Authors
Caplan, K. A., Chen, K., Wang, Y., Wienisch, M., Chai, R., Kang, I.-H., Chen, C. S., del Rosario, R., Krienen, F., Wang, F., Feng, G.
Abstract
To enable genetic access without genetically engineered animals, we developed and benchmarked enhancer-AAVs for selectively targeting dopamine (DA) neurons across mice and marmosets. 12 DA enhancer candidates were nominated through integrated single-nucleus RNA-seq/ATAC-seq analysis of marmoset ventral midbrain tissue. We identified two leads (cjDAE8, cjDAE4) by systemic AAV delivery and dual-color fluorescence screening in mice. Local co-injection of both AAVs in marmoset confirmed DA-restricted expression, with cjDAE8 exhibiting higher specificity. However, antibody amplification of reporter fluorescence exposed weakly labeled off-target cells (leaky expression). We therefore engineered next-generation AAV backbones for cjDAE8 to strengthen expression while limiting leakiness. Quantitative histology comparing natural versus antibody-amplified fluorescence defined AAV doses for achieving both high efficiency and greater than 90-95% specificity of DA neuron labeling across injection routes. We demonstrate applications for (i) retrograde targeting of projection-defined DA populations in marmoset, (ii) fiber-photometric recording of divergent DA-axonal dynamics in mouse striatal subregions, and (iii) driving optogenetic VTA DA self-stimulation. Our results provide a resource for interspecies DA targeting and two practical guidelines: backbone context critically shapes enhancer performance, and antibody-amplified readouts rigorously assesses specificity.
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bioRxiv
The authors list and abstract were imported from bioRxiv on 09 Nov 2025.
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