Intercellular barriers to and transcellular transfer of albumin in the fetal sheep brain.

The nature of the barriers that keep proteins out of the developing brain has been studied in tissues obtained from fetal sheep in experiments conducted under controlled physiological conditions. In anaesthetised pregnant ewes, 60 day gestation fetuses (term is 150 days) were exposed to human albumin injected intravenously for periods up to 6 h. The immunocytochemical distribution of exogenous human albumin was compared with that of endogenous sheep albumin at both the light and electron-microscopical level. Immunogold labelling of ultracryosections suggests that a tubulocisternal endoplasmic reticulum system in immature choroid-plexus epithelial cells is the route by which albumin crosses from blood to cerebrospinal fluid (CSF) in the developing brain. The integrity of the blood-brain barrier, the blood-cerebrospinal fluid barrier and the cerebrospinal fluid-brain barrier to protein, was confirmed. In addition, at the outer surface of the developing brain there also appears to be a restriction on the passage of albumin from CSF into the brain. These observations support earlier proposals that the immature brain develops within an internal environment from which proteins in plasma and CSF are largely excluded.

Ontogenetic development of diffusional restriction to protein at the pial surface of the rat brain: an electron microscopical study.

Blood-brain, blood-CSF and ventricular CSF-brain barriers to protein are present very early in brain development. In order to determine whether the outer pial surface of the brain also restricts free penetration of macromolecules, the dorso-lateral part of the sensorimotor cortex from rats at embryonic day 12 (E12), 14, 16, and 18, the day of birth (P0), and adult rat, was studied by electron microscopical techniques. Potassium ferrocyanide, Ruthenium Red and immunogold labelling of endogenous albumin were used to investigate junctional structures and the sites of restriction to albumin diffusion. At E12, large fenestrated sinusoids were present in the pia-arachnoid and the brain surface was formed by an incomplete layer of neuroepithelial and presumptive radial glial end feet, but capillaries in the pia-arachnoid showed no fenestrations at E14 or later. From E14, we observed the progressive appearance of distinct junctional structures between the glial end feet which, to our knowledge, have not been described before. Analysis of albumin distribution from E16 to P0 suggests that the junctions may contribute to restriction of diffusion between the subarachnoid space and the brain extracellular fluid. The restriction to the penetration of protein at both the pial and the ependymal surfaces may ensure the isolation of the neural environment during a critical phase in development of the nervous system. The changes in the structure of the junctions between E12 and P0 suggests a transitional series of embryonic junctional types, which eventually give way to the mature junctions of the adult. Parallels between the embryonic glial junctions and junctions described in adult invertebrate brain, suggest some interesting parallels in junctional development in phylogeny and ontogeny.

Ongoing activity of RNA polymerase II precludes chromatin collapse and DNA fragmentation in Chinese hamster ovary cells.

The role of ongoing RNA synthesis in chromatin organization in Chinese hamster ovary cells was examined upon exposure to the transcription inhibitor alpha-amanitin. Treatment with alpha-amanitin led to pleomorphic nuclei with chromatin heavily condensed and with the remaining ribonucleoprotein aggregated in large compact granular masses around the margins of the nuclear periphery. Concommitant with the changes in nuclei morphology transient focal dilatation of the rough endoplasmic reticulum was observed while other cytoplasmic organelles appeared structurally unaffected. The morphological changes occurred after complete inhibition of RNA polymerase II mediated transcription. The molecular integrity of isolated DNA was monitored in parallel with the structural analysis. Fragmentation of cellular DNA occurred in a time-dependent fashion and well after the complete inhibition of RNA synthesis. Characteristic oligonucleosomesized DNA fragments of about 187 base pairs in length was produced in a cotemporal time-dependent fashion. Our findings indicate that ongoing transcription and the structural state of chromatin are very closely integrated, and provide further evidence that RNA is a structural component of the nuclear matrix, which in turn is involved in keeping chromatin physically dispersed and decondensed.

Synaptogenesis in the neocortical anlage and early developing neocortex of rat embryos.

