Rat Brain Cell Cultures Research Articles

Effects of macrophages on myelination, demyelination and remyelination in aggregating rat brain cell cultures

Effects of macrophages on myelination, demyelination and remyelination in aggregating rat brain cell cultures

Brain neurons and glial cells express Neu differentiation factor/heregulin: a survival factor for astrocytes.

Neu differentiation factor (NDF, also called heregulin) was isolated from mesenchymal cells on the basis of its ability to elevate phosphorylation of ErbB proteins. Earlier in situ hybridization analysis showed that NDF was transcribed predominantly in the central nervous system during embryonic development. To gain insights into the role of NDF in brain we analyzed its distribution by immunohistochemistry and in situ hybridization. Late-gestation (day 17) rat embryos displayed high NDF immunoreactivity in both motor (e.g., putamen) and limbic (e.g., septum) regions. Lower levels of the factor were exhibited by adult brain, except for the cerebellum, where NDF expression was increased postnatally. Both neurons and glial cells were identified by immunohistochemistry as NDF-producing cells (e.g., pyramidal neurons in the cerebral cortex and glial cells in the corpus callosum). By establishment of primary cultures of rat brain cells we confirmed that NDF was expressed in neurons as well as in astrocytes. In addition, by using such primary cultures we observed that NDF treatment exerted only a limited mitogenic effect, which was accompanied by significant acceleration of astrocyte maturation. Furthermore, long-term incubation with the factor specifically protected astrocytes from apoptosis, implying that NDF functions in brain as a survival and maturation factor for astrocytes.

Inhibitors of N-linked oligosaccharide processing glucosidases interfere with oligodendrocyte differentiation in culture.

Previous studies have demonstrated that inhibitors of glycoprotein processing glucosidases interfere with the development of oligodendrocyte properties in primary cultures of embryonic rat brain cells (Bhat, J Neurosci Res 20:158-164, 1988). The present study examines the effect of castanospermine, an inhibitor of the processing glucosidases, on the development and differentiation of isolated oligodendrocyte progenitor cells. Treatment of oligodendrocyte progenitors with castanospermine did not affect the developmental progression of the precursors to become committed oligodendrocytes as revealed by comparable increases in the percentages of cells positive for galactocerebroside (a surface marker for terminally differentiated oligodendrocytes) in control and drug-treated cultures. On the other hand, there was an impairment of the expression of differentiated properties of oligodendrocytes [i.e., sulfolipid synthesis, myelin basic protein (MBP)] and 2'3'-cyclic nucleotide 3'-phosphohydrolase in the drug-treated cultures. Immunocytochemical analysis with anti-MBP antibodies revealed a reduced number of MBP-positive cells in inhibitor-treated cultures. Furthermore, a majority of MBP-positive cells in such cultures displayed immunoreactive MBP in their cell body and not the processes, unlike in control cultures where both cell body and the processes of oligodendrocytes stained intensely for MBP. The strong inhibitory effect of castanospermine on the expression of oligodendrocyte-specific activities was contrasted with a relatively smaller effect of swainsonine, a mannosidase inhibitor on oligodendrocyte differentiation. Both castanospermine and swainsonine, however, effectively blocked the formation of complex-type oligosaccharides, suggesting thereby a lack of correlation between the inhibition of the formation of complex-type oligosaccharides and oligodendrocyte differentiation. It is suggested, therefore, that early trimming reactions involving the removal of glucose residues from the high mannose oligosaccharides in the endoplasmic reticulum may be essential for the cell surface localization and function of glycoproteins critically involved in surface interactions of oligodendrocytes with each other and/or with the substratum.

