A 48 kilodalton intermediate filament associated protein (IFAP) in reactive-like astrocytes induced by dibutyryl cyclic AMP in culture and in reactive astrocytes in situ.

In this paper we demonstrate that the 48 kilodalton (kDa) intermediate filament associated protein (IFAP), previously reported to be present in normal astrocytes, is also present in reactive astrocytes in situ and in reactive-like astrocytes induced by dibutyryl cyclic AMP in vitro. This IFAP is detectable by antibodies in normal rabbit serum (F2N) and is closely associated with glial fibrillary acidic protein-containing intermediate filaments (IF). The expression of 48 kDa IFAP is related to the acquisition of stellate shape by normal and reactive-like astrocytes in vitro. It is proposed that 48 kDa IFAP may be responsible for cross-linking IF into bundles and is thereby associated with cell process formation.

Role of colony stimulating factor-1 in brain damage caused by ischemia.

A marked effect of colony stimulating factor-1(CSF-1) on microglial response and neuron survival in cerebral cortex ischemic damage was observed. In osteopetrotic op/op mice, which lack systemically functional CSF-1 microglia do not respond to ischemic damage to the cerebral cortex, and the infarcts are considerably larger than in CSF-1 producing mice with similar vascular impairment. Delivery of extraneous CSF-1 to op/op mice alleviates the functional deficiency of the microglia and potentiates neuron survival in ischemic lesion. Delivery of extraneous recombinant CSF-1 to normal CSF-1 producing mice does not increase either the number or degree of activation of microglia, but does further potentiate neuronal survival. We found that neurons in the cerebral cortex have active CSF-1 receptors, and we therefore propose that neuronal rescue in cerebral cortex ischemic damage is linked to activation of the CSF-1 receptor on neurons.

Astrocyte cell lineage. I. Astrocyte progenitor cells in mouse neopallium.

The astrocyte cell lineage during postnatal development of the neopallium of Swiss mice was studied, using a colony culture assay method in which dissociated neopallial cells form discrete colonies in culture. It was found that immature epithelial-like cells that from type A colonies in culture come primarily from the subventricular zone but also from other regions of the neopallium. In culture, cells of type A colonies from type C colonies consisting of cells, which although still epithelial like, differ morphologically from the type A colony-forming cells. In the presence of dibutyryl cyclic adenosine monophosphate (dBcAMP) the type C colonies form cells rich in glial fibrillary acidic protein (GFAP) and stainable with Cajal's gold chloride sublimate, a stain specific for astrocytes. Therefore, it is proposed that type A colony-forming cells are astrocyte progenitor cells resembling the "pale" cells found in the subventricular zone (Blakemore and Jolly, '72), the "large glioblasts" (Sturrock, '76) and the free subependymal cells (Privat, '70; Paterson et at., '73) in the corpus callosum. The sequence of the lineage, i.e., cells forming type A colonies give rise to cells forming type C colonies which eventually differentiate into astrocytes, takes place in situ as well as in culture. As postnatal development of the neopallium progresses the number of colony-forming cells decreases in the subventricular zone and in other parts of the neopallium. The astrocyte progenitor cells migrate from the subventricular zone to other parts of the neopallium and progress through the lineage of differentiation in all regions of the neopallium.

Organization of microfilaments in astrocytes that form in the presence of dibutyryl cyclic AMP in cultures, and which are similar to reactive astrocytes in vivo.

This paper deals with changes in the arrangement of microfilaments at various stages during the transformation of astroblasts into reactive astrocytes in the presence of dibutyryl 3',5'-cyclic adenosine monophosphate in vitro. When cultures of two-week-old mouse astroblasts are treated with dibutyryl cyclic AMP, drastic changes occur in cell shape and in the organization of microfilaments, resulting in cells that closely resemble reactive astrocytes in vivo. A thick, prominent ring of microfilaments in such cells which stains strongly with 7-nitrobenz-2 oxa-1,3-diazole-phallacidin, delineates the perikaryon. Electron microscope examination showed that the ring is composed of many smaller bundles of microfilaments running parallel to each other. Prominent bundles of microfilaments radiate from the cell body into the cell processes. Based on the observation of intermediate forms, we propose that the microfilament ring may be important in the development of cell processes in reactive astrocytes.

Contractile units in stress fibers of fetal human astroglia in tissue culture.