The recent finding of synapses in Monodelphis domestica (South American grey short-tailed opossum) before the establishment of the cortical plate raises the question of whether this finding is species-specific. Therefore, the establishment of the first synapses in the developing neocortex has been studied in the sensorimotor cortex of rat fetuses with a gestational age ranging from embryonic day 12 (E12) to birth. At E14, we found well-defined synapses with postsynaptic thickening and containing several vesicles in the presynaptic element in the primordial plexiform layer, before the appearance of the cortical plate. The postsynaptic elements were probably dendrites of Cajal-Retzius cells in the primordial plexiform layer and/or differentiating dendrites of presumptive cortical plate neurons. At E16, in addition to the presence of axodendritic and very few axosomatic synapses in the marginal and the subplate zones, synapses of both types were present within the cortical plate. Thus, this paper provides evidence for the presence of synapses in the developing rat brain both at an earlier stage and with a wider distribution than previously reported.

Development of spinal cord in the isolated CNS of a neonatal mammal (the opossum Monodelphis domestica) maintained in longterm culture.

The CNS of the newly born opossum removed in its entirety survives and maintains its electrical excitability in suitable culture media for up to ten days at 25 degrees C. The structure of the developing neonatal spinal cord has been studied in the intact animal and in the cultured CNS. The differentiation and survival of individual cells and subcellular structures were followed at the light and electron microscopic level. The expression of cell markers in neuronal and glial cells was studied immunocytochemically using commercially available antibodies. Both mono- and polyclonal antibodies raised against antigens from several other species cross-reacted with Monodelphis antigens. The spinal cord of preparations removed from three-day-old-animals showed many neuron specific enolase-positive large neurons in the ventral horn as well as vimentin- and glial fibrillary acidic protein-positive radial glial cells and numerous small diameter unmyelinated axons, abundant dendrites and synaptic structures. From post natal day 5 to post natal day 8 continued differentiation of neurons and differentiation of radial glial cells into astrocytes were apparent. Radial glial fibres and astrocytes reacted positively to antibodies against glial fibrillary acidic protein. Myelin had not appeared at 8 days. A comparison of material obtained from postnatal day 3-postnatal day 4 preparations fixed immediately after dissection and from postnatal day 3-postnatal day 4 preparations fixed after 5 days in culture showed growth with continued mitotic activity of the neuroepithelial cells and further neuronal and glial maturation in the spinal cord especially in the more rostral end. In successful experiments in vitro, the preservation of individual cells, organelles, membranes and synapses was similar in the freshly dissected and cultured preparations apart from a distinct loss of the youngest and some of the oldest neurons in the spinal cord. Also the main fibre tracts (dorsal, lateral and ventromedial funiculus) survived. Virtually all preparations that had not been damaged or injured showed these results. Possible reasons for the death or survival of individual neuronal or glial cell populations in these preparations are discussed.

Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry.

Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG-gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase-isomaltase. The labelling efficiency of RPGG was compared to that of protein A-gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12 nm particles, 10.3 and 6.2 particles/micron of length of microvillar membrane, 3.5 and 1.0 particles/micron2 of Golgi profile and 5.9 and 2.0 particles/micron2 of multivesicular body profile; and for the 6 nm particles, 49.6 and 15.7 particles/micron of length of microvillar membrane, 24.4 and 5.0 particles/micron2 of Golgi profile and 25.4 and 3.4 particles/micron2 of multivesicular body profile. Controls showed very little non-specific gold labelling (less than 0.02 gold particles/micron2 of section).

The subcellular distribution of transferrin in rat choroid plexus studied with immunogold labelling of ultracryosections.

Choroid plexus epithelium from third ventricle choroid plexus of 2-3-week-old rats was examined for transferrin-like immunoreactivity. In 5 microns paraffin sections most epithelial cells exhibited a pronounced immunoperoxidase staining for transferrin. The ultrastructure of the epithelium in question was examined by conventional electron microscopy. Immunolabelling of ultracryosections with IgG-gold, protein-A gold or protein-A gold-antiprotein-A protein-A gold showed an intense labelling of the basal extracellular space. The lateral intercellular space and the luminal surface showed a more variable labelling; no labelling of the tight junction zone was seen. Intracellularly a distinct labelling of the 'uptake and disposal pathway' (the endosomal-lysosomal system) was observed, but also the synthetic machinery (rough endoplasmic reticulum, stacked Golgi membranes) showed a characteristic labelling. Thus it seems likely that both uptake and synthesis of transferrin occur in choroid plexus epithelial cells.