Ethanol‐Induced Increase in Catalase Activity in Reaggregation Cultures of Rat Brain Cells is Due to Increased Oligodendrocyte Differentiation

Recent studies have shown that ethanol exposure of reaggregation cultures of fetal rat brain cells causes an increased activity and amount of catalase, and also an increased activity of the oligodendrocyte marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). In the present study, reaggregation cultures were grown in the presence of 40 mM ethanol during 17 days, corresponding to a period in vivo from gestational day 17 to postnatal day 12. The activities of catalase, the peroxisomal marker enzyme acyl-coenzyme A oxidase and CNP were increased in ethanol-treated cultures. Immunofluorescence experiments confirmed that catalase mainly resided in oligodendrocytes. By immunoperoxidase staining for CNP followed by image analysis, it was observed that the proportion of CNP-positive cells, as well as the intensity of staining, was increased in ethanol-treated cultures. Thus, the previously reported increase in catalase protein and activity in ethanol-treated cultures might be due to a stimulation, or earlier onset, of oligodendrocyte differentiation.

Borrelia burgdorferi and Escherichia coli lipopolysaccharides induce nitric oxide and interleukin-6 production in cultured rat brain cells.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, infects the central nervous system (CNS), but the factors that mediate inflammation and neurologic dysfunction are not known. Sonicated B. burgdorferi stimulated in a concentration-dependent manner the production of nitric oxide (NO) in glial-enriched primary cultures of neonatal rat brain cells via induction of NO synthase activity. Lipopolysaccharide (LPS) of Escherichia coli also stimulated nitrite accumulation in a concentration-dependent manner. Stimulation of NO production by B. burgdorferi sonicate and E. coli LPS was associated with increased levels of mRNA coding for the cytokine-inducible form of NO synthase. B. burgdorferi sonicate also stimulated release of interleukin-6, with a concentration-response relationship similar to that for its stimulation of nitrite production, as did E. coli LPS. A competitive antagonist of E. coli LPS, Rhodopseudomonas sphaeroides lipid A, inhibited LPS-induced stimulation of NO synthase activity but markedly potentiated that of B. burgdorferi, indicating that the initial triggering mechanism of B. burgdorferi is distinct from that of E. coli LPS. Induction of NO synthase by bacterial agents within the brain may represent a common pathway of CNS inflammation and neurotoxicity.

Thyroidal stimulation of tubulin and actin in rat brain cytoskeleton

In cultures of neonatal rat brain cells, labeled with 35S-methionine in the presence or absence of triiodothyronine (T3), the hormone promoted a significant enhancement of labeled tubulin and actin in the insoluble fraction (30,000 g pellet) of cell homogenate. To identify the specific sub-cellular fraction associated with this induction, organ cultures of 1 day rat cerebra were labelled with 35S-methionine in the presence and absence of T3 and the insoluble fraction (30,000 g pellet) was subfractionated into mitochondria, plasma membrane and cytoskeleton. Analysis of the labeled proteins by SDS-PAGE, autoradiography and densitometry revealed a T3-induced increase of 50–80% for both tubulin and actin, only in the cytoskeleton fraction without any significant effect on the other fractions. Similar results were obtained when plasma membrane or cytoskeleton were isolated directly from labeled cerebrum by conventional methods instead of fractionating from the 30,000 g pellet. Analysis of relative stimulation of labeled tubulin and actin by T3 in cytoskeleton fraction derived from primary cultures of neuronal (N) and glial (G) cells labeled with 35-methionine show that the stimulatory effect is predominantly on the N cells. Studies on the kinetics of induction of labeled tubulin and actin by T3 in the cytoskeleton fraction prepared from cerebra labeled with 35S-methionine for 2, 8 and 18 hrs revealed no significant difference at 2 hrs; at 8 hrs, an increased incorporation into both tubulin and actin was reproducibly seen in the controls relative to T3-treated samples. However by 18 hrs, this pattern reversed and an enhanced accumulation of both labeled tubulin and actin was observed under the influence of T3. The mechanism of this apparently intriguing effect of T3 on the kinetics of association of tubulin and actin with the cytoskeleton has been discussed in the light of the dual effect of the hormone on tubulin viz. enhancing its stability as well as rate of synthesis. The overall results indicate that the thyroid hormones play a major role in the cytoskeletal transport of tubulin and actin from their site of synthesis to that of assembly thus facilitating axodendritic outgrowth and morphological differentiation.