The interaction between myosin and F-actin requires the enzyme, myosin light chain kinase (MLCK), as well as Ca(2+)-calmodulin and the calmodulin binding protein, caldesmon, which also binds to F-actin. Using immunofluorescence staining, we have demonstrated that in human fetal astroglia as in mouse astroglia (Abd-El-Basset et al., 1991) the stress fibers contain these contractile elements: F-actin, myosin, tropomyosin and caldesmon. F-actin extends continuously along the stress fibers, whereas myosin, tropomyosin and caldesmon are localized discontinuously in a periodic pattern. In addition, we have demonstrated that fetal human astroglia have the enzyme MLCK and calmodulin. The association of the contractile elements listed above together with calmodulin and MLCK constitutes what may be termed 'contractile units', suggesting that the stress fibers in astroglia may be contractile. Contractile stress fibers would enable astroglia to exert tension on the matrix surrounding them, thus facilitating rapid changes in cell shape.

Astrocyte proliferation in culture following exposure to potassium ion.

Following brain injury astrocytes swell acutely and proliferate thereafter. To investigate the direct relationship between swelling and proliferation, cultured astroglial cells were exposed to 60 mM K+ ion or hypo-osmolar (205 mOsm) electrolyte solution for 1 h to cause transient swelling. Proliferation was assessed by bromodeoxyuridine (BrdU) uptake 24 and 96 h later. The cells were double-labelled with antibodies to glial fibrillary acidic protein (GFAP) and BrdU. At 96 h the GFAP immunoreactive cells exposed to K+ exhibited 44.9% uptake versus 27.0% and 21.1% for control and hypo-osmolar exposed cells, respectively. We conclude that swelling of astroglia in vitro does not induce cell proliferation but transient depolarization following exposure to high K+ does.

Temporal relationship between the appearance of vimentin and neural tube development.

Intermediate filaments of the vimentin type that were initially identified within mesodermally derived cells have recently been demonstrated within several immature cell types derived from neuroectoderm, such as astroblasts and early stage neuroblasts. The objective of the present study was to determine the earliest developmental stage at which vimentin could be detected in the mouse neural tube. Vimentin was not detectable in the newly formed neural tube in E8 embryos. In the E9 neural tube the first positively labeled processes were observed in the ventrolateral region of the cervical neural tube with the processes having the distribution and appearance of those of radial glial cells. Between E9 and E10 there was a significant increase in the vimentin content of the neural tube as labeled filamentous bundles were observed throughout the ventricular cell layer and in the forming mantle layer. The distribution of labeled filaments in the E11 neural tube was similar to that of the E10 tissue although staining intensity was greater in the mantle layer in the E11 tissue. This work identifies the temporal relationship between the appearance of vimentin and neural tube development.

Isolation and identification of neuroblast precursor cells from mouse neopallium.

In this paper we describe the isolation of mouse neopallial neuroblast precursors using colony cultures and density gradients. The differentiation of neuroblast precursors was studied in tissue culture and after transplantation to the cerebellums of neonatal mice. In culture, survival of these cells is dependent on astroblasts, and their differentiation is incomplete. In the cerebellum, however, the cells give rise to neurons that correspond closely to the pyramidal cells and interneurons of mouse cerebral cortex according to morphometric measurements.

Spectrin does not redistribute with actin during dBcAMP-induced changes in astrocytes in vitro.

Cells of the astrocyte lineage obtained from mouse neopallium and grown in colony culture have been investigated for a correlation between distributions of F-actin and the common subunit of an erythrocyte actin binding protein, alpha-spectrin (brain fodrin). The cells of the astrocyte lineage at the astroblast stage have F-actin organized in the form of prominent, linearly arranged microfilament bundles. We have demonstrated that spectrin in these cells forms a fine reticulum lining the cell cortex. During the dibutyryl cyclic (dBcAMP)-induced transition from astroblasts to reactive astrocytes, actin-containing microfilaments undergo the dramatic rearrangement from a predominantly linear to a predominantly circumferential spatial organization. remains in the form of a fine reticulum lining the cellular cortex. These remains in the form of a fine reticulum lining the cellular cortex. These findings support the recent notion that spectrin in non-erythroid cells is not essential for maintaining the organization and plasma membrane membrane anchorage of the prominent microfilament bundles.

Fibrous astrocytes and reactive astrocyte-like cells in transplants of cultured astrocyte precursor cells.