Cell junctions and membrane specializations in the ventricular zone (germinal matrix) of the developing sheep brain: a CSF-brain barrier.

Cell junctions in the ventricular zone (germinal matrix) of the embryonic and foetal sheep brain were examined with thin-section and freeze-fracture electron microscopy. Neuroependymal cells in the early ventricular zone (days 19-40 of embryonic development, gestation period 147 days) exhibit a novel arrangement of cell junctions that connect adjacent neuroependymal cells at their lateral cell membranes next to the ventricular system. Small but typical gap junctions were also identified from the earliest stages examined. In serial thin sections and using a goniometer with a tilting device, the cell contacts showed a tight junction-like appearance of close and continuous fusion between neighbouring cell membranes. However, they were not arranged in a belt-like fashion close to the ventricular surface, but spiralled from the ventricular pole of the cells along the lateral cell membrane towards the deeper parts of the ventricular zone. Their freeze fracture appearance was different from that of single-stranded tight junctions in that the dimensions of their ridges and grooves were generally greater and the E-face grooves contained many particles. The junctions were especially prominent where more than two cells made contact. At mid-gestation they were less prominent than earlier and at 125 days gestation the neuroependymal layer was replaced by a mature-looking normal ependymal layer in which individual ependymal cells were connected by zonulae adherentes and large gap junctions; orthogonal arrays were also prominent. The close contact between gap junctions and single-stranded junctions found early in gestation suggests that there may be some developmental relation between these two membrane specializations. The transient single-strand junctions presumably form the morphological basis for a recently described CSF-brain barrier in the early foetal sheep brain. They may also have some mechanical function in anchoring neighbouring cells together in the region of the developing brain where cells are continuously dividing and migrating.

Epithelial membrane polarity: a stable, differentiated feature of an established human breast carcinoma cell line MCF-7.

Carcinoma cells typically show little or no polarity as compared to normal, differentiated epithelial cells. We have studied polarity in two established human breast carcinoma cell lines, T47D and MCF-7, by various techniques (electron microscopic enzyme- and immunocytochemistry, freeze-fracture) and show that one of them (MCF-7) is characterized by a high degree of polarity. Thus, in contrast to T47D cells, MCF-7 cells in monolayer culture form apical tight junctions, do not allow a ricin-horseradish peroxidase conjugate, which binds to terminal galactose residues on the apical surface, to stain the basolateral membrane domain, and express a surface antigen (MFGM-A) only in the apical surface membrane domain, as do normal mammary epithelial cells in vivo. This polarization is independent of a basement membrane, since it is maintained when MCF-7 cells, which do not deposit type IV collagen themselves, are grown directly on plastic. Moreover, even though MCF-7 cells express estrogen receptors rather homogeneously, estrogen has no effect on this polarity, neither in vitro nor after transplantation to nude mice. We conclude that polarity is a stable, differentiated feature of MCF-7 cells.

Microglia in the hypendyma of the rat subcommissural organ following brain lesion with serotonin neurotoxin.

The population of microglial cells in the subependymal layer of the subcommissural organ is sparse in normal adult rats. The number of microglial cells was substantially increased in this area following intraventricular injection of the serotonin neurotoxin 5,6-dihydroxytryptamine (5,6-DHT). In sections of plastic embedded material, 1 micron thick, the majority of phagocytic cells scattered in the subependymal layer had an appearance similar to that described in classical studies of microglial cells. At the electron microscopic level microglial cells exhibited the characteristic elongate nucleus with peripheral chromatin condensation. The perikaryon was scanty, containing strands of rough endoplasmic reticulum. The abundant organelles in the processes included Golgi complexes, mitochondria, rough and smooth endoplasmic reticulum as well as dense and multivesicular bodies. In addition, the processes contained phagocytosed axon terminals originating from the dense serotoninergic input to the subcommissural organ, which had degenerated on accumulating the serotonin neurotoxin. A fraction of the phagocytosed material was contained in subependymal subcommissural organ cells, astrocytes and oligodendrocytes. At the light microscopic level the phagocytosed terminals were visualized histochemically with Schmorl's reaction, which resulted in Prussian Blue precipitates. This allowed screening of microglial cells in complete series of sections through the well-defined subependymal layer of the subcommissural organ.