Abstract
Purpose :
Excitatory retinal amacrine cells use VGLUT3 to concentrate cytosolic glutamate into synaptic vesicles. These amacrine cells can be genetically marked in mice where vGluT3 promoter drives expression of a fluorescent reporter or Cre recombinase. It is known that some GCL neurons are also marked. The purpose of the study is to investigate the identity of these GCL neurons.
Methods :
VgluT3-Cre (JAX-028534) and Ai9 mice (Jax-007909) were mated and genotyped according to online protocols available in JAX website. F1 mice double positive for VgluT3-Cre and Ai9 were used. Flat-mounted retinas were perfused in carbogenated mammalian Ringer’s solution and tdTomato positive GCL cells were patch clamped to record intrinsic membrane properties (IMPs) and internally dialyzed with and stained for biocytin. Dendritic structure was traced in Neurolucida 360 to obtain morphometric data. Dendritic stratification level in the inner plexiform layer (IPL) was determined in reference to cholinergic plexus in a customized algorithm programmed in Python. A hierarchical cluster analysis was used to classify the cells, which were validated using immunohistochemistry against RGC markers such as RBPMS, melanopsin, CART, Osteospondin, TBR2, FOXP2, POU6F2, BRN3B, and BRN3A.
Results :
Approximately 5,000 GCL cells per retina in the VgluT3-Cre/Ai9 mice have recombinase activity and all are positive for the pan-RGC marker RBPMS. The dendrites of labels cells stratify to various IPL levels and have diverse dendritic morphology. All cells are positive for at least one of the aforementioned RGC markers. The labeled cells possess stereotypic IMPs that are linked to each cell's unique dendritic morphology and IPL stratification depth, allowing them to be reliably identified during recording. While diverse, we routinely encountered three RGC types: the ON-OFF direction selective ganglion cells, the F-minioff (mini-J), and an ON-RGC whose dendrites co-stratify with the ON-cholinergic plexus.
Conclusions :
This study finds that many RGC types have Cre activity in the VgluT3-Cre mouse and that genetic marking together with type-specific IMPs enable reliable RGC identification during recording. Expanding this approach to additional Cre mouse lines has the potential to reveal full RGC diversity in mouse to guide future investigations.
This is a 2020 ARVO Annual Meeting abstract.