Development and differentiation of microglial cells in rat brain cell culture

Development and differentiation of microglial cells in rat brain cell culture

COMT inhibitors and metabolism of fluorodopa enantiomers in aggregating cell cultures.

Organotypic primary cell cultures of fetal rat brain were used as a model system to study the effect of COMT inhibitors on the cerebral metabolic conversions of fluoro-DOPA enantiomers. The selective COMT inhibitors OR 486 and CGP 28014 were used in conjunction with 5F-L-DOPA, 6F-L-DOPA and 6F-D-DOPA as substrates. Methylation can be clearly reduced by application of OR 486 at nanomolar level, without inhibition of AADC and MAO. The uptake of the substrate is unchanged. CGP 28014, already known to be active only in vivo, has no influence on the metabolic conversion rates of the fluoro-DOPA isomers. These results show that use of this culture system allows statement concerning the in vitro activity of COMT inhibitors. It has not been possible to show an increase of absolute levels of decarboxylation products due to inhibition of COMT, however, but the reduction in levels of methylated product itself may have significance for PET studies of the human brain.

Increase in catalase activity in developing rat brain cell reggregation cultures in the presence of ethanol

The aim of this investigation was to study the effects of ethanol on antioxidant enzymes in the developing brain, using reaggregation cultures of fetal rat brain cells as a model. The cultures were grown in the presence of 20 and 40 mM ethanol from day 2 until day 44 of the culture period, corresponding to a period in vivo from gestational day 17 to postnatal day 37. The catalase (EC 1.11.1.6) activity was consistently increased at all observation periods, from culture day 11 to day 44, by both doses of ethanol, and an immunoblot showed that the amount of catalase protein was markedly increased. The activities of manganese and copper-zinc superoxide dismutase (EC 1.15.1.1) and glutathione peroxidase (EC 1.11.1.9) were largely unaffected.

Cystine uptake and glutathione level in fetal brain cells in primary culture and in suspension.

The glutathione level and the factors affecting this level were investigated in fetal rat brain cells in a primary culture. Early in the culture, the glutathione level of the brain cells decreased, but after 5 h it began to increase. This increase was not observed in a cystine-free medium and was prevented by excess glutamate. Cystine was taken up in freshly isolated brain cell suspensions, and its rate increased during the culture. The cystine uptake was mediated by a Na(+)-independent, glutamate-sensitive route previously found in various types of cells and designated as system X-c. The uptake of cystine is a crucial factor in maintaining the glutathione level of the cells under culture, because it provides cysteine for the cells for glutathione synthesis. Cysteine was undetectable in the medium before the culture, but it appeared, though at a very low level, when the brain cells were cultured there. The source of this cysteine was the cystine in the medium. Presumably the decrease in the glutathione level of the cells in the early stage of the culture resulted from the fact that the medium did not contain cysteine. The enhancement of the cystine uptake during culture may constitute a protective mechanism against the oxidative stress to which the cultured cells are exposed. Regulation of the glutathione level in fetal brain cells in vivo by the transport of cystine and cysteine is discussed.

Rapid phosphorylation of phospholipase C gamma 1 by brain-derived neurotrophic factor and neurotrophin-3 in cultures of embryonic rat cortical neurons.

Phospholipase C gamma 1 (PLC-gamma 1) is involved at an early step in signal transduction of many hormones and growth factors and catalyzes the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate to diacylglycerol and inositol trisphosphate, two potent intracellular second messenger molecules. The transformation of PC12 cells into neuron-like cells induced by nerve growth factor is preceded by a rapid stimulation of PLC-gamma 1 phosphorylation and PI hydrolysis. The present study analyzed the effects of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on phosphorylation of PLC-gamma 1 in primary cultures of embryonic rat brain cells. BDNF and NT-3 stimulated the phosphorylation of PLC-gamma 1, followed by hydrolysis of PI. The stimulation of PLC-gamma 1 phosphorylation occurred within 20 s after addition of BDNF or NT-3 and lasted up to 30 min, with a peak after 4 min. ED50 values were similar for BDNF and NT-3, with approximately 25 ng/ml. Phosphorylation of PLC-gamma 1 by BDNF and NT-3 was found in cultures from all major brain areas. K-252b, a compound known to inhibit selectively neutrophin actions by interfering with the phosphorylation of trk-type neutrophin receptors, prevented the BDNF- and NT-3-stimulated phosphorylation of PLC-gamma 1. Receptors of the trk type were coprecipitated with anti-PLC-gamma 1 antibodies. The presence of trkB mRNA in the cultures was substantiated by northern blot analysis. The action of BDNF and NT-3 seems to be neuron specific because no phosphorylation of PLC-gamma 1 was observed in cultures of nonneuronal brain cells. The results provide evidence that developing neurons of the cerebral cortex and other brain areas are responsive to BDNF and NT-3, and they indicate that the transduction mechanism of BDNF and NT-3 in the brain involves rapid phosphorylation of PLC-gamma 1 followed by PI hydrolysis.

