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Ching-Kang Chen, Yin-Peng Chen, Hung-Ya Tu, Viet Chau, Adam McQuiston, Chuan-Chin Chiao; Cre-expressing retinal ganglion cells in a Pcp2-Cre transgenic mouse. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3389.
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© ARVO (1962-2015); The Authors (2016-present)
The availability of mouse lines expressing fluorescent markers or recombinases provides opportunities to distinguish different retinal ganglion cell (RGC) types in mice. Cre expression were noted by Ivanova et al. (Neuroscience, 2010) in certain gangalion cell layer (GCL) cells of a Pcp2-Cre transgenic mouse line but the true identities of these cells remained unclear. This study systematically characterized these GCL cells in this Pcp2-Cre mouse line.
Male Pcp2-Cre (Jax-006207) and female Ai9 mice were genotyped according to online protocols found in Jax website. F1 mice double positive for Pcp2-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 properties, while being dialyzed internally with a biocytin-containing and K-gluconate based solution. Morphology of recorded cells was visualized by streptavidin-Alexa-488 staining and confocal microscopy. Dendritic structures were traced in Neurolucida and analyzed for soma and dendritic field sizes, dendritic density, asymmetry index and stratification levels in the inner plexiform layer. Morphometric parameters were analyzed by a hierarchical cluster analysis to categorize these cells. Immunohistochemistry were performed to examine expression of parvalbumin, melanopsin, SMI-32 and CART in labelled GCL cells.
Approximately 2~3 % of GCL cells in Pcp2-Cre mice express Cre recombinase. We found that all labelled cells spike robustly when depolarized beyond action potential threshold, suggesting that they all are RGCs and with distinctive intrinsic properties, different soma sizes and dendritic stratifications. Immunohistochemical studies further reveal that none of these labeled cells are positive for melanopsin, but some are positive for SMI-32 and CART, while most are positive for parvalbumin. Akin to Cre expressing cells in Grik4-Cre mice, cells with similar morphological features tend to have different intrinsic membrane properties, allowing them to be further divided into different subgroups.
Only a unique subset of RGCs expresses Cre recombinase in this Pcp2-Cre mouse line. Genetic marking combined with intrinsic membrane properties enable reliable RGC identification prior to experimentation. Expanding this approach to other Cre lines thus have the potential to reveal the real diversity of RGCs in mouse retina.
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