Abstract
Purpose :
Retinal ganglion cell (RGC) subtype susceptibility has been demonstrated in several optic neuropathies, including glaucoma. The Thy1-Yellow Fluorescent Protein-H (Thy1-YFP-H) transgenic mouse (Jackson Laboratories, ME) expresses YFP in only 0.2% of RGCs. This reduced expression enables single RGC imaging with visualisation of the entire dendritic arbour and provides valuable clues on the impact of experimental glaucoma on RGCs. However, to ensure there is non-selectivity in YFP expression among subtypes, a characterisation study must be carried out. To date, there is no study supporting the notion of non-selective labelling of RGC structural subtypes in this strain.
Methods :
Retinas from Thy1-YFP-H mice were processed for immunohistochemistry (ChAT) and fluorescence imaging (Z-stacks, LeicaTCSSP8 confocal microscope). Dendritic arbours were traced with the filaments Tree Autopath Algorithm (plugin, Imaris), and Sholl analysis (plugin, Imaris) performed on each 3D RGC filament. Sholl profiles were plotted and the area under the curve (AUC), normalised AUC, peak number of intersections, distance to peak number of intersections, soma size and dendritic field, were quantified for each RGC. Projection images were created (LAS X) and classified as ON, OFF, or ON-OFF depending on the extension of synapses within the IPL sublaminas.
Results :
The Thy1-YFP-H mouse expresses YFP in ON, OFF and ON-OFF RGCs (Fig.1A). The AUC calculated from Sholl profiles was 37.1% and 25.6% higher in ON RGCs compared to OFF and ON-OFF cells, respectively (Fig.1B). Whereas there was only a 9.2% AUC difference between OFF and ON-OFF cells. The Sholl profiles showed that all subtypes had similar PNI (Fig.1C.D). PD profiles were similar between OFF and ON-OFF cells (Fig1.C.D), whilst ON RGCs PD was over 50% higher. ON RGCs displayed an increased soma and DF size, compared to OFF which had a similar SS but 48% smaller DF. ON-OFF RGCs had the smallest SS but with a 13.6% higher DF compared to OFF cells (Fig.1D). Of note, we also observed displaced RGCs regardless of whether they were ON or OFF (Fig.2).
Conclusions :
Our findings indicate that ON, OFF and bistratified ON-OFF RGCs are labelled non-selectively in the Thy1-YFP-H strain. The findings are consistent with previous studies both in the variety of morphological variables, as well as how these variables characterise subtypes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.