Mouse neopallium was disaggregated at 3 developmental stages (E15, E18, PO) and grown in colony cultures for 7 days. On the seventh day of culturing the colonies of cells were transplanted into the cerebellums of neonatal mice. After 3 weeks the astrocytes within the transplants were identified with GFAP immunoperoxidase staining and by morphometric nuclear measurements of the GFAP positive cells. Cultures of E15 and E18 disaggregated neopallium gave rise to typical fibrous astrocytes in the transplants which were similar to fibrous astrocytes in the cerebral white matter of adult mice. Cultures of PO disaggregated neopallium gave rise to reactive astrocyte-like cells in the transplants which stained intensely for GFAP and had nuclei significantly larger than the astrocytes in the cerebral white matter of adult mice and in transplants of the E15 and E18 cultures.

Assembly of glial intermediate filament protein is initiated in the centriolar region.

Assembly of glial intermediate filament protein (GFP) into intermediate filaments (IF) was first detected by immunofluorescence in the perinuclear region of astrocytes differentiating in colony cultures before the rest of the cytoplasm was labeled. Double labeling with antisera specific for centrioles indicated that this site corresponds to the centriolar region. These studies suggest that the centriolar region plays an important role in the assembly of some types of IF as well as microtubules.

Astrocyte cell lineage. II. Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin- and GFP-containing intermediate filaments.

When cells from mouse neopallium are grown in colony cultures for 10-12 days, small cells with many processes, resembling normal fibrous astrocytes, form on top of the astrocyte precursor cells independently of the presence of dBcAMP in the culture medium. These cells are distinctly different from the much larger, previously described reactive astrocytes which also form in colony cultures and whose maturation is greatly enhanced by the presence of dBcAMP in the culture medium. Immunofluorescence studies showed that both vimentin-containing and glial filament protein (GFP)-containing intermediate filaments (IF) are present in the small normal fibrous astrocytes as well as in the larger reactive astrocytes. The vimentin-containing IF are assembled first in astrocyte precursor cells, whereas GFP-containing IF are assembled later toward the final stages of astrocyte differentiation both in vivo and in vitro. Thus in respect to the expression of the two types of IF, astrocyte differentiation in vitro closely resembles that in vivo. Parallel studies by electron microscopy showed that the vimentin-positive but GFP-negative astrocyte precursor cells contain single IF or small groups of IF, whereas in the more differentiated normal fibrous astrocytes and reactive astrocytes which are also GFP-positive, additional IF arranged in large bundles are present.

Development of mouse spinal cord in tissue culture-III. Quantification of neuron development in neural tube microfragment cultures.

Mouse neural tube development in vitro was examined following the isolation and culturing of specific regions of the neural tube. Developmental characteristics of neuron formation and differentiation were assessed both quantitatively and qualitatively. A 1 mm length of embryonic day 10 mouse neural tube was cut into 32 microfragments of equal size and cultured on collagen coated cover-slips. Neuronal cells were observed to emerge after 3 days in culture and to migrate away from the fragment upon an immature astroglial precursor cell layer that had begun to form within the first 24 h of culturing. The extent of neuronal migration, the density and total number of neurons per outgrowth zone, and the size distribution of neurons was quantitated after 21 days in culture. Three distinct patterns of neuronal outgrowth (Types II, III and IV) could be observed with a fourth pattern (Type I) best described as being neuron free. Neuron free outgrowth zones (Type 1) were comprized totally of glial cells and represented approximately 10% of the outgrowth zones examined. Characteristics of Type II outgrowth zones were a relatively restricted neuronal migration and a predominance (85%) of small neurons (< 10µm diameter) scattered around the outgrowth zone. In contrast the area of Type III outgrowth zones was twice that of the Type II, with a neuronal density 2.5 times greater, composed of a near equal proportion of small to medium sized (10-18 µm) neurons. The neuronal density and extent of migration of Type IV zones was similar to that of Type II zones; however the neurons of this fragment type were primarily medium and large sized neurons. In a second experiment the brachial region of E10 neural tube was sectioned into dorsal and ventral halves and microfragments from each half were cultured separately. Dorsal fragments produced primarily Type I and Type II outgrowth zones, and never a Type IV. In contrast, ventral fragments produced primarily Type III and Type IV zones with a small proportion of Type II zones. The results indicate that neuronal precursor cells in vitro maintain an adherence to patterns of neurogenesis programmed in vivo during early stages of neural tube development. The present culture system thus provides a means for the study of the expression in vitro of specific patterns of neuronal development and of the possible environmental influences (epigenetic factors) upon these patterns.

Effects of prenatal alcohol exposure on neural cells in mice.

