To reveal information about neuronal structure, various silver staining methods and numerous modifications have been developed that all trace their origin to the “dark reaction” introduced by Golgi in 1875. However, resolving the morphology and spatial distribution of the dendritic and axonal arbors of neurons has been a topic of interest for many years in the belief that knowledge of neuron structure is important in understanding neuron function. While these various methods, and others not mentioned here, have provided important insights into fundamental aspects of neuronal connectivity, they have offered relatively limited information about the detailed structure of the dendritic and axonal arbors of the cells involved. Subsequently, however, the selective staining of degenerating axons as a method for studying connectivity in the CNS was all but replaced by methods that exploited the capability of neurons to transport various biomolecules anterogradely and retrogradely.Īmong the many transported molecules that have been used to trace neural connections are radioactive amino acids, horseradish peroxidase, conjugates of horseradish peroxidase (HRP) such as wheat germ agglutinin-HRP –, biocytin, , cholera toxin –, and Phaseolus vulgaris leucoagglutinin. Several years later, Fink and Heimer refined this selective silver staining method to resolve the terminal boutons of degenerating axons. Nearly 60 years ago, Nauta and Gygax modified Bielschowsky's method to demonstrate that metallic silver could be used to impregnate degenerating axons selectively. In 1908, Bielschowsky introduced a silver staining method for axons and neurofibrils that is still used today by neuropathologists. In these instances, anterogradely labeled axons and retrogradely labeled dendrites were resolved in fine detail, approximating that which can be achieved with intracellularly injected compounds such as biocytin or fluorescent dyes.Įxperimental methods have been used to study neurons and their connectivity in the central nervous system (CNS) for many decades.

However, the detailed morphology of axon arbors and distal dendrites was evident only under optimal conditions for BDA labeling that take into account the: molecular weight of the BDA used, concentration and volume of BDA injected, post-injection survival time, and toning of the resolved BDA with gold and silver. Following the extracellular injection of BDA into the visual cortex, labeled cells and axons were observed in both cortical and thalamic areas of all animals studied. Specifically, we compared the effect on labeling sensitivity of: (a) using 3,000 or 10,000 MW BDA (b) injecting different volumes of BDA (c) co-injecting BDA with NMDA and (d) employing various post-injection survival times. To overcome this limitation, we varied several experimental parameters associated with the BDA labeling of neurons in the adult rat brain in order to improve the sensitivity of the method. However, BDA labeling often has proved to be inadequate to resolve the fine structural details of axon arbors or the dendrites of neurons at a distance from the site of BDA injection. Typically, BDA labels axons and cell somas in sufficient detail to identify their topographical location accurately. Biotinylated dextran amine (BDA) has been used frequently for both anterograde and retrograde pathway tracing in the central nervous system.

Axon dendrite labeled