Brain-wide projection-specific gene expression using an intersectional viral strategy
Xiaonan Sun, Anup Khanal, Simon Musall, Anne Churchland
Introduction: Genetically accessing projection pathways using viral approaches facilitates circuit-specific understanding of neural connectivity and behavior, carrying clinical and therapeutic implications for neuropsychiatric and movement disorders. While significant advances have been made in viral vector engineering, targeting of broadly-distributed projection pathways remains an ongoing challenge.
Objectives: By combining retrograde and systemic viral vectors, we implemented an intersectional viral strategy to express transgenes efficiently in corticostriatal and corticothalamic circuits, whose cell bodies are distributed across all cortical regions.
Methods: We tested two promising retrograde vectors - retrograde AAV (AAV-2-retro) and canine adenovirus-2 (CAV-2) - expressing Cre recombinase to activate and amplify Cre-dependent transgenes transduced by the intravenously-administered AAV-9 capsid variant AAV-PHP.eB. This strategy was applied by stereotactically targeting the striatum or the thalamus to express the fluorescent calcium indicator protein GCaMP7s. Gene expression was characterized histologically using immunofluorescence and in skull-cleared awake behaving mice using wide-field epifluorescence microscopy.
Results: Both AAV-2-retro and CAV-2 drive cortex-wide GCaMP7s expression in corticostriatal neurons, where expression levels and labeled neuron density are higher with CAV-2, which also demonstrates reduced anterograde uptake in the striatum. With thalamic targeting, AAV-2-retro drives robust expression in both cortex and thalamus. Lastly, we tracked neural activity through GCaMP7s using wide-field calcium imaging to generate visually-evoked cortical maps.
Conclusions: This work demonstrates an efficient two-component strategy for cortex-wide functional transgene expression specified by target selection where CAV-2 internalization is more selective at axon terminals, as opposed to cell bodies. Our current research leverages this technique towards understanding cortex-wide projection-specific neural representations of perceptual decision-making in rodents.