Pregnant DBA/1J mice were treated orally with 1.0 or 2.0 g/kg ethanol from days 11-17 of gestation to determine whether ethanol can perturb normal brain development. On gestational day 18 the fetuses were removed and fetal growth parameters were determined. The cerebrums from one group of fetuses were subsequently analyzed for cell number and protein content. The remaining cerebrums were assayed for their ability to grow in an in vitro cell culture system. Prenatal ethanol exposure decreased fetal body and brain weights and crown-rump length. The brain was particularly affected as indicated by a decreased brain: body wt ratio. The percentage of affected and marginally affected fetuses increased in a dose-dependent manner. While the number of cells/brain was unaffected, the number of cells/g cerebrum and the number of cells/mg cerebral protein was increased. Prenatal ethanol exposure decreased the ability of cerebral cells to grow in culture as demonstrated by the reduced plating efficiency and reduced colony size. The data from the present study suggest that ethanol induces a two-fold effect on mouse brain development. First, since the total number of cells/brain was not appreciably affected by prenatal ethanol treatment, it is possible that the reduction in brain size is due to a decreased amount of neuropil. This putative effect on the neuropil was manifested in vitro by decreased colony area. Second, the decreased plating efficiency of cells from brains of affected fetuses suggests that these cells are not functionally normal. These effects may be important in the pathogenesis of central nervous system anomalies associated with the Fetal Alcohol Syndrome.

The hematopoietic cytokine, colony-stimulating factor 1, is also a growth factor in the CNS: congenital absence of CSF-1 in mice results in abnormal microglial response and increased neuron vulnerability to injury.

In this study we used op/op mice, which are deficient in the hematopoietic cytokine, colony-stimulating factor 1 (CSF-1), to determine the effect of CSF-1 on neuronal survival and microglial response in injury. In normal mice microglia express the CSF-1 receptor and are primarily regulated by CSF-1, produced mainly by astrocytes. The CSF-1 deficiency in op/op mice results in a depletion in the number of monocytes and macrophages but does not affect the number or morphology of microglia. We produced an ischemic lesion in the cerebral cortex of mice by disrupting the pia-arachnoid blood vessels in a defined area. Using Nissl stain and astrocyte- and microglia-specific antibodies, we determined the number of viable neurons in such injury and the intensity of glial reaction. The cellular response to injury on the operated side of op/op mice was compared to that on the non-operated contralateral side and to the cellular response in similar lesions in CSF-1 producing C3H/HeJ mice. We found that the systemic lack of CSF-1 in op/op mice results in a significant increase in neuron vulnerability to ischemic injury and considerably reduced microglial response to neuron injury. Remedying the CSF-1 deficiency, either by grafting CSF-1 secreting astroglia into the brain or by implanting encapsulated CSF-1 secreting fibroblast-like cells into the peritoneum, partially restores the microglial response to neuron injury and significantly potentiates neuronal survival in cerebral cortex ischemic lesions. Astroglial reaction was approximately the same in the lesions in op/op mice, grafted and implanted op/op mice and C3H/HeJ mice, indicating that CSF-1 modulates microglia, but not the response of astrocytes to injury. The degree of neuronal survival was not correlated to the degree of microglial proliferation and intensity of their reaction. We report some indications that CSF-1, in addition to modulation of microglia, may also act directly on neurons.

Macrophage-like cells originate from neuroepithelium in culture: characterization and properties of the macrophage-like cells.

Cultures of astroglia from C3H/HeJ mice, which are resistant to bacterial cell wall polysaccharide (LPS), initiated from embryos of Theiler stage 14 (9 days of gestation) up to Theiler stage 25 (17 days of gestation) as well as newborn animals, when subjected to nutritional deprivation, i.e. non-feeding of cultures, form large numbers of macrophage-like cells. These cells express Mac-1, Mac-3, F4/80 and Fc antigens. The cells are negative for GFAP, positive for vimentin, express Ia antigen and take up DiL-Ac-LDL. They are positive to non-specific esterase, secrete lysozyme and are phagocytic. Their morphology and ultrastructure closely resemble those of macrophages. Cultures initiated from neuroepithelium of Theiler stage 13 (8.5 days of gestation), before vascularization, when subjected to nutritional deprivation, also produce macrophage-like cells. Using spleen colony assay and methyl cellulose cultures, we were unable to detect the presence of hemopoietic (macrophage) precursor cells in astroglia cultures. This supports the hypothesis that the macrophage-like cells are of neuroectodermal origin and probably correspond to resident microglia of the CNS. Using nutritionally deprived astroglia cultures, a procedure was developed for isolation of macrophage-like cells and production of highly enriched macrophage-like (microglia) cultures.

