Did you choose appropriate tracer for retrograde tracing of retinal ganglion cells? The differences between cholera toxin subunit B and Fluorogold

Abstract

Cholera toxin subunit B (CTB) and Fluorogold(FG) are two widely utilized retrograde tracers to assess the number and function of retinal ganglion cells (RGCs). However, the relative advantages and disadvantages of these tracers remain unclear, which may lead to their inappropriate application. In this study, we compared these tracers by separately injecting the tracer into the superior Colliculi (SC) in rats, one or 2 weeks later, the rats were sacrificed, and their retinas, brains, and optic nerves were collected. From the first to second week, FG displayed a greater number of labeled RGCs and a larger diffusion area in the SC than CTB; The number of CTB labeled RGCs and the diffusion area of CTB in the SC increased significantly, but there was no distinction between FG; Furthermore, CTB exhibited more labeled RGC neurites and longer neurites than FG, but no difference was evident between the same trace; The optic nerves labeled using CTB were much clearer than those labeled using FG. In conclusion, both CTB and FG can be used for the retrograde labeling of RGCs in rats at 1 or 2 weeks. FG achieves retrograde labeling of a greater number of RGCs than CTB, whereas CTB better delineates the morphology of RGCs. Furthermore, CTB seems more suitable for retrograde labeling of some small, non-image forming nuclei in the brain to which certain RGC subtypes project their axons.

Nonviral Fluorescent Retrograde Tracers

“…Retrograde tracing techniques allow us to: (1) assess neuronal connections between a target population of neurons and their inputs throughout the nervous system, (2) identify the connection from their source through to their point of termination . In general, retrograde tracing can be achieved using viral neuronal tracer (VNT), such as rabies virus (RV) and pseudo RV that are transported to the cell body via axonal dynein . However, VNT can spread quickly and cause degradation of neural tissue.…”

“…There are two main structurally important working mechanisms: (1) small organic molecular tracers: it forms a DNA complex in the nucleus with fluorescence enhancement after endocytosis, (2) dextran‐based tracers: a specific molecular weight (10 kDa) of dextran tends to stay in the intracellular cytosol of neural cells. Retrogradely injected tracers use axonal transport to move from the distal region (axonal branches responsible for signal transduction) of neural cell to the cell body …”

“…4 In general, retrograde tracing can be achieved using viral neuronal tracer (VNT), such as rabies virus (RV) and pseudo RV that are transported to the cell body via axonal dynein. 5 However, VNT can spread quickly and cause degradation of neural tissue. It can also infect the nervous system, which can lead to undesirable outcomes.…”

“…Retrogradely injected tracers use axonal transport to move from the distal region (axonal branches responsible for signal transduction) of neural cell to the cell body. 5,15,16 Unlike the small molecular tracers, the Fluoro-series (FG, FE, and FR) expands the emission wavelength to a longer region that can avoid the autofluorescence effect coming from intrinsic fluorophores. 17 In relation to the biological optical window, the development of fluorescent antero-/retrograde tracers have recently been trending.…”

Piggybacking on the Cholera Toxin: Identification of a Toxoid-binding Protein as an Approach for Targeted Delivery of Proteins to Motor Neurons

“…Indeed, the ability of B5 toxoids to undergo retrograde transport in neurons has led to extensive use of CTBconjugates as neuronal tracers. [16][17][18] Tinker et al previously demonstrated that by mimicking the full-length holotoxin, fluorescent CT chimeras could be produced by replacing the toxic A1 domain with a fluorescent protein. 19 Chen et al expanded on this work by expressing fusions with heat-labile enterotoxin IIA, and showing that these could be successfully delivered to the cytosol of neurons in mammalian cell culture.…”