce brain cells in Xenopus ( Yamaguchi et al, 2018 ). Vaccinia virus and vesicular stomatitis virus (VSV) have been used for gene expression in Xenopus ( Wu et al, 1995 ; Mundell et al, 2015 ; Yamaguchi et al, 2018 ), but expression from Vaccinia is transient and VSV transduction is inefficient (A. Yamaguchi, personal communication). Therefore, we investigated applications of rabies virus in Xenopus .…”

Generation, Coordination, and Evolution of Neural Circuits for Vocal Communication

“…Temporal and spectral differences in Xenopus vocalizations across species are innate or genetically determined, rather than learned Yamaguchi et al (2018). (Tobias et al, 2011(Tobias et al, , 2014.…”

“…Viral tracing methods in Xenopus (Fig. 8; Yamaguchi et al, 2018) should facilitate the analysis of this entire social communication circuit at mesoscale (Mitra, 2014).…”