Contribution of organic and inorganic osmolytes to volume regulation in rat brain cells in culture

In this work we examined the time course and the amount released, by hyposmolarity, for the most abundant free amino acids (FAA) in rat brain cortex astrocytes and neurons in culture. The aim was to evaluate their contribution to the process of cell volume regulation. Taurine, glutamate, and D-aspartate in the two types of cells, beta-alanine in astrocytes and GABA in neurons were promptly released by hyposmolarity, reaching a maximum within 1-2 min. after an osmolarity change. A substantial amount of the intracellular pool of these amino acids was mobilized in response to hyposmolarity. The amount released in media with osmolarity reduced from 300 mOsm to 150 mOsm or 210 mOsm, represented 50%-65% and 13%-31%, respectively, of the total amino acid content in cells. In both astrocytes and neurons, the efflux of glutamine and alanine was higher under isosmotic conditions and increased only marginally during hyposmotic conditions. 86Rb+, used as tracer for K+, was released from astrocytes, 30% and 11%, respectively, in hyposmotic media of 150 mOsm or 210 mOsm but was not transported in neurons. From these results it was calculated that FAA contribute 54% and inorganic ions 46% to the process of volume regulation in astrocytes exposed to a 150 mOsm hyposmotic medium. This contribution was 55% for FAA and 45% for K+ and Cl- in cells exposed to 210 mOsm hyposmotic solutions. These results indicate that the contribution of FAA to the process of cell volume regulation is higher in astrocytes than in other cell types including renal and blood cells.

Oligodendrocyte differentiation and progenitor cell proliferation are independently regulated by cyclic AMP.

Oligodendrocytes, the glial cells specialized to synthesize myelin in the central nervous system, differentiate in primary rat brain cell cultures on a schedule similar to that observed in vivo. The schedule of oligodendrocyte differentiation and the rate of oligodendroglial progenitor cell proliferation in vitro are both modulated by 3',5'-cyclic AMP (cAMP). A 24-hour exposure to 1 mM N6,2'O-dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) induced a wave of oligodendrocyte differentiation but inhibited proliferation of oligodendroglial progenitors, and reduced by 30-fold the proliferation of progenitors in response to platelet-derived growth factor (PDGF). When cells were grown in the presence of maximally stimulating concentrations of PDGF, the inhibitory effect of cAMP on progenitor cell proliferation was abolished while the stimulatory effect of cAMP on oligodendrocyte differentiation remained, demonstrating that these two cAMP-regulated events are independent.

Effects of nerve growth factor on X-irradiated reaggregation cultures of rat brain cells.

The effects of exogenously added nerve growth factor (NGF) on reaggregation cultures of foetal rat brain cells after X-irradiation with 2 Gy were studied. Irradiation caused decreased protein and DNA levels, which was not prevented by NGF. The activities of the cholinergic marker enzymes choline acetyl transferase and acetylcholine esterase were increased in irradiated cultures. However, no difference in the activities of these enzymes was found between irradiated and unirradiated NGF-treated cultures. Irradiation did not affect the activity of the marker enzyme for oligodendrocytes (2',3'-cyclic nucleotide 3'-phosphodiesterase), but caused an increase in the astrocyte marker (glutamine synthetase) activity. This effect on astrocytes was prevented by NGF.

