Large numbers of dying cells are found in proliferating tissues, suggesting a link between cell death and cell division. We detected and quantified dying cells during pre-and early postnatal development of the rat cerebral cortex using in situ end labeling of DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)] and electron microscopy. The proliferative zones that give rise to the neuronal and glial cell types of the cortex, the ventricular and, to a larger extent, the subventricular zones showed higher incidence of cell death than other regions of the developing cortex during the period of neurogenesis. Gel electrophoresis of DNA isolated from the subventricular zone of newborn animals showed a ladder pattern that is characteristic of apoptosis. The number of apoptotic cells remained high in this zone for at least 2 weeks, during which period cells continued to divide. The correlation between cell division and cell death was studied in the subventricular zone of newborn rats; cumulative labeling with bromodeoxyuridine showed that 71% of TUNEL-labeled cells had taken up this S-phase marker before undergoing cell death. Using bromodeoxyuridine and [ 3 H]-thymidine in succession to identify a cohort of proliferating cells, we found that the clearance time of TUNEL-positive nuclei was 2 hr and 20 min. A comparison between the number of mitotic figures and that of TUNEL-positive nuclei showed that cell death affects one in every 14 cells produced by dividing ventricular zone cells at embryonic day 16 and one in every 1.5 cells produced in the subventricular zone of newborn rats. In addition, we found that most of TUNEL-positive cells were in the G1 phase of their cell cycle. We conclude that apoptosis is prominent in the proliferating neuroepithelium of the developing rat cerebral cortex and that it is related to the progression of the cell cycle.

Two different modes of cell division are adopted by progenitor cells to generate the neurons and glia of the cerebral cortex: they either divide symmetrically to generate other progenitors or a pair of postmitotic cells or divide asymmetrically to generate both a progenitor and a postmitotic cell. In this study we used a lineage marker, the BAG retrovirus, in embryonic day 16 rats in combination with bromodeoxyuridine (BrdU) to identify patterns of cell generation in the cerebral cortex, and investigated the relationship between the phenotype of cells and the history of their lineages. The location, phenotype and birth order of clonally related cells were studied in the subsequent 3 weeks. Only pyramidal neurons and/or astrocytes formed discrete clusters in which several generations of family members were present, whereas nonpyramidal neurons were found exclusively in pairs or as single cells. Analysis of BrdU levels in these cells showed that nonpyramidal neurons were originally part of larger clones and were found dispersed in the neocortex because of tangential migration of their progenitors, dispersion of postmitotic cells, or death of clonal relatives. These results suggest that both symmetrical and asymmetrical division can be adopted by progenitor cells to generate cortical neurons and glial cells and that cell extrinsic events contribute to the isolation of nonpyramidal neurons.

Basic fibroblast growth factor (bFGF) has been shown to influence the survival, proliferation and differentiation of a variety of cell types in the nervous system. In this investigation we have examined the action of bFGF on: (i) the rate of proliferation; (ii) cell cycle parameters; (iii) the maintenance of cell division; (iv) the recruitment of quiescent cells; and (v) the degree of differentiation of cortical progenitor cells in cultures prepared from E16 rat embryos. The proliferation rate (labelling index) of cortical progenitor cells doubled in the presence of bFGF over 48 h. However, the lengths of the cell cycle phases were unchanged. Clones marked with a recombinant retrovirus on the first day in vitro (DIV) grew significantly larger in the presence of bFGF. Furthermore, many of the clones examined in control cultures had ceased to divide after a maximum of four cell cycles, whereas almost all clonally related cells were still dividing in the presence of bFGF 4 days later, i.e. for at least six cell cycles. Basic FGF also stimulated the division of quiescent progenitor cells, which otherwise would have differentiated or undergone cell death. The degree of neuronal and glial differentiation was studied after 5 DIV using MAP-2 and GFAP immunocytochemistry. In the presence of bFGF, the percentage of MAP-2-labelled cells was less than half that of control cultures, whereas the number of cells immunoreactive for nestin (a marker of progenitor cells) remained very high. Cells immunoreactive for GFAP were present in bFGF-treated cultures, yet were extremely rare in control conditions. These experiments show that bFGF, a potent mitogen for cortical progenitor cells, has no effects on the parameters of their cell cycle but extends their proliferative capability, promotes their survival and delays their differentiation into neurons.

Using histochemical, immunohistochemical and biochemical techniques, noradrenaline-, neuropeptide Y-, vasoactive intestinal polypeptide-, substance P- and calcitonin gene-related peptide-containing nerve fibres were studied in the uterine artery of virgin, progesterone-treated and pregnant guinea-pigs. Morphological changes following hormone treatment or in pregnancy were also evaluated in a quantitative study on semithin sections of the uterine artery. In late pregnancy, the number of noradrenaline-containing nerve fibres, which formed the densest plexus in virgin animals, was significantly decreased, a finding supported by a significant reduction in noradrenaline levels. This reduction was not mimicked by systemic progesterone treatment. In contrast, the innervation of the uterine artery by neuropeptide Y-containing nerve fibres was increased in pregnancy, while the other peptidergic nerves and peptide levels were unchanged after progesterone treatment and in pregnancy. These changes led to a predominance of innervation by neuropeptide Y- rather than noradrenaline-containing nerve fibres in late pregnancy. No morphological changes were detected following progesterone treatment, but pregnancy led to a marked increase in the cross-sectional area of the vessel accompanied by an increase in the thickness of the media.

This study was designed to investigate the effects of multiple denervation procedures on calcitonin gene-related peptide- and substance P-immunoreactive neurons in sympathetic and sensory cranial ganglia and in selected targets. Sympathectomy by long-term guanethidine treatment induced a pronounced increase in calcitonin gene-related peptide-immunoreactive and substance P-immunoreactive nerve fibres in all the tissues investigated, in contrast to a significant reduction of immunoreactive cell bodies. Neonatal capsaicin treatment abolished substance P immunoreactivity in many targets and caused a dramatic reduction of substance P-immunoreactive sensory nerve cell bodies; calcitonin gene-related peptide-immunoreactive nerve density was decreased, but the number of immunoreactive nerve cell bodies was unchanged. Guanethidine treatment of capsaicin-injected rats reversed the loss of calcitonin gene-related peptide-immunoreactive nerves, but not that of substance P-immunoreactive neurons. In the iris, capsaicin treatment had little effect on calcitonin gene-related peptide- and substance P-immunoreactive nerves, suggesting that in rats the majority of these fibres originate from capsaicin-insensitive neurons. The results suggest that the denervation procedures used in this study alter the synthesis and transport of neuropeptides in sensory neurons in conjunction with changes in the number of nerve fibres.