An optimized method for simultaneous demonstration of neurons and myelinated fiber tracts for delineation of individual trunco- and palliothalamic nuclei in the mammalian brain

The truncothalamic complex has long been considered to be a nuclear group with 'non-specific' projections. More recently, it is suggested that these thalamic nuclei play an important role in regulating distinct basal ganglia circuits. To further analyze the exact biological function of individual nuclei of the truncothalamic complex a simple and reliable technique for an exact delineation of distinct nuclei is desirable. Therefore, we evaluated and optimized several potential procedures for a combined visualization of neurons and myelinated fibers. Fiber staining with gold toning or immunocytochemical visualization of myelin basic protein shows high contrast and precision but precludes sufficient demonstration of neuronal cell bodies. When the most common technique for the simultaneous visualization of both structures, the Kluver-Barrera procedure, is used, demonstration of myelinated fibers is restricted when the technique is applied to cryostat or vibratome sections. In the present report this limitation was abolished. The final protocol includes lipid extraction prior to the incubation with Luxol Fast Blue and uses carefully characterized staining conditions for Luxol Fast Blue and cresyl violet rendering microscopically controlled differentiation steps unnecessary. The optimized Kluver-Barrera technique results in high precision localization of individual axons and cell bodies and thus permits an exact and simple delineation of individual nuclei in the vertebrate thalamus.