Neuroscience

The process of moving proteins and organelles along the axon is essential for neuronal survival and function, ensuring proper communication between the cell body and distant synapses. The efficient and precise delivery of proteins via axon transport is critical for processes ranging from synaptic plasticity and neurotransmission to neuronal growth and maintenance. However, the identities of all the transported proteins have only recently begun to be investigated. Retinal ganglion cells (RGCs) provide a unique opportunity for access to central nervous system (CNS) axons as the retina is located outside the brain in the eye, with long axonal projections (~1 cm in mouse) that innervate the brain. We have developed and optimized methods for unbiased in vivo protein labeling in rodent RGC somata with intravitreal N-hydroxysuccinimido (NHS)-biotin and subsequent visualization of transported proteins along the optic nerve using confocal microscopy. Here, we describe these procedures in detail.

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.

The retina is sensitive for light damages, because of direct light exposure, especially intense blue and UV light, which increase level of ROS and other toxic phototransduction products in photoreceptor cells. In our previous work (Damulewicz et al., 2017a and 2017b), we used 8-oxo-deoxyguanosine (8-OHdG) as a marker for oxidative stress to investigate the role of heme oxygenase in DNA protection against UV light. In this protocol, we showed how to determine the level of DNA damages in the retina using immunohistochemical staining.