DEATH OF NEURONS IN THE NEONATAL RODENT AND PRIMATE GLOBUS-PALLIDUS OCCURS BY A MECHANISM OF APOPTOSIS

We have examined the developing rat, mouse and marmoset globus pallidus for evidence of cells dying by a process of ''naturally occurring'' or programmed cell death. We have demonstrated that cells in the developing mammalian globus pallidus die by a process of apoptosis and that by day 7 after birth many of the apoptotic cells possess a neuronal phenotype. Light microscopic and ultrastructural evidence of apoptotic cell death included cell shrinkage, blebbing of the extracellular membrane and condensation of the nuclear chromatin. Additionally we used an in situ nick translation method to assess the integrity of the DNA within the dying cells. This revealed that cells with the morphological characteristics of apoptosis also possessed Fragmented DNA typical of cells undergoing Type 1 programmed or apoptotic cell death. The lack of lysosomal enzyme activity within the dying cells and the frequent observations of phagocytosis by neighbouring cells also suggests that the form of programmed cell death is apoptosis and not Type 2 autophagic degeneration. We found no evidence for cells dying by Type 3 non-lysosomal degeneration since all dying cells examined under the electron microscope possessed intact intracellular organelles and cell membranes. We developed a sensitive silver stain which detected balls of condensed chromatin within the apoptotic cells. This enabled identification of apoptotic cells in the developing globus pallidus at low magnification and so allowed us to map the numbers and distribution of dying cells with time. The incidence of apoptotic cells in the neonatal globus pallidus was greatest at birth and then declined such that few cells were detected at one week and none was seen in the adult rat. Although the loss of large numbers of cells in the developing nervous system is a well documented phenomenon, there are only a limited number of reports of the mechanism by which neuronal cells die, and few of these are in the developing mammalian brain. There are at least four different morphological categories of neuronal cell death which are discriminated on morphological and biochemical criteria. Our analysis suggests that apoptotic or Type 1 cell death is the major form of programmed cell death occurring in the mammalian globus pallidus in the first week of life. This report also describes the use of two methods for the ready identification of apoptotic cells at the light microscope level. Because these methods are suitable for use on tissue sections they provide a means to assess the incidence of apoptotic cell death, in parallel with other analyses of the expression of gene products which control cell fate.