Embryonic and fetal development: Fundamental reseatch

Much progress has been made over the past decades in the development of in vitro techniques for the assessment of chemically induced effects in embryonic and fetal development. In vitro assays have originally been developed to provide information on the mechanism of action of normal development, and have hence more adequately been used in fundamental research. These assays had to undergo extensive modification to be used in developmental toxicity testing. The present paper focuses on the rat whole embryo culture system, but also reviews modifications that were undertaken for the in vitro chick embryo system and the aggregate cultures of fetal rat brain cells. Today these tests cannot replace the existing in vivo developmental toxicity tests. They can, however, be used to screen chemicals for further development or further testing. In addition, these in vitro tests provide valuable information on the mechanisms of developmental toxicity and help to understand the relevancy of findings for humans. In vitro systems, combined with selected in vivo testing and pharmacokinetic investigations in animals and humans, can thus provide essential information for human risk assessment.

Stimulation of phosphatidylinositol hydrolysis by brain-derived neurotrophic factor and neurotrophin-3 in rat cerebral cortical neurons developing in culture.

Phosphatidylinositol (PI) breakdown represents a powerful system participating in the transduction mechanism of some neurotransmitters and growth factors and producing two second messengers, diacylglycerol and inositol trisphosphate. The transformation of PC12 neuroblastoma cells into neuron-like cells induced by nerve growth factor (NGF) is preceded by a rapid stimulation of PI breakdown; however, it was not known whether PI breakdown mediates actions of other members of the neurotrophin family. The present study analyzed the effects of NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) on PI breakdown in primary cultures of embryonic rat brain cells. Cultures were grown for 7 days; PI was then labeled by incubating cultures with myo-[3H]inositol, which then were exposed acutely to growth factors. BDNF and NT-3, but not NGF, elevated the levels of labeled inositol phosphates within 10-15 min after addition to the cultures in a dose-dependent manner. ED50 values for BDNF and NT-3 were 12.4 and 64.5 ng/ml, respectively. Comparable effects were found in cultures of cortical, striatal, and septal cells. The actions of BDNF and NT-3 probably reflect actions on neurons, because no effects were seen in cultures of nonneuronal cells. In contrast, basic fibroblast growth factor induced a marked stimulation of PI breakdown in cultures of nonneuronal cells. K252b, which selectively blocks neurotrophin actions by inhibiting trk-type receptor proteins, prevented the PI breakdown mediated by BDNF and NT-3. The findings suggest that rapid and specific induction of PI breakdown is involved in the signal transduction of BDNF and NT-3, and they provide evidence that cortical neurons are functionally responsive to BDNF and NT-3 during development.

Neurotrophin-induced trk receptor phosphorylation and cholinergic neuron response in primary cultures of embryonic rat brain neurons.

Tyrosine phosphorylation of trk type neurotrophin receptors in primary cultures of embryonic rat brain cells was studied by immunoprecipitation and immunoblotting. In cultures containing basal forebrain cholinergic neurons, but not in cultures of cerebral cortex, nerve growth factor (NGF) treatment for 4 min induced tyrosine phosphorylation of trk family proteins. Stimulation with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), resulted in a very robust phosphorylation signal in basal forebrain and cortical cultures, suggesting actions of these neurotrophins not only on cholinergic cells but probably on most embryonic brain neurons. Trk tyrosine phosphorylation was completely abolished by 5 microM K-252b. Inhibition was rapid, being evident by 30 s following addition of the drug. Corresponding stimulatory and inhibitory effects were seen for phospholipase-C gamma 1 (PLC gamma 1) and extracellular signal-regulated kinase 1 (Erk1), two enzymes involved in second messenger mechanisms. Our findings indicate involvement of trk receptor activation in the NGF response of basal forebrain cholinergic cells and provide evidence for widespread presence of BDNF and NT-3 responsive neurons in the embryonic brain.

