Title: Local Transcriptional Variation across Subtypes of Cortical Synapses

RNA localization and local translation in neuronal synapses are necessary for biological processes such as synapse formation and synaptic plasticity (Holt and Schuman, 2013). Alterations to RNA localization have been implicated in multiple neurological conditions (Fernandopulle et al., 2021; Lu et al., 2021). For example, many genes shown to cause Amyotrophic Lateral Sclerosis (ALS) encode RNA-binding proteins, with suggested roles in neuronal RNA-trafficking (Coyne et al., 2014, Sahadevan et al., 2021). Although these proteins are ubiquitously expressed throughout the body, ALS leads to the selective loss of corticospinal and spinal motor neurons for unknown reasons. We hypothesize that RNAs are differentially targeted to distinct synapse types in neurons, and that these differences in local RNA content contribute to the selective vulnerability of specific cell types and synapse types in disease. However, whether different synapse types contain different complements of local RNA, in health or disease, is poorly understood. In this study, we therefore tested whether two major, functionally distinct excitatory synapse types in the mouse cortex have different complements of local RNA: 1) depressing synapses formed by Layer 5 Pyramidal Neurons (L5PNs) and 2) facilitating synapses formed by Layer 6 Corticothalamic Neurons (L6CThNs). To perform this test, we developed a synaptosome sorting and sequencing workflow that can be applied broadly to study the molecular contents of genetically accessible synapse types. We find that 335 unique RNA isoforms are differentially expressed between L5PN and L6CThN synapses (Wald test, corrected p < 0.05), including RNAs that regulate synaptic release properties, synapse formation and plasticity, and transcription. These results provide evidence for synapse-type-specific RNA localization in neurons, and serve as a basis for future studies of synapse-type-specific disruption of RNA localization in disease.