Neuronal apoptosis studied by a sequential TUNEL technique: a method for tract-tracing.

ABSTRACT

A novel tract-tracing procedure by using a sequential in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling of DNA fragments (TUNEL) is described. This method identifies fragmented DNA transported into neuronal fibers in tissue sections of gerbil hippocampal CA1 neurons following transient forebrain ischemia. The transported DNA has been confirmed by another method, fluorescence DNA detection technique by DAPI. Many methods have been developed to study the neuroanatomical connections in the central nervous system. Principally, these techniques are based on tract-tracing studies using xenobiotics into the central nervous system. Our tract-tracing method is originated from an intrinsic marker that is produced during the apoptotic process of neurons. Furthermore, the advantage of this method is that only the selected cells undergoing apoptosis are recognized and traced to the end of the related neuronal fiber. Usually, apoptotic cells possess intact intracellular metabolic mechanisms until completion of cell death. Thus, apoptotic neurons retain the axonal transport mechanisms which enables us to detect fragmented DNA moving from nuclei to distal terminals of neuronal fibers. Since TUNEL-positive DNA movement within neuronal fibers occurs only during a limited period, it is essential that a time-course of the TUNEL technique is used to study tract-tracing of apoptotic neurons. Although this method can identify only the apical dendrites of cells that are undergoing apoptosis during the limited period, some projections of the gerbil hippocampal CA1 neurons undergoing apoptosis are clearly demonstrated.