Abstract
Axons of retinal ganglion cells (RGCs) relay visual information from the retina to lateral geniculate nucleus (LGN) and superior colliculus (SC), which are two major image-forming visual nuclei. Wiring of these retinal projections completes before vision begins. However, there are few studies on retinal axons at embryonic stage due to technical difficulty. We developed a method of embryonic intravitreous injection of dyes in mice to visualize retinal projections to LGN and SC. This study opens up the possibility of understanding early visual circuit wiring in mice embryos.
Keywords: Embryo, Intravitreous injection, Retinal projection, Retinal axons, Uterus
Background
Retinal axons begin to project to LGN and SC as early as embryonic day 14.5 (E14.5) in mice. To investigate axon projections in embryos, dyes need to be injected intravitreally while keeping the embryos alive for at least 10 h prior to injection surgery. Previous studies showed embryos can be cultured in vitro using mouse serum with oxygen. However, issues such as nutrition diversity, oxygen saturation and temperature regulation impinge the physiological condition of the embryo. In our research, we conducted intravitreous injection through uterine wall and eyelids in embryos and kept them in uterine after injection. Retinal axon projections to LGN and SC were nicely labeled from E15.5 to E18.5.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Analysis of retinal projection was conducted as previously described (from our lab) (Diao et al., 2018). Briefly, the background of all images was subtracted in ImageJ (NIH, USA). Quantifications of fluorescently labeled areas, such as the fraction of contralateral projections and sizes of LGN and SC, were performed using Matlab (Mathworks Inc, USA). For 20 μm brain slices, a threshold of 30% (fluorescence intensity of pixels above 30% of the maximum intensity) is applied in the calculation of fraction of contralateral projections. CTB-555 and DAPI fluorescent signals helped outlining the territory of LGN and SC. Note: The birth-dating of embryos could be differed by ± 0.5 days. It is necessary to use sufficient number of pregnant females to obtain sufficient embryos for analysis.
Recipes
Acknowledgments
The authors declare no competing interests. This protocol was adapted from Diao et al. (2018). We thank the following funding agencies for supporting this work: the NSF of China (31421091 and 31422025), the Young 1000 Plan and Ministry of Science and Technology of the People’s Republic of China (2015AA020512).
References
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