Sergey Fedoroff

Kinetic characteristics of the glutamate uptake into normal astrocytes in cultures

Kinetics for uptake and release of glutamate were measured in normal, i.e., nontransformed, astrocytes in cultures obtained from the dissociated, cortexenriched superficial parts of the brain hemispheres of newborn DBA mice. The uptake kinetics indicated a minor, unsaturable component together with an intense uptake following Michaelis-Menten kinetics. TheKm (50 μM) was reasonably comparable to the corresponding values in brain slices and in other glial preparations. TheVmax (58.8 nmol min−1 mg−1 protein) was, however, much higher than that observed in glial cell lines or peripheral satellite cells, and also considerably higher than that generally reported for brain slices. The release of glutamate was much smaller than the uptake, and only little affected by an increase of the external glutamate concentration, suggesting a net accumulation of glutamate rather than a homoexchange. Such an intense accumulation of glutamate into normal astrocytes may play a major role in brain metabolism and may help keep the extracellular glutamate cohcentration below excitatory levels.

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.

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.

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 of 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 strocyte‐ 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 annd 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.

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.

Astrocyte cell lineage. III: The morphology of differentiating mouse astrocytes in colony culture

SummaryDisaggregated cells of newborn DBA/1J mouse neopallium were grown in colony cultures, and colonies of cells at various stages of differentiation along the astrocyte cell lineage were examined after 3 days, 1, 2 and 4 weeks by electron microscopy and by NBD-phallacidin which demonstrates the distribution of microfilaments. The earliest astrocyte precursor cells or glioblasts are closely apposed epithelial cells that rarely have junctions. Their scanty cytoplasm contains many free ribosomes but few microfilaments. The cells in the next stages of astrocyte lineage or proastroblasts are flat and are separated from each other to a variable degree. They have intercellular junctions associated with microfilaments and contain singly dispersed intermediate filaments. The proastroblasts gradually differentiate into astroblasts which have a similar morphology except that in addition to the singly distributed intermediate filaments they also contain intermediate filaments arranged into bundles of various sizes. The mature fibrous astrocytes have well-defined processes and distinct perikarya. They form from astroblasts in culture and also contain numerous bundles of intermediate filaments. The dibutyryl-cyclic AMP (dBcAMP)-induced astrocytes in culture in contrast are large stellate cells similar to reactive astrocytes found around sites of injury in the brain. On the basis of these and previous immunocytochemical studies of the formation and distribution of intermediate filaments in the cytoplasm of differentiating astrocytes, criteria are proposed for identification of different cells along the astrocyte lineage.

CELLULAR LOCALIZATION OF STEM CELL FACTOR AND C-KIT RECEPTOR IN THE MOUSE NERVOUS SYSTEM

We have characterized the cellular localization of stem cell factor (SCF) and c‐kit receptor (c‐kitR) in the adult mouse nervous system in situ and in culture by using immunocytochemistry. We found that SCF is largely confined to the neuronal population in normal brain, whereas c‐kitR is expressed by glial cells as well as some neurons. We also found that astroglia at an early stage of culture (7 days in vitro) are strongly SCF positive and weakly c‐kitR positive. Microglia in cultures express both SCF and c‐kitR, but the immunostaining of SCF is weak and diffuse when microglia are cultured in the presence of colony stimulating factor‐1. Northern blot analysis confirmed the expression of mRNAs of c‐kit and SCF in cultured neurons, astroglia, and microglia. The addition of recombinant SCF to astroglia in culture upregulates the expression of mRNAs of nerve growth factor, brain derived neurotrophic factor, and ciliary neurotrophic factor. These observations suggest that SCF/c‐kitR signaling is involved in neuron‐neuron as well as neuron‐glia interactions. J. Neurosci. Res. 47:1–15, 1997.

Microglia and astroglia have a common progenitor cell

Disaggregated neopallial cells from newborn C3H/HeJ mice were cloned in Grenier hybridoma tissue culture dishes, and culture wells that contained only one cell were marked. After 8–10 days of culturing, the cultures were fixed and double immunolabeled for microglia with Mac‐1 antibody and for astroglia with antibody to GFAP. Each marked well containing a clone was identified as either a microglia, astroglia, mixed microglia–astroglia, or an unlabeled clone. The effect of LM cell line conditioned medium (LM‐CM), which contains colony‐stimulating factor‐1, on the development of mixed microglia–astroglia clones was determined. Formation of mixed clones was dose dependent (P < 0.0001). We concluded that microglia and astroglia have a common progenitor cell and that the development of mixed clones is LM‐CM dependent. J. Neurosci. Res. 50:477–486, 1997.

