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Gap junctions are clusters of intercellular channels that connect the interiors of coupled cells. In the brain, gap junctions function as electrotonic synapses between neurons and as pathways for the exchange of metabolites and second-messenger molecules between glial cells. Astrocytes, the most abundant glial cell type coupled by gap junctions, are intimately involved in the active control of neuronal activity including synaptic transmission and plasticity. Previous studies have suggested that astrocytic-neuronal signaling may involve gap junction-mediated intercellular connections; this issue remains unresolved. In this study, we demonstrate that second-trimester human fetal hippocampal neurons and astrocytes in culture are coupled by gap junctions bidirectionally; we show that human fetal neurons and astrocytes express both the same and different connexin subtypes. The formation of functional homotypic and heterotypic gap junction channels between neurons and astrocytes may add versatility to the signaling between these cell types during human hippocampal ontogeny; disruption of such signaling may contribute to CNS dysfunction during pregnancy., (Copyright 2002 S. Karger AG, Basel)

Toxoplasma gondii is an intracellular parasite that is a common opportunistic infection of AIDS patients where it causes a severe and often fatal encephalitis. Toxoplasmic encephalitis in AIDS patients results from a reactivation of the cyst stage of Toxoplasma gondii in the brain. A previous study found an association of host cell intermediate filaments with parasitophorous vacuoles and some studies have suggested the host cell cytoskeletal elements are incorporated into the cyst wall. In this study, the interaction of glial filaments with Toxoplasma gondii cysts was studied in cysts derived in vitro in mouse astrocytes and in cysts isolated from mouse brains. Glial filaments, detected by immunostaining of the glial fibrillary acidic protein, were found to accumulate around the perimeter of the cysts as they developed in mouse astrocytes. Transmission electron microscopy revealed a layer of glial filaments was wrapped around the cytoplasmic side of the cyst. The glial filaments were present in close apposition to the cyst wall and arranged around the cysts in a concentric layer, measuring 5-10 microns in thickness. The layer of glial filaments excluded host cell mitochondria and endoplasmic reticulum from the cytoplasmic surface of the cyst. Colocalisation of glial fibrillary acidic protein and the cyst wall via confocal and immunoelectron microscopy, confirmed that there was no glial fibrillary acidic protein present within the cyst wall. The cyst wall of cysts isolated from mouse brains were also found to be negative for glial fibrillary acidic protein. In conclusion, we found no evidence of structural integration of the host cell intermediate filaments in the cyst wall, but glial filaments were found to encase the cysts in the host cell during cyst development in host cells in vitro. The glial filaments wrapping of cysts may play a role in bradyzoite differentiation and/or cyst stabilisation in the host cell cytoplasm.

Toxoplasma gondii (T. gondii) is one of the most common opportunistic infections affecting the central nervous system (CNS) in AIDS patients. Disease results from a reactivation of a latent infection in the brain resulting in a severe and necrotizing encephalitis. In this study we infected a primary culture from human fetal brain with T. gondii and studied the behavior of both the active and latent stages in this culture system. We found that the active (tachyzoite) stage of T. gondii can infect both astrocytes and neurons. However, a higher percentage of astrocytes were infected than neurons. Additionally, astrocytes were found to support more replication of T. gondii than did neurons. Both astrocytes and neurons also supported the cyst stage, found in the latent infections. These data indicate that astrocytes are the host cells supporting most of the replication of T. gondii in the brain in reactivated infections, but both host cell types may be able to support the cyst stage in latent infections. However, evidence indicates that cysts formed in astrocytes may be distinct from neuronal cysts. These findings may have relevance to reactivation of latent T. gondii infections in AIDS patients.

