Cell Type Specific Enhancers for Dorsolateral Prefrontal Cortex.

Abstract

The dorsolateral prefrontal cortex (DLPFC) is crucial to primate cognitive functions, but a paucity of cell type specific tools limits studies of DLPFC neurocomputational principles. Therefore, we set out to identify enhancers that fit inside Adeno-Associated Virus (AAV) vectors and that elicited functional, cell type specific gene expression in the non-human primate (NHP) DLPFC. We used single nucleus RNA-Seq and ATAC-Seq from rhesus macaque tissue samples to define DLPFC cell types and their associated open chromatin regions (OCRs). We trained machine learning (ML) models to recognize the unique regulatory grammar associated with each DLPFC neuron type, performed in silico screening of all OCRs, and identified candidate enhancers most likely to elicit cell type specific transgene expression in each neuron type. For layer 3 pyramidal neurons (L3PNs) and layer 5 extratelencephalic neurons (L5ETs), we cloned the top twelve identified candidates into AAVs and injected them into NHP DLPFC. In situ observation of enhancer-driven expression revealed the best performers, RMacL3-01 and RMacL5ET-01. We validated RMacL3-01 and RMacL5ET-01 using one-at-a-time injections in NHP DLPFC. RMacL3-01 restricted GFP expression to pyramidal neurons in layers 2 and 3, whereas RMacL5ET-01 restricted expression to POU3F1+ neurons in layer 5. RMacL3-01 elicited functional levels of channelrhodopsin expression that enabled optical activation of single- and multi-unit activity in NHP DLFPC. Together, these results and resources establish a solid foundation to study cell type specific principles of primate cognitive functions.