Confocal imaging of organotypic brain slices for real time analysis of cell death

Programmed cell death (PCD) is a widespread phenomenon during normal brain development and also occurs in numerous neuropathological conditions. Among the several forms of PCD occurring in neurons, apoptosis is a gene-regulated process whereby affected cells undergo a stereotyped sequence of morphological and biochemical changes eventually leading to death. One of the most relevant features of this type of PCD is the very rapid clearance of apoptotic cells from tissue, this rendering their direct microscopic visualization a very demanding task. We describe here a procedure to realtime monitoring apoptosis in organotypic cerebellar cultures. By the use of a custom-designed microincubator that is mounted over the stage of an upright confocal microscope, we have been able to follow the activation of the apoptotic machinery in individual neurons that were labeled with a fluorescent tag. The incubation chamber allows for changing the culture medium without interfering with the observation, thus enabling the experimenter to follow the fate of individual neurons over long periods of time (days-weeks) and after challenge with different pharmacological agents.