Primary astrocytes were cultured from forebrains of 1-day-old rats. Immunofluorescence microscopy showed that approximately 80% of the cells were positive for glial fibrillary acidic protein (GFAP) and greater than 80% were stained with an antiserum to the molecular weight 58,000 fibroblast intermediate filament protein (vimentin). Gel electrophoresis of Triton-insoluble cytoskeleton preparations from these cultures revealed three major bands having molecular weights of 58,000, 51,000, and 42,000, together with some prominent lower-molecular-weight species. The protein of molecular weight 51,000 was not present in preparations from fibroblasts. Each of the three major astrocyte proteins was subjected to limited proteolysis, while two of the proteins were cleaved by cyanogen bromide. The electrophoretic peptide patterns of the 58,000 protein were similar to those of vimentin isolated from NIL-8 fibroblasts, and the patterns of the 51,000 protein were similar to those of GFAP isolated from rat spinal cord. The patterns of the protein of molecular weight 42,000 resembled those of muscle action. Rocket immunoelectrophoresis showed that the 51,000 astrocyte protein reacted with an antiserum to bovine GFAP, but the 58,000 and 42,000 proteins failed to react. We conclude that the major proteins of cytoskeleton preparations from cultured primary astrocytes are vimentin (58,000), GFAP (51,000), and actin (42,000), and that our data show no obvious structural relationship among them.

We have examined the effects of dibutyryl-cyclic AMP (dBcAMP) on the organization and expression of filamentous proteins in astroglia. The drug produced several effects on astrocytes grown in primary cultures. Cultures ceased to grow, and cells changed shape to a contracted form, displaying thin cytoplasmic processes. Cellular levels of the intermediate filament (IF) proteins, vimentin and glial fibrillary acidic protein (GFAP), and actin, insoluble in Triton X-100, were examined by polyacrylamide gel electrophoretic analysis. The cellular content of both of the IF proteins increased concurrently, approximately doubling during a 2-week course of treatment. The content of actin associated with the Triton residue decreased, however, a biochemical alteration which correlated with a loss of stress fibers in treated cells. Treatment with sodium butyrate did not change either cell shape or cytoskeletal protein content. Filament protein expression in astrocytes can, therefore, be modulated via cAMP-dependent mechanisms. The effects do not, however, appear specific for the GFAP-type of intermediate filament.

We previously reported that the cytoskeleton of rat astrocytes in primary culture contains vimentin, glial fibrillary acidic protein (GFAP), and actin. These proteins were found in a fraction insoluble in Triton X-100 and thought to be assembled in filamentous structures. We now used primary astrocyte cultures to study the kinetics of synthesis and turnover of these cytoskeletal proteins. The intermediate filament proteins were among the most actively synthesized by astrocytes. High levels of synthesis were detectable by the third day of culture in the early log phase of growth, and the pattern of labeling at day 3 was similar to that at 14 days when the cultures had reached confluency. In short-term incorporation experiments vimentin, GFAP, and actin in the Triton-insoluble fraction were labeled within 5 min after exposure of the cultures to radioactive leucine. We did not detect any saturation of labeling for up to 6 h of incubation. The turnover of filament proteins studied by following the decay of radioactivity from prelabeled vimentin, GFAP, and cytoskeletal actin displayed biphasic decay kinetics for all three proteins. In the initial phase a fast-decaying pool with a half-life of 12-18 h contributed about 40% of the total activity in each protein. A major portion, about 60%, of each protein, however, decayed much more slowly, exhibiting a half-life of about 8 days.

In either actively or passively transferred experimental autoimmune encephalomyelitis (EAE), increased immunocytochemical staining of glial fibrillary acidic protein (GFAP) in astrocytes was detected early in the disease process in both the gray and white matter of the spinal cord. Staining was not restricted to areas of perivascular mononuclear infiltration, and was observed at all levels of the cord. This enhanced staining pattern was delayed in rats in which clinical signs of EAE had been suppressed by treatment with the alpha 1-adrenoceptor antagonist prazosin. This glial reaction in EAE was not accompanied by increased GFAP synthesis, as measured by in vitro labeling of spinal cord slices, nor an increase in GFAP content, as measured by densitometry of intermediate filament fractions separated by polyacrylamide gel electrophoresis. Total protein synthesis was increased, with vimentin being labeled especially heavily; in prazosin-treated EAE animals, the increase in total protein synthesis was reduced and delayed.

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.

We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 X 10(5)/cm2. At initial densities above 1 X 10(5) the population increased to reach confluency by 10-12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2-0.4 X 10(5)/cm2, cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.