Multiplex Neural Circuit Tracing With G-Deleted Rabies Viral Vectors

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 (RV∆G) comprises a powerful approach for studying the structure and function of neural circuits. Pseudotyping of RV∆G 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. Transcomplementation 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 RV∆G 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 RV∆G 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 RV∆G 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.

In Vivo Whole-Cell Patch-Clamp Methods: Recent Technical Progress and Future Perspectives

“…They profiled the sensory response properties and determined the monosynaptic connectivity in the cortico-cortical or cortico-thalamic loop mediating neocortical neural computations relevant to sensory perception [ 172 ]. The gene manipulation-aided identification of synaptic connectivity impinging onto the patched cell bridges the gap between the anatomical and physiological properties of neural networks [ 173 , 174 , 175 , 176 , 177 , 178 , 179 ].…”

Cell type‐ and layer‐specific convergence in core and shell neurons of the dorsal lateral geniculate nucleus

“…RVΔG was generated as previously described (Osakada et al, 2011; Osakada & Callaway, 2013; Suzuki et al, 2020). Briefly, RVΔG was recovered in B7GG cells by transfection with the plasmids pcDNA‐B19G, pcDNA‐B19N, pcDNA‐B19P, pcDNA‐B19L, or pSADΔG‐GFP.…”

“…AAVs were generated in HEK293T cells using triple‐transfection methods as previously described (Suzuki et al, 2020). Briefly, AAV2/9 was produced by the transfection of HEK293T cells with pHelper (Cell Biolabs, San Diego, CA, USA), rep/cap vector (pAAV2.9), and pAAV genomic vector.…”

“…mCherryexpressing axon arbors of the AAV-infected dLGN neurons were distributed around Layers 4 and 6 of the mouse V1 (Figure 1 To identify presynaptic cells in the dLGN core region in mice, we combined a RVΔG trans-synaptic tracing system with the iontophoretic injection system (Osakada et al, 2011;Osakada & Callaway, 2013;Wickersham et al, 2007). First, to locally express TVA (oTVA-L), the EnvA receptor, and the rabies glycoprotein (oG) in the dLGN region (Kim et al, 2016;Suzuki et al, 2020), we iontophoretically injected a mixture of three AAVs into the dLGN under the above conditions: AAV2/9-hSyn-iCre or AAV2/9-eSyn-iCre, AAV2/9-CAG-FLEX-oTVA-L-mCherry, and AAV2/9 expressing both histone-tagged BFP (nuclear hBFP) and oG under the CAG promoter (AAV2/9-CAG-hBFP-P2A-oG) ( Figures 1(c,d)). Next, we injected the EnvApseudotyped G-deleted RV expressing GFP (EnvA-RVΔG-GFP) into the dLGN by air pressure injection 2-3 weeks after iontophoretic injection of the AAV mixture (Figure 1 monosynaptic circuit tracing procedure from the dLGN core region of adult mice.…”

Golgi staining-like retrograde labeling of brain circuits using rabies virus: Focus onto the striatonigral neurons

“…The failure of rCVS-N2c-M-GFP to provide satisfactory staining per se did not allow us to achieve such an objective. Recently, multiplex G-deleted RABV tracing has been developed to simultaneously label and characterize multiple circuits in single animals (Suzuki et al, 2020).…”