In vivo visualization of callosal pathways: a novel approach to the study of cortical organization

I describe here the successful visualization of interhemispheric callosal connections in the live mammalian cortex. The development of this method was prompted by the finding that fluorescent tracer labeling of groups of cortical neurons, when done under optimal conditions, is sufficiently intense to be visible even in the whole brain preparation. The new approach could provide a useful tool for enhanced precision in localizing cortical modules in vivo. In a typical experiment, rats had their left cortical hemisphere extensively injected with the fluorescent tract-tracer bisBenzimide (BB). After appropriate survival, the right cortical hemisphere was illuminated with UV light and the fluorescing callosal pattern could be discerned under the network of blood vessels even with the unaided eye. The pattern, although diffuse, was grossly similar to the pattern of callosal connections as seen in flattened, sectioned cortex. Features that could be discerned were: the main callosal band straddling the lateral border of area 17, several rings and bands in extrastriate areas 18a and 18b, and a major band straddling the lateral border of area 3. The vitally visualized callosal pattern was used to guide injections of either wheat germ agglutinin conjugated to HRP (WGA-HRP) or rhodamine-labeled microspheres (RLM) into precisely localized sites in occipital cortex. There were numerous instances of doubly labeled neurons stained both with BB and WGA-HRP or RLM, suggesting that uptake of BB combined with UV exposure did not hinder the ability of stained neurons to take up and transport a second tracer. It is suggested that vital tract tracing be used as a tool for enhanced precision in studies of cortical connectivity.