Mutations in genes involved in synaptic function have been identified as risk factors for autism spectrum disorder (ASD), yet how risk-alleles encoding ubiquitous synaptic proteins affect the structure and function of synaptic circuits across the brains’ many cell populations remains unknown. Brain imaging in individuals with ASD and detailed functional characterization in animal models suggests large-scale changes in cell-type-specific synaptic connectivity relationships, yet characterizing such changes at scale remains a challenge.

In the current project, Arpiar Saunders plans to develop a novel in vivo experimental framework in which cell-autonomous, variant-specific molecular and synaptic connectivity phenotypes can be identified across cell types and developmental time periods in mice in a highly parallelized and scalable manner. The proposed framework combines next-generation viral tools and high-throughput single-cell RNA sequencing (scRNA-seq) to encode then read out (1) temporal and variant-specific manipulations; (2) quantitative reconstructions of cell-type-specific synaptic connectivity; and (3) gene expression information, all from the cellular RNA of each of many thousands of spatially intermingled cells.

To infer synaptic relationships and molecular profiles simultaneously from the same cells, Saunders and colleagues developed a technology called Synaptic Barcode Analysis by Retrograde Rabies ReadOut (SBARRO), which allows thousands of directional, monosynaptic networks to be reconstructed from scRNA-seq analyses¹. SBARRO works by tracking synaptic infectivity paths of individual rabies virus clones as they spread from the dendrites of one cell into the axons of many pre-synaptic partner cells.

As a proof-of-concept, Saunders proposes to characterize how human GRIN2B and SYNGAP1 ASD variants expressed in mice synergize with developmental stage to influence gene expression and synaptic connectivity properties in primary somatosensory cortical cell types. The proposed datasets will not only demonstrate how variant-specific cellular and connectivity phenotypes can be efficiently screened within bona fide mammalian cell types, but will also address a critical unknown of ASD pathophysiology: Do ASD mutations associated with different synaptic proteins, each widely expressed, change the molecular and synaptic properties of cortical circuits in similarly specific ways? Discovering shared cell or circuit endophenotypes across distinct classes of ASD-associated mutations while simultaneously discovering their developmental sensitivity may offer new and consolidated points of therapeutic invention.