ORIGINAL RESEARCH article
Front. Neural Circuits
Tunable dual-AAV sparse labeling of PV+ retinal ganglion cells enables single-neuron projection by fMOST
Provisionally accepted
- 1Eye Center, Renmin Hospital of Wuhan University, Wuhan, China
- 2Zhongmou Therapeutics, Wuhan, Hubei, 430075 P. R. China, Wuhan, China
Select one of your emails
You have multiple emails registered with Frontiers:
Notify me on publication
Please enter your email address:
If you already have an account, please login
You don't have a Frontiers account ? You can register here
Sparse and bright labeling of defined retinal ganglion cell (RGC) subtypes is critical for linking single-cell morphology to brain-wide visual pathways. This study aims to develop a celltype-specfic sparse labeling strategy for parvalbumin-expressing retinal ganglion cells (PV+ RGCs) in the transgenic mouse retina using recombinant adeno-associated virus (rAAV), and to map the whole-brain projection patterns of single PV-RGC by using fluorescence micro-optical sectioning tomography (fMOST). We employ a celltype specific dual AAV system by co-packaging of a Cre-dependent Flpo plasmid and Flpo-dependent enhanced yellow fluorescent protein (EYFP) plasmid, optimizing different parameter such as mixing ratio, AAV serotype, and gene copy number of Flpo & EYFP. By systematically comparing the transduction efficiency and sparsity under different experimental parameters of AAV packaging ratios for the core plasmids (1/100–1/1000), gene copy number (single vs. double), and different serotypes (AAV2.2 vs AAV2.NN), we found that the sparseness and signal intensity of labeled RGCs varied with changes in the ratio of the core plasmid, AAV serotype and gene copy number of Flpo & EYFP. We conducted the whole-retina-brain imaging of AAV2.2-double-1/1000 by employing fluorescence micro-optical sectioning tomography (fMOST) system, reconstructed full axonal trajectories of individual PV+ RGCs from retina to their targets, including the superior colliculus (SC), dorsal and ventral lateral geniculate nuclei (dLGN/vLGN), and visual cortex. Engineered AAV2.NN increased transduction and labeling density under equivalent conditions, facilitating morphological subclassification of PV+ RGCs (ON, ON-OFF, OFF) by stratification relative to ChAT bands. This platform overcomes the classical trade-off between sparsity and signal intensity, provides a robust route to single-RGC projection, and offers a practical foundation for mechanistic and therapeutic studies of RGC subtype-specific vulnerability.
Keywords: aav, fMOST, PV+ RGC, Single cell reconstruction, Sparse labeling
Received: 06 Nov 2025; Accepted: 16 Dec 2025.
Copyright: © 2025 Zhou, Tan, Li, Yuan, Jin, Zhang, Wang and Shen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Yin Shen
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.