Myelin gene expression during demyelination and remyelination in aggregating brain cell cultures

Remyelination can be studied in aggregating rat brain cell cultures after limited demyelination. Demyelination was induced using a monoclonal antibody against myelin/oligodendrocyte glycoprotein (MOG mAb), in the presence of complement. De- and remyelination were assessed by measuring myelin basic protein (MBP). Two days after removing the MOG mAb, MBP levels reached 50% of controls and after 7 days 93%. During this period, cell proliferation determined by [ 14C]thymidine incorporation was similar in remyelinating and control cultures. Hormones and growth factors were tested for possible stimulatory effect on remyelinating cultures. Bovine growth hormone (bGH), triiodothyronine (T 3), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) did not improve remyelination. Only epidermal growth factor (EGF) increased the level of remyelination. PDGF increased the rate of cell proliferation in both control and remyelinating cultures. A significant proportion of oligodendrocytes entered the cell division cycle and were not available for remyelination. The results obtained with PDGF and FGF (inhibition) support the idea that a pool of progenitor cells was still present and able to proliferate and differentiate into myelinating oligodendrocytes. The levels of myelin protein mRNAs were investigated during de- and remyelination. During demyelination, myelin protein mRNA levels decreased to approximately 50% of control cultures and returned to normal during remyelination. These preliminary results indicate that normal levels of gene transcription are sufficient to meet the increased need for newly synthesized myelin proteins during remyelination.

Metabolism of 5-fluoro-DOPA and 6-fluoro-DOPA enantiomers in aggregating cell cultures of fetal rat brain

The cerebral metabolism of enantiomers of 5-fluoro-DOPA (5F-DOPA) and 6-fluoro-DOPA (6F-DOPA) was characterized in organotypic cell cultures of fetal rat brain. This system permits the investigation of metabolic processes in brain tissue exclusively, without the effects of peripheral metabolism and transport. Metabolic profiles for each substrate were determined in comparison with those of l-DOPA and d-DOPA. The uptake of DOPA and fluoro-DOPA in aggregating brain cell cultures is strongly preferential for l-enantiomers. Decarboxylation by aromatic l-amino acid decarboxylase is an active step: the major products are dopamine (DA) or 6F-DA and their corresponding products of oxidative deamination, i.e. dihydroxyphenylacetic acid (DOPAC) or 6F-DOPAC, respectively. Decarboxylation products of d-enantiomers occur in lower amounts, and 5F- d-DOPA is not decarboxylated. However, 5F-DOPA is O-methylated to a great extent, and levels of 3-O-methyl-5F-DOPA are higher after incubation with 5F- d-DOPA than with 5F- l-DOPA. These data may serve as a support for more detailed modeling of [ 18F]F-DOPA metabolism than can be applied to the evaluation of the cerebral biochemistry of the DA system with positron emission tomography in vivo

Glutamate differentially inhibits the expression of class II MHC antigens on astrocytes and microglia.

Abstract MHC molecules are required for Ag recognition by T cells. Inasmuch as cells in the central nervous system do not express MHC constitutively, appearance of MHC, in inflammatory and degenerative diseases of the brain, may indicate local Ag presentation and subsequent immune response. Although both astrocytes and microglia are capable of class II MHC expression in vitro, in vivo studies failed to show the presence of significant amounts of class II on astrocytes compared to microglia. Our study is designed to clarify possible regulatory mechanisms that can explain the differences in inducibility of class II MHC between astrocytes and microglia in vivo. Using dissociated rat brain cell cultures, we have found that glutamate, an excitatory neurotransmitter, exerted a profound inhibitory effect on IFN-gamma-induced expression of class II on astrocytes, but not on microglia. Both glutamate and norepinephrine, a neurotransmitter previously reported to down-regulate class II on astrocytes, inhibited the induction of class II on astrocytes by eliminating accumulation of class II MHC mRNA. The kinetics of class II mRNA induction by IFN-gamma in the presence of glutamate suggested that glutamate may act as a transcriptional inhibitor. It is likely that class II induction on astrocytes in vivo may be selectively down-regulated by neurotransmitters such as glutamate and norepinephrine.