Numerous neuroanatomical tract-tracing techniques have been reported to demonstrate the origin, course, and termination of neural pathways. New techniques have been developed to achieve higher specificity and efficiency. Early tract-tracing studies at the microscopic level used non-specific staining, for example, by tracing fiber bundles of normal nervous tissue using myelin staining. However, when combined with neurodevelopment or degeneration, myelin staining provides important information regarding the major pathways, even in the early years. Impregnation techniques, including the Golgi method, have contributed to the demonstration of connections between individual neurons. Specific staining for degenerating myelin and axons has established most of the basic knowledge of the major pathways described in classical neuroanatomical textbooks. Since the 1970s, tract-tracing techniques using axonal transport have opened a new era of more detailed and selective connectivity analyses. They show normal morphology of neural pathways, including synaptic contact with target cells. Various tracer substances have been reported that can be injected into the nervous tissue and transported anterogradely or retrogradely through axons. Neurotropic viruses that can be transported trans-synaptically are particularly useful for analyzing the chains of neuronal connections. Introducing genes encoding tracer substances or reporters using various techniques, including electroporation, lipofection, and viral vectors, can yield higher intracellular concentrations of these molecules and provide high-contrast images of the entire dendritic tree and axonal arborization of labeled neurons. Since gene manipulation allows us not only to visualize neurons but also to control their functions, we can now conduct integrative research on neuronal morphology and function.
