Retrograde Single Labeling System

What is Retrograde Tracing?

The normal anatomical connectivity between brain regions is the structural basis for functional interactions in the brain. Therefore, it has been of considerable interest to characterize fiber tracts linking different brain regions in detail and identify their neurotransmitters. Anterograde and retrograde tracing with various tracers are the major neuroanatomical tools used to identify pathways connecting brain areas. For retrograde transport, the tracer materials are applied to a fiber tract or a terminal field of innervation, becomes incorporated into the cell axons (usually by the process of endocytosis), and are then carried back to the parent cell body. Retrograde tracing is a powerful technique for identifying cells that give rise to neuronal pathways.

Fig.1 Anterograde and retrograde labeling with static and trans-synaptic tracers. (Saleeba, 2019)

Retrograde Single Labeling System

The retrograde tracing techniques enable one to map connections between cell populations and regions. Retrograde single labeling systems are typically used to ascertain the cells of origin that innervate brain structure and the projection target of the axons of a given population of neurons. The development of sensitive retrograde tracers has provided many new approaches for analyzing neuronal circuits. These retrograde tracers can be made of chemical compounds, glycoproteins, radioactively tagged amino acids, or fluorescently conjugated beads that can be transported along axon fibers in either direction.

Commonly single labeling used retrograde tracers include horseradish peroxidase (HRP), cholera toxin B fragment (CTB), Fluoro-gold (FG), and retro-beads. Subsequently, many new substances have been found that are useful for retrograde single labeling tracing, such as fluorescent dyes of various colors, which can be combined in the same experiment for multiple retrograde labeling.

• Single Labeling HRP System

The enzyme HRP, an effective retrograde tracer, was found in the early 1970s and taken up into neurons non-selectively by passive endocytosis. It can be used as a single labeling system and taken up by axons and transferred back to cell somata by active transport. This single enzyme reaction may then stain the cell body and its major dendrites. In addition, conjugation of HRP to the plant lectin wheat germ agglutinin (WGA) significantly improved both uptake and transport within neurons. WGA has been used as a trans-synaptic tracer that can be genetically targeted and can cross synapses in retrograde (i.e., from postsynaptic to presynaptic side) directions, providing extensive labeling of the neurons.

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Reference

1. Saleeba, C.; et al. A student’s guide to neural circuit tracing. Frontiers in Neuroscience. 2019, 897.

For Research Use Only. Not For Clinical Use.