A microRNA-based viral strategy for targeting and classifying neuron subtypes in the rodent brain
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In order to advance understanding of the brain, more specific and widely applicable methods are needed for genetically defining and labelling neuron subtypes in vivo. This dissertation describes the development and application of a novel strategy for designing viral gene delivery vectors that target and classify specific neurons in the adult rodent brain based primarily on endogenous microRNA (miRNA) expression profiles. We call these vectors microRNA-guided neuron tags (mAGNETs). We first demonstrate the feasibility of using neuron-type-specific miRNA profiles to guide viral-mediated transgene expression. Using lentiviral (LV) vectors bearing “signature” miRNA recognition sites, we explore several microRNA cassette design principles from a synthetic biology perspective and demonstrate the feasibility of preferentially targeting inhibitory (GABA+) neurons in the mouse cortex. Next, we maximize interneuron targeting specificity by altering the strength of the constitutive promoter driving transgene expression and switching the viral packaging platform from LV to adeno-associated virus (AAV), producing a GABA mAGNET that can label cortical interneurons with 98% targeting specificity in the mouse brain. The utility of this GABA mAGNET is highlighted by demonstrations of inhibitory neuron targeting in a Ube3a 2X Tg mouse model of autism, cross-species functionality in the rat cortex and hippocampus, and viral-mediated dual-color optogenetic manipulation of two separate neural subsets in the mouse cortex. Finally, we design and implement a mAGNET vector to characterize a novel neuron subtype: calcium/calmodulin dependent protein kinase II alpha (CamKIIα) neurons with low expression of miR-128. Calcium imaging in behaving mice, slice physiology and immunofluorescence characterization reveal that low-miR-128-CamKIIα+ (Lm128C) cells exhibit several unique biophysical properties, and resemble fast-spiking interneurons. This work highlights the potential of mAGNETs to help define neuron subtypes, and characterize neural activity and physiology. Overall, this dissertation presents a novel platform for viral-mediated, neuron-type specific labelling based upon endogenous miRNA expression, provides a useful tool for targeting cortical interneurons in the rodent brain with high specificity, and demonstrates the classification of a previously uncharacterized neuron subtype.