RECOMBINASE-ORTHOGONAL MONOSYNAPTIC RABIES VIRUS TRACING TOOLS FOR INTRA-BRAIN SYNAPTIC NETWORK COMPARISONS: A CASE STUDY OF SYNGAP1 HAPLOINSUFFICIENCY

Normal development of neural circuitry requires carefully coordinated molecular and neural activity programs that guide, stabilize, and maintain synaptic wiring. Mutations that confer risk for neurodevelopmental disorders frequently disrupt synaptic proteins and may alter the formation of synaptic networks across the brain’s many neuron types. Rabies virus-based monosynaptic tracing is a powerful strategy to characterize cell type-specific connectivity, yet current methods cannot directly compare presynaptic networks arising from populations of neurons with and without conditional gene deletions in a single brain.

Here, we report a recombinase-orthogonal monosynaptic tracing system that overcomes this limitation. Our approach combines optimized Cre- or Flpo-dependent AAVs that selectively functionalize “starter” neurons to express TVA- or TVB. EnvA- or EnvB-pseudotyped N2c glycoprotein-deleted rabies viruses selectively label presynaptic inputs to these two parallel, genetically defined populations of postsynaptic starter neurons. Subcellular targeting of GFP and RFP reporters further allows unambiguous discrimination among four experimental conditions within a single animal. When paired with transgenic mouse models harboring recombinase-sensitive floxed alleles, this platform enables direct, within-brain comparisons of presynaptic network organization arising from wild-type versus mutant postsynaptic neurons, enabling sensitive and internally controlled analysis of cell autonomous mutational effects. We apply this system to interrogate synaptic network alterations in a mouse model of SYNGAP1-related intellectual disability, in which haploinsufficiency of SYNGAP1 leads to transient defects in neuronal morphology and persistent disruptions in adult cortical processing.