The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.

18Neocortex contains a multitude of cell types segregated into layers and functionally distinct regions. To 19investigate the diversity of cell types across the mouse neocortex, we analyzed 12,714 cells from the 20 primary visual cortex (VISp), and 9,035 cells from the anterior lateral motor cortex (ALM) by deep single-21cell RNA-sequencing (scRNA-seq), identifying 116 transcriptomic cell types. These two regions represent 22 distant poles of the neocortex and perform distinct functions. We define 50 inhibitory transcriptomic cell 23 types, all of which are shared across both cortical regions. In contrast, 49 of 52 excitatory transcriptomic 24 types were found in either VISp or ALM, with only three present in both. By combining single cell RNA-25 seq and retrograde labeling, we demonstrate correspondence between excitatory transcriptomic types and 26 their region-specific long-range target specificity. This study establishes a combined transcriptomic and 27projectional taxonomy of cortical cell types from functionally distinct regions of the mouse cortex. 28 29 1

Summary Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV) — a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers, and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations.