Anterograde Multi-level Labeling System

Neurons connect with others through synapses, wiring to form complex but precise networks. Among certain neuron circuits that are the basis of brain functions, information can be processed and transmitted. Understanding the full brain connectivity by mapping neuronal circuits is vital for neuroscience research. Mapping these neuronal circuits requires proper tracers to label the neurons in the initial area and then transmit them to the upper or downstream connected areas.

Anterograde Tracing

Tracing is managed through a phenomenon called axonal transport or axoplasmic flow. Conventional tracing methods rely on cellular markers transported in axons in the anterograde or retrograde direction from the neuronal cell body. Minute quantities of these markers are injected into a specific peripheral or CNS site. Then, the animals are perfused with fixatives, and the distribution of the marker is studied in tissue sections after a sufficient time has elapsed for uptake and transport to occur. Considerable amounts of connectional data have been collected in this manner.

A series of tracers has been developed as anterograde tracing, which means that the labeling begins at the cell bodies and dendrites. The tracers are transported out to the axons and their terminals according to the preferential direction of their transport in the axon. Many classes of tracer tools are available, including fluorescent dyes (e.g., RITC), plant lectins [Phaseolus vulgaris leucoagglutinin (PHA-L)], dextrans [biotinylated dextran amine (BDA)].

Multi-level Labeling System

There is little doubt that modern tract-tracing protocols will continue to be very useful in basic neuroanatomical research. The further study of the complexity of brain circuits requires effective multi-labeling procedures to visualize several projections simultaneously because single-staining approaches may often not provide enough detail. Multiple-labeling strategies using different fluorescent dyes in vivo or in fixed tissues can be used to study divergent or convergent projections. A suite of tracers has been generated that could enable simultaneous multicolor labeling and tracing. Moreover, this procedure has advantages over the fluorescent markers nowadays commonly used in multiple neuroanatomical tract-tracing methods, such as the stability of the staining and compatibility with EM.

Fig.1 Multidimensional experiment combining in a dual-anterograde tracing with PHA-L (blue) and BDA (red). (Lanciego, 2020)

Multi-level Labeling for Tracing in Spinal Cord Injury (SCI) Research

Deficits in function after SCI are attributable to the interruption of motor axonal tracts in the spinal cord. The labeling within the spinal cord to the brain or periphery is an important concern for disease research. In anterograde tracing technology, multiple tracers are transported at different speeds from the soma to the axon terminal to identify axonal connections in the normal or injured spinal cord and determine axonal projections from grafted neurons into the lesion site.

Multi-level labeling strategies can also be combined with various other techniques and can therefore be widely applied in the SCI research field. In particular, in vivo labeling with fluorescent tracers allows the definition of axonal sorting according to their anatomical connections and makes them readily accessible to a wide range of additional approaches. These approaches include multiple labeling systems using different fluorescent dyes in vivo or fixed tissues to label different fiber tracts. When combined with another labeling system, anterograde tracers may be used to establish specific neural networks.

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Reference

1. Lanciego, J. L.; Wouterlood, F. G. Neuroanatomical tract-tracing techniques that did go viral. Brain Structure and Function. 2020, 225(4), 1193-1224.

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