Abstract
Neural circuits interconnect to organize large-scale networks that generate perception, cognition, memory, and behavior. Information in the nervous system is processed both through parallel, independent circuits and through intermixing circuits. Analyzing the interaction between circuits is particularly indispensable for elucidating how the brain functions. Monosynaptic circuit tracing with glycoprotein (G) gene-deleted rabies viral vectors (RVΔG) comprises a powerful approach for studying the structure and function of neural circuits. Pseudotyping of RVΔG with the foreign envelope EnvA permits expression of transgenes such as fluorescent proteins, genetically-encoded sensors, or optogenetic tools in cells expressing TVA, a cognate receptor for EnvA. Trans-complementation with rabies virus glycoproteins (RV-G) enables trans-synaptic labeling of input neurons directly connected to the starter neurons expressing both TVA and RV-G. However, it remains challenging to simultaneously map neuronal connections from multiple cell populations and their interactions between intermixing circuits solely with the EnvA/TVA-mediated RV tracing system in a single animal. To overcome this limitation, here, we multiplexed RVΔG circuit tracing by optimizing distinct viral envelopes (oEnvX) and their corresponding receptors (oTVX). Based on the EnvB/TVB and EnvE/DR46-TVB systems derived from the avian sarcoma leukosis virus (ASLV), we developed optimized TVB receptors with lower or higher affinity (oTVB-L or oTVB-H) and the chimeric envelope oEnvB, as well as an optimized TVE receptor with higher affinity (oTVE-H) and its chimeric envelope oEnvE. We demonstrated independence of RVΔG infection between the oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems and in vivo proof-of-concept for multiplex circuit tracing from two distinct classes of layer 5 neurons targeting either other cortical or subcortical areas. We also successfully labeled common input of the lateral geniculate nucleus to both cortico-cortical layer 5 neurons and inhibitory neurons of the mouse V1 with multiplex RVΔG tracing. These oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems allow for differential labeling of distinct circuits to uncover the mechanisms underlying parallel processing through independent circuits and integrated processing through interaction between circuits in the brain.
Highlights
The function of the nervous system arises from complex interactions between networks of neurons composed of multiple cell types
Deleting native envelope glycoprotein genes from the rabies genome and pseudotyping the RV∆G with a foreign glycoprotein, such as the EnvA derived from avian sarcoma leukosis virus (ASLV), allows targeting of viral vectors to specific cell populations that express the receptors of foreign glycoproteins such as TVA (Mebatsion et al, 1996; Etessami et al, 2000; Wickersham et al, 2007; Osakada and Callaway, 2013; Figure 1A)
GFP-positive and H2B-tagBFP-negative neurons were observed outside layer 5 of the cortex. This is because leak expression of optimized tumor virus subgroup E (TVE) receptor with higher affinity (oTVE-H) derived from AAV-DIO vectors induced oEnvERV∆G-GFP infection. These results suggest that the 2A-system permits labeling and detection of the starter neurons with a fluorescence reporter and that only a small amount of oTVE-H is needed for the infection of oEnvE-RV∆G even at a barely visible expression level of its reporter fluorescence
Summary
The function of the nervous system arises from complex interactions between networks of neurons composed of multiple cell types. The wiring patterns of individual cell types underlie how neural circuits process and represent information. Detailed information on the cell types and their connectivity, in addition to the spatiotemporal patterns of activity in neural circuits, is essential for understanding how the brain functions. The combination of parallel processing through independent circuits and integrated processing through interaction between circuits is the fundamental principle of neural circuits and computations in the brain. Despite the advances in methods for linking cell types to neural circuits (Arenkiel and Ehlers, 2009; Luo et al, 2018), a major impediment in elucidating how information processing is integrated in the nervous system is lack of means for the dissection of the complex interactions between neural circuits
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