Expression of colony stimulating factor-1 receptor (CSF-1R) by CNS neurons in mice†

We report that neurons in the central nervous system express colony stimulating factor‐1 receptor (CSF‐1R) mRNA and protein and that the expression has regional specificity. The presence of CSF‐1R in neurons was demonstrated by the use of four different types of antibodies to CSF‐1R and the presence of CSF‐1R mRNA by in situ hybridization using oligonucleotide probe. In the steady state in most areas of the brain, CSF‐1R is weakly expressed in only a few neurons. In the cerebellum, brainstem, and spinal cord, however, CSF‐1R is expressed constitutively in greater numbers of neurons. After cerebral cortex ischemic injury, neurons in the area next to the ischemic lesion markedly upregulate CSF‐1R. It is also upregulated in the contralateral cortex and in many other areas of the brain and spinal cord. We demonstrated that in cultures the ligand CSF‐1 binds to its receptor (CSF‐1R) in neurons and that reduction of the number of apoptotic neurons and potentiation of neuron survival is CSF‐1 dose dependent. We propose that CSF‐1/CSF‐1R signaling is an important regulatory pathway between neurons, microglia, and astrocytes. J. Neurosci. Res. 57:616–632, 1999.

Biology and pathology of astrocyte-neuron interactions

Metabolic and Ionic Astrocyte-Neuron Interactions -- Metabolic Interactions between Neurons -- The Perinodal Astrocyte: Functional and Developmental Considerations -- Anoxia-Induced Extracellular Ion Shifts in Mammalian CNS White Matter -- Hyperexcitability of Neurons and Astrocytes in Epileptic Human Cortex -- Inter-Cellular Signalling by Nitric Oxide -- Production of Nitrosyl Mediators in Astrocytes -- Regulation of Astrocyte-Neuron Interactions -- The Possible Roles of Astrocytes in Energy Metabolism of the Brain -- Astroglia: Receptors, Second Messengers, and Function -- Neurotrophic Factors Produced by Astrocytes Involved in the Regulation of Cholinergic Neurons in the Central Nervous System -- Olfactory Ensheathing Cells: Factors Influencing the Phenotype of these Glial Celss -- Role of Peroxidase-Positive Astrocytes in Estradiol-Related Hypothalamic Damage -- Regulation of Gene Expression in Astrocytes -- GFAP Gene Expression in Normal and Reactive Astrocytes -- Trophic Astrocyte-Neuron Interactions -- Astrocytes Can Act as Permissive Subtrates for the Growth of NFG-Sensitive Acons in Vivo -- Glial-Neuronal Interactions Exemplified by the Synthesis and Actions of Ciliary Neurotrophic Factor -- How Does Thrombin Cause Neurite Retraction? -- Neuronal Control of Astrocyte Proliferation -- The Role of 5-HT1A Receiptors in Development and Adult Plasticity of the Serotonergic System -- Effects of Cytokines on Neural Cells -- The Role of Substance P in Cytokine Production by Glial Cells -- Sources and Targets of Cytokines in the Central Nervous System -- Regulation of Tumor Necrosis Factor-Alpha Gene Expression in the Astrocyte -- Paracrine and Autocrine Signalling in Regulation of Microglia Survival -- Antigen Presentation at the Blood-Brain Barrier: A Role for Astrocytes? -- Oligodendrocytes and the Immune System -- Astrocyte Response to Injury -- Perineuronal Glial Reactions in Regeneration of Motoneurons -- Regulation of Type III Intermediate Filament Protein Genes in Astrocytes during Development and after Injury -- Heterogeneity of Reactive Astrocytes -- Inhibition of GFAP Synthesis with Antisense Nucleic Acid Constructs -- X-Irradiation for Promoting Recovery in Lesioned Adult Mammalian CNS -- Migration and Fate of Transplanted Astrocytes -- Astrocyte Response to Disease -- Morphology of Astroglial Swelling in Culture and in the Edematous Brain: An Adaptive Response to a Disturbed Microenvironment -- Glial Activation as a Common Denominator in Neurodegenerative Disease: A Hypothesis in Neuropathophysiology -- Downs’s Syndrome and S-100 Protein -- Astrocyte/Oligodendrocyte Interaction in Association with Reactive Gliosis -- Astroglial Response to Liver Failure -- Possible Roles for Astroglia and Microglia in the Pathogenesis of Unconventional Slow Infections -- Abstracts -- Contributors.