Retrograde Multi-Level Labeling System

By providing customized solutions and high-quality services, Creative Biolabs is your reliable partner. Our professional team develops custom retrograde multi-level labeling systems for your neuroscience research.

Retrograde tracing is a powerful tool to understand the neuronal circuit and structure/function of brain communication networks in a broad range of animals including rodents and primates. The presence and extent of collaterals in a set of pathways can be examined with multiple methods, including antidromic stimulation, single axon reconstruction, or multi-label retrograde tracing. The multi-label retrograde tracing, in which different tracers are injected into targets of interest and then individual cells are examined for the presence of multiple tracers, is a common approach made popular by the growing number of fluorescent tracers.

Different tracers have different transport characteristics and labeling efficacy; hence, each tracer has specific applicability. Thus, to achieve multi-labeling, it is necessary to screen the available tracers for cut nerve exposure from various common retrograde tracers. Here are some different neuronal retrograde tracers.

Wheat Germ Agglutinin (WGA)-Conjugated HRP

Scientists have compared the in vivo retrograde transport of horseradish peroxidase (HRP) covalently linked with glutaraldehyde to WGA, with the transport of free HRP. They have observed that WGA-HRP is transported through retrograde axoplasmic transport from the rat tongue to the hypoglossal nucleus, or from the submandibular gland to the superior cervical ganglion; and that WGA-HRP is a much more sensitive tracer than free HRP.

WGA-Apo HRP Gold

WGA-apo HRP gold is a retrograde tracer of colloid gold labeling, which is made by coupling colloid gold to WGA and then to enzyme inactive horseradish peroxidase (apo HRP). This tracer has the advantages of long retention time in cells, limited diffusion, high sensitivity, good histocompatibility with a variety of aldehydes fixators, and easy detection. In addition, since these tracers contain inactive forms of HRP molecules, they can be used simultaneously with fluorescence and HRP based immunocytochemistry and tracing techniques. WGA can also be conjugated with a fluorophore such as Alexa Fluor 555 or 647.

Bacterial Toxin Fragments

Bacterial toxin fragments (cholera toxin subunit B, CTB) and tetanus toxin have also been explored as a combination of HRP or fluorescent labels such as Alexa Fluor 555 or 488. The CTB-HRP has several advantages as a neuronal tracer. It is highly sensitive due to receptor-mediated absorption. It binds to the monosialoanglioside (GM1) ganglioside of the nerve cell membrane, labels a large number of neurons, and allows immunohistochemical detection with less diffusion from labeled neurons than natural HRP.

Fluorescent Retrograde Tracers

Fluoro-Gold (FG) and Fluoro-Ruby (FR) appear very robust as well as extremely photobleaching resistant. Its intense and bleach-resistant labeling has contributed to raising FG to the level of 'gold standard' for fluorescent retrograde labeling in rodents, particularly for multiple labeling in combination with other tracers. FR is a highly sensitive tracer; it is the most appropriate tracer for multiple labeling in combination with other tracers as its fluorescent excitation.

Because of their spectral characteristics, fluorescent retrograde markers serve as near-ideal tracers to study axon collateralization in double-labeling experiments and, in a multi-dimensional setting in combination with immunofluorescence, to study the neurochemical identity of traced fiber projections.

Fig.1 Monosynaptically restricted retrograde tracing using glycoprotein-deleted rabies. (Saleeba, 2019)

Dual Viral Tracing

Unlike most traditional chemical retrograde tracers, viral retrograde tracers move reliably from one neuron to another primarily through synapses. What's more, they replicate in the infected neurons, leading to a strong amplification of signals. Many neurotropic viruses have been used for this trans-synaptic tracing, namely, herpes simplex virus type 1 (HSV-1), pseudorabies (PRV), and rabies virus (RV).

To further elucidate the complex neural networks in the CNS, it is useful to combine double retrograde labeling with viral cross-synaptic tracing. Two recombinant virus strains are used as trans-synaptic tracers, each expressing a unique marker protein. Using this method, two distinct neuronal circuits can be labeled, and the origin of the two bifurcated circuits can be visually shown by the co-expression of the two labeled proteins (double labeling).

To achieve efficient double labeling, the timing of the two injections should be adjusted according to the specific's conditions of the two virus strains so that the two virus strains reach the neurons of interest in a short time.

Fig.2 Dual viral tracing using genetically modified virus. (Ohara, 2009)

With the careful experimental design, the multi-labeling method is likely to provide the best approach for characterizing the projection patterns of many cells, and of revealing projection patterns that characterize a minority of cells in a population. Creative Biolabs is specialized in the custom development of multi-level retrograde labeling systems for neuroscience research. Please contact us for more information. We look forward to finding a solution that best suits your needs.

References

1. Saleeba, C.; et al. A student’s guide to neural circuit tracing. Frontiers in Neuroscience. 2019: 897.
2. Ohara, S.; et al. Untangling neural networks with dual retrograde transsynaptic viral infection. Frontiers in Neuroscience. 2009, 3: 32.

For Research Use Only. Not For Clinical Use.