Viability of dopaminergic neurones is influenced by serum and astroglial cells in vitro.

Transplantation of embryonic nigral grafts into the striatum of Parkinson's disease patients is not optimal, mainly due to low survival of grafted neurones. Current strategies focus on enhancing neuronal survival by transplanting enriched neuronal cell populations. There is growing evidence for the importance of astroglia in neuronal survival.To characterise the effects of glial cells on dopaminergic neurones, 5-fluoro-2'-deoxyuridine was added to embryonic rat ventral mesencephalic cultures in the presence or absence of serum. The survival and morphology of glial fibrillary acidic protein immunopositive astroglia and tyrosine hydroxylase immunopositive dopaminergic neurones was examined. In serum-containing medium, astroglial cells predominated and 5-fluoro-2'-deoxyuridine had no significant effect on either astroglia or dopaminergic neurone survival. In serum-free medium, astroglial growth was attenuated and numbers were significantly lower in 5-fluoro-2'-deoxyuridine treated compared with untreated cultures. There was no significant difference in the numbers of dopaminergic neurones between 5-fluoro-2'-deoxyuridine treated and untreated cultures. However, by the eighth day in vitro, there were differences in the morphology of these neurones between treated and untreated cultures. This study shows that the use of 5-fluoro-2'-deoxyuridine and serum-free medium can produce a neurone-enriched culture. However, the dopaminergic neurone population present in these cultures appeared to be morphologically dissimilar to those found in control cultures as neurites were retracted and the cell somas of these cells appeared enlarged. These results provide information on the effects of astrocytes on dopaminergic neurones in ventral mesencephalic cultures and thus have implications for transplantation in Parkinson's disease.

Estimation of nuclear volume as an indicator of maturation of glial precursor cells in the developing rat spinal cord: a stereological approach.

Studies on nuclear volume have shown that it is an indication of the state of differentiation of cells. This study provides evidence indicating increasing nuclear volume during cell maturation. Using unbiased stereological techniques, nuclear volume of both proliferating and non-proliferating glial cells was analysed in the developing spinal cord. Proliferating glial precursor cells were identified using a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. The nuclear volume of BrdU-labelled cells and unlabelled cells was determined in both periventricular regions and the white matter of the cord at different embryonic ages. In the periventricular region BrdU-labelled nuclei were smaller than unlabelled nuclei at all ages examined. These labelled cells represent dividing undifferentiated progenitors. The unlabelled neighbouring cells with larger nuclei represent a more differentiated population. In the white matter BrdU-labelled nuclei were of similar volume to the unlabelled nuclei. Both of these groups represent glial precursor cells that have migrated from deeper regions and are at similar stages of differentiation, perhaps with different proliferative potential. These findings indicate that the nuclear volume of early glial cells increases as these cells migrate and differentiate.

Proliferation and migration of glial precursor cells in the developing rat spinal cord.

The mechanisms that control the production and differentiation of glial cells during development are difficult to unravel because of displacement of precursor cells from their sites of origin to their permanent location. The two main neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. Considerable evidence supports the view that oligodendrocytes in the spinal cord are derived from a region of the ventral ventricular zone (VZ). Some astrocytes, at least, may arise from radial glia. In this study a 5-Bromo-2'-deoxyuridine (BrdU) incorporation assay was used to identify proliferating cells and examine the location of proliferating glial precursor cells in the embryonic spinal cord at different times post BrdU incorporation. In this way the migration of proliferating cells into spinal cord white matter could be followed. At E14, most of the proliferating cells in the periventricular region were located dorsally and these cells were probably proliferating neuronal precursors. At E16 and E18, the majority of the proliferating cells in the periventricular region were located ventrally. In the white matter the number of proliferating cells increased as the animals increased in age and much of this proliferation occurred locally. BrdU labelling showed that glial precursor cells migrate from their ventral and dorsal VZ birth sites to peripheral regions of the cord. Furthermore although the majority of proliferating cells in the spinal cord at E16 and E18 were located in the ventral periventricular region, some proliferating cells remained in the dorsal VZ region of the cord.

The regional distribution of myelin oligodendrocyte glycoprotein (MOG) in the developing rat CNS: an in vivo immunohistochemical study.

