Light and electron microscopic evidence of transneuronal labeling with WGA‐HRP to trace somatosensory pathways to the thalamus
Authors: Peschanski Marc , Ralston Henry J. 
Published: January 2005

Horseradish peroxidase conjugated to wheat‐germ lectin is being used with increasing frequency as an anterograde label to trace pathways in the nervous system, owing to the sensitivity of the method and ease of use. However, it has been suggested that horseradish peroxidase conjugated to wheat‐germ lectin may be transneuronally transported, thus affecting the ease of interpretation of the results. The present study used the projections of the dorsal column nuclei and spinal cord to the thalamus as a model system to determine whether transneuronal transport could be demonstrated and whether the degree of such transport was related to the size of the injection site.

Light microscopic observation of sections incubated with tetramethyl benzidine after large injections (1 μL of a 10% solution of horseradish‐peroxidase‐conjugated wheat‐germ lectin in water) in the dorsal column nuclei demonstrated the presence of labeled neurons in the nucleus reticularis thalami, which is not known to receive afferents from or project to these nuclei. The electron microscopic study, although based upon the use of the chromogen benzidine dihydrochloride, less sensitive than tetramethyl benzidine, revealed the existence of labeled neurons in the thalamic ventrobasal complex. This is unlikely to be due to retrograde labeling and is therefore interpreted as a result of transneuronal, perhaps transsynaptic, transport. Glial and perivascular cells also contained granules of reaction product in some cases. Smaller injections (100 nL) in the dorsal column nuclei, on the other hand, did not produce this apparent transneuronal labeling.

After small injections (100 nL) in the spinal cord, anterograde labeling was observed mainly in the thalamic ventrobasal complex in the rat, and in the posterior group in the cat, and the nuclei centralis lateralis and submedius in both species, as has been described in numerous other studies. After large injections, additional labeled areas were observed in the posterior intralaminar region (parafascicular‐center median complex), in the medial thalamus (nuclei reuniens, rhomboid and paraventricular), and in the cat, in the ventroposterolateral nucleus. In the rat, experiments were performed in which a kainic acid injection was made to induce neuronal loss in the nucleus reticularis gigantocellularis of the medulla, which is a relay of the spinoreticulothalamic pathway, known to project to some of these thalamic areas. After such lesions and large spinal injections of horseradish‐peroxidase‐conjugated wheat germ lectin, labeling was clearly decreased in some of these additional sites of projection and not in the areas which contained labeling after small injections, thus demonstrating that nucleus reticularis gigantocellularis neurons are necessary for some of the additional labeling. N‐methyl DL aspartic acid was applied to the spinal cord in other rats, destroying dorsal horn neurons but not axons of other systems. Horseradish‐peroxidase‐conjugated wheat germ lectin injected into the spinal cord of these animals resulted in retrograde labeling of brain stem neurons, but the loss of labeled fibers in the thalamus, which suggested that collaterals of brain stem neurons are not the source of transport of the tracer from cord to medial thalamus.

The results of the present study suggest the possibility of significant transneuronal labeling when horseradish‐peroxidase‐conjugated wheat germ lectin is used to trace anterograde transport in somatosensory pathways, especially when large volumes of it are injected. Thus, it is necessary to interpret the results of these tract‐tracing experiments with caution. Some mechanisms which may subserve such transneuronal labeling are proposed.