Immunolocalization of the alpha isoform of smooth muscle actin in mouse astroglia in cultures.

In mouse astroglia colony cultures, cells display dramatic changes in their 'social behavior' during differentiation. The non-motile glioblasts progress through highly motile pro-astroblast and astroblast stages before becoming non-motile fibrous astrocytes. These changes in the behavior of astroglia are paralleled by changes in the organization of their microfilaments. Immunofluorescence staining of the astroglia colony cultures with rhodamine phalloidin, which binds to actin filaments (F-actin) formed by any of the 6 actin isoforms and with monoclonal antibody to alpha-smooth muscle (alpha-sm) actin (an actin isoform typical of smooth muscle cells), shows that the majority of the cells are positive for both alpha-sm actin and F-actin. Approximately 15% of the cells, however, were negative for alpha-sm actin but stained positively for F-actin, indicating that the actin of these cells belongs to isoforms other than the alpha-sm isoform.

Isolation and transplantation of oligodendrocyte precursor cells.

Newborn DBA/1J mouse neopallium was disaggregated and grown in high cell densities in tissue culture. In culture, the oligodendrocyte cell precursors are recognized as small refractile cells which use astrocyte precursor cells as a substratum. Using metrizamide density gradients, the oligodendrocyte precursor cells were separated from the astroblasts after 7 days in culture and then transplanted into the cerebellums of neonatal mice. The differentiation of the cultured oligodendrocyte precursors was analyzed in the transplants by nuclear morphometry, light and electron microscopy and immunocytochemistry. Analysis of the experiments indicated that the oligodendrocyte precursor cells, initially grown in culture, differentiated and myelinated host neuronal processes after transplantation. Moreover, the ultrastructure of the transplanted oligodendrocytes resembled mature oligodendrocytes in situ.

The acute and subchronic effects of organophosphorus and carbamate pesticides on cholinesterase activity in aggregate cultures of neural cells from the foetal rat brain.

The biochemical and morphological effects of five organophosphorus and carbamate pesticides were examined in aggregate cultures of neural cells from rat brains. Acute cholinesterase inhibition assays with carbofuran were performed on culture days 5, 10 and 17. No significant age-related differences in the median inhibitory concentrations (IC(50)s) were detected. When the cholinesterase IC(50) values for malaoxon, fenitrothion, carbaryl and carbofuran, determined on culture day 17, were plotted against the previously published median lethal dose (oral LD(50)s) of the same compounds in rats and mice, there was a good linear relationship between IC(50) and LD(50) values. In a 14-day subchronic study with fenitrothion, total protein levels in the cultures were significantly lower than control values at all doses tested (4, 8 and 20 mug/ml medium) while cholinesterase activity was unaffected at all but the highest dose. Central necrosis was observed at the intermediate dose, while cultures exposed to the highest dose exhibited extensive cell death and abnormal cell arrangement within the aggregate.

Cholinesterase inhibition by organophosphorus and carbamate pesticides in aggregate cultures of neural cells from the foetal rat brain: The effects of metabolic activation and pesticide mixtures.

The effects of pesticide mixtures on cholinesterase activity in aggregate cultures of neural cells were investigated; we also determined whether exogenous rat-liver microsomal fraction (S-9) might be used in conjunction with the cultures to mimic the in vivo activation of pesticides such as malathion. Studies of the effects of pesticide mixtures on the cholinesterase activity of cultures demonstrated that a hepatic microsomal fraction (S-9) played a major role in the nature of the interaction between combinations of malathion and fenitrothion or carbofuran. In the absence of S-9, malathion potentiated the anti-cholinesterase effect of fenitrothion, while neither synergistic nor antagonistic interactions occurred with mixtures of carbofuran and malathion. When S-9 was added to cultures with the pesticide mixtures, malathion's interaction with fenitrothion was antagonistic, and a synergistic response was observed for the mixtures of malathion and carbofuran. The antagonistic interaction of mixtures of fenitrothion and carbofuran was demonstrated to be independent of exogenously added S-9. Neither antagonistic nor synergistic interactions were observed for mixtures of triallate and fenitrothion or carbofuran. The data indicate that the addition of exogenous S-9 may be used to mimic certain aspects of the in vivo biotransformation of pesticides in aggregate cultures of neural cells from rat brain. Furthermore, the effects on cholinesterase activity of several of the pesticide mixtures tested were dependent upon the presence of exogenous S-9.