Neural tract tracing using Di-I: a review and a new method to make fast Di-I faster in human brain

The use of Di-I in tract-tracing is briefly reviewed and a novel delayed-fi xation approach to neural tract-tracing in the postmortem human adult brain is reported. Using the new approach, fast Di-I, a highly lipophilic fluore scent dye was injected into a particular region or nucleus and labelled tra cts were followed for distances of some 20-40 mm. The procedure required ap proximately 36 h, yielding dye penetration rates of 1.0 mm/h or more. This contrasts with previous Di-I, silver impregnation, and horseradish peroxida se protocols, where the tracer penetration rate is typically 0.003 mm/h or less, and the distance traversed amounts to only a few mm even after months of incubation. The new method hinges on the simple consideration that alde hyde fixation, which is normally employed prior to administration of the ma rker, crosslinks membrane proteins and impedes dye diffusion. The short pos tmortem samples used in our protocol permit delaying fixation until after t he dye has had time to penetrate, dramatically increasing the length and sc ope of neural circuits that can be traced. Using these methods, for example , we have confirmed the presence of an ipsilateral olivocerebellar climbing fiber projection in the human. (C) 2000 Elsevier Science B.V. All rights r eserved.