A genetic approach to characterize the Robo3+ amacrine cell

Abstract

Purpose : The murine retina contains over one hundred and twenty neuronal subtypes with distinct molecular profiles. Amacrine Cells (ACs), the inhibitory interneurons of the inner retina, are disproportionately diverse, comprising around half of the neuronal subtypes but only one percent of the total neuronal population in the retina. Many AC subtypes are understudied due to a lack of genetic tools to prospectively identify them, leaving a large gap in our understanding of retinal function. Here we describe a new genetic approach using a Robo3^CreERT2 knock-in mouse line to genetically label a previously undescribed AC subtype.

Methods : We used RNAscope to define the temporal expression pattern of Robo3 in a population of mouse ACs. We crossed Robo3^CreERT2 mice with Cre-dependent reporter lines to genetically label Robo3-expressing ACs (RACs), which allowed us to analyze their single-cell morphology and population distribution across the retina.

Results : Robo3 is expressed transiently in the first three postnatal weeks in a small population of ACs. RACs have somas exclusively in the Inner Nuclear Layer and are GABAergic. RAC dendrites stratify in sublamina S3 and S4 of the Inner Plexiform Layer, with an average arbor area of 5666μm², without significant variation across the retina. Population labeling of RACs with high doses of Tamoxifen showed an even distribution of RACs across the entire retina.

Conclusions : The Robo3^CreERT2 line allows for genetic access to a subtype of uncharacterized ACs in the murine retina. Titration of Tamoxifen enables analysis of individual RACs or the overall population. Ongoing electrophysiological studies will define RAC circuit partners, and uncover their functional role in the retina.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.