Using an immunohistochemical approach we have characterized the in vivo developmental distribution of myelin oligodendrocyte glycoprotein within the rat CNS. Myelin oligodendrocyte glycoprotein expression emerged in a non-uniform manner during the first 3 postnatal weeks. Although it was absent throughout the CNS of the newborn rat at postnatal day 0(P0), it had appeared in the spinal cord and brainstem by P7. The forebrain and cerebellum remained devoid of immunoreactivity until after P14. Myelin oligodendrocyte glycoprotein emerged at different times within the closely associated fasciculi of the dorsal funiculus. It appeared in the fasciculus cuneatus during the first postnatal week and in the fasciculus gracilis and corticospinal tracts during weeks 2 and 3 respectively. Myelin oligodendrocyte glycoprotein expression developed along a caudo-rostral gradient from spinal cord to forebrain and along an antero-posterior gradient within the CNS in general. The relationship between the onset of myelin oligodendrocyte glycoprotein expression and myelinogenesis was also investigated. In most regions, myelin oligodendrocyte glycoprotein expression lagged behind the initial appearance of myelin basic protein and Luxol Fast Blue-stained myelin by at least 1 week. These observations support the idea that myelin oligodendrocyte glycoprotein is the latest myelin protein to appear in development, only being expressed during the final stages of oligodendrocyte differentiation. Furthermore, the pattern of staggered expression within the dorsal columns indicates that localized, region-specific interactions may comprise a key element in the control of the terminal phases of oligodendrocyte differentiation.

Distribution and fine structural analysis of undifferentiated cells in the primate subependymal layer.

The subependymal layer (SEL) of the postnatal marmoset, a simian primate, has been investigated by histological and electron microscopic techniques. Although well documented in rodents, little is known about this layer in primates. The distribution of the SEL in marmosets is most extensive at birth around the anterior lateral ventricles, where the layer is generally 5-10 cells deep; however, there is considerable regional variation. With age the size of the SEL decreases dramatically, becoming very poorly demarcated in adult animals. Ultrastructurally, many subependymal cells in neonatal and young brains display the features of undifferentiated cells, although neurons and glia are also present. Cells displaying features intermediate between astrocytes and undifferentiated SEL cells are occasionally encountered. In adults undifferentiated cells are seen rarely and the former SEL is composed primarily of glial and neuronal processes. Thus the layer in primates probably represents a site of continued cellular differentiation in the postnatal brain and as such must play an important role in the final stages of cortical development.

Isolation and characterisation of proliferating cells from the telencephalic vesicles of gestational-day 13 rats.

A method of isolating in culture a specific subpopulation of cells in the "S" phase of the mitotic cycle, from embryonic forebrain, is described. Different enzyme treatment and different substratum pretreatment are compared quantitatively to ensure optimal yield. These cells, isolated from the telencephalic vesicles of embryonic day (E) 13 rat embryos, were maintained in culture and their morphological and immunocytochemical characteristics were investigated at one and eight days in vitro.

Cell proliferation in the subependymal layer of the postnatal marmoset, Callithrix jacchus.

Cells of the subependymal layer (SEL) have been shown to be capable of continued postnatal cell division throughout life in rodents. To determine if the primate brain behaves similarly, proliferative activity in the SEL of the marmoset has been investigated by tritiated thymidine autoradiography and bromodeoxyuridine immunocytochemistry. Both methods revealed the presence of DNA-synthesizing cells at all postnatal ages studied. The labelling index (LI), low at birth, reached a peak of almost 4% at one month but decreased gradually thereafter. In animals older than two years the LI was extremely low and labelled cells were rarely seen anywhere in the brain. The cell density of the SEL, in contrast to the low LI, was highest in neonates and decreased linearly with increasing age. Bromodeoxyuridine immunoreactivity revealed the distribution of proliferating cells in the SEL and neighbouring regions. Such cells were most abundant around the anterior lateral ventricle where the SEL was most evident. Proliferating cells were numerous in neonates, though not adjacent to the ependyma where counts for the LI were made, and were mainly located dorsally and ventrally at the junctions of the corpus callosum and caudate nucleus.

The distribution of glial fibrillary acidic protein and vimentin in postnatal marmoset (Callithrix jacchus) brain.

Antisera to glial fibrillary acidic protein (GFAP) and vimentin were used to elucidate the distribution of these intermediate filament proteins in postnatal marmoset brains of various ages. The ependyma of the lateral ventricles was unique in being equally immunoreactive for both GFAP and vimentin at all ages. Vimentin alone was consistently demonstrated in endothelial and leptomeningeal cells at all ages. In neonates, vimentin immunoreactivity greatly exceeded that of GFAP and was located primarily in radial glia in the subependymal plate of the anterior cerebrum. Their vimentin-positive processes formed thick fascicles in the corpus callosum but separated into fine fibres on entering the cortex. GFAP immunoreactivity in these cells and processes was very limited. With age, GFAP-positive cells increased in number and displayed the typical stellate appearance of astroglia. The vimentin-positive radial glial population decreased considerably during this period and by 6 months had virtually disappeared. The GFAP reaction in adult brain was even more widespread, largely due to the increased number of positive astrocytes in the white matter. Vimentin immunoreactivity in the adult was greatly diminished and positive radial glia were not detectable. A major change in intermediate filament protein expression, therefore, occurs in the early postnatal period and probably reflects phases in the differentiation of radial glial precursors into astrocytes.