Life-Long Genetic and Functional Access to Neural Circuits Using Self-Inactivating Rabies Virus

Abstract

SummaryNeural networks are emerging as the fundamental computational unit of the brain and it is becoming progressively clearer that network dysfunction is at the core of a number of psychiatric and neurodegenerative disorders. Yet, our ability to target specific networks for functional or genetic manipulations remains limited. Monosynaptically restricted rabies virus facilitates the anatomical investigation of neural circuits. However, the inherent cytotoxicity of the rabies largely prevents its implementation in long-term functional studies and the genetic manipulation of neural networks. To overcome this limitation, we developed a self-inactivating ΔG-rabies virus (SiR) that transcriptionally disappears from the infected neurons while leaving permanent genetic access to the traced network. SiR provides a virtually unlimited temporal window for the study of network dynamics and for the genetic and functional manipulation of neural circuits in vivo without adverse effects on neuronal physiology and circuit function.

The BRAIN Initiative Cell Census Consortium: Lessons Learned toward Generating a Comprehensive Brain Cell Atlas

“…For retrograde monosynaptic tracing, i.e., labeling neurons directly presynaptic to a defined population, the original rabies virus -based system (Wickersham et al, 2007b) has been refined in ways that range from improvements in the complementing helper viruses (Kim et al, 2016; Kohara et al, 2014; Liu et al, 2017; Miyamichi et al, 2013; Wall et al, 2010) to the use of a rabies virus strain with reduced toxicity (Reardon et al, 2016) and the addition of a destabilization domain to the viral nucleoprotein (Ciabatti et al, 2017). Although there are fewer tools available, anterograde monosynaptic tracing – i.e., labeling neurons directly postsynaptic to a starting population – based on the vesicular stomatitis virus or herpes simplex virus has been described (Beier et al, 2011; Zeng et al, 2017).…”

Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics

“…Additional modifications to the SADB19 rabies dG genome have given rise to less toxic viruses for prolonged expression. Rabies dGL (deletion of G and L genes) and self-inactivating rabies viruses (SiR dG) are promising new tools for studying neural network organizations (Chatterjee et al, 2018;Ciabatti et al, 2017). Rabies L protein is essential for viral genome transcription and protein production.…”

“…Neurons infected with SADB19 dGL appear intact and functional for many months. The self-inactivating (SiR dG) rabies genome is engineered to contain a PEST protein sequence that targets viral protein for destruction (Ciabatti et al, 2017). Degradation of viral proteins removes toxicity and allows for long-term expression of genes for neuroanatomical studies.…”

“…Please note that packaging and pseudotyping cell lines and plasmids for SADB19 rabies SiR dG and CVS-N2C rabies dG can be obtained from the laboratories of Dr. Marco Tripodi and Dr. Andrew Murray, who developed the protocols and created the cell lines, respectively (Ciabatti et al, 2017;Reardon et al, 2016). Necessary transfer vectors and packaging plasmids have been generously provided by authors and deposited in Addgene.…”

“…A tremendous body of work was published by Didier Trono and Patrick Salmon describing details of lentiviral packaging and titering methods (Salmon & Trono, 2007). The extensive retrograde activity of rabies dG, its characteristics, and production method were first described by the Ed Callaway team and developed further by the laboratories of Thomas Jessell, Marco Tripodi, and Ian Wickersham (Chatterjee et al, 2018;Ciabatti, Gonzalez-Rueda, Mariotti, Morgese, & Tripodi, 2017;Osakada & Callaway, 2013;Reardon et al, 2016). Protocols described in this unit are adapted from their published work to provide a side-by-side comparison between production efforts and effective use of each viral type.…”