Cerebral Cortical Cells Research Articles

Role of glutathione in neuroprotective effects of mood stabilizing drugs lithium and valproate

Mood stabilizing drugs lithium and valproate are the most commonly used treatments for bipolar disorder. Previous studies in our laboratory indicate that chronic treatment with lithium and valproate inhibits oxidative damage in primary cultured rat cerebral cortical cells. Glutathione, as the major antioxidant in the brain, plays a key role in defending against oxidative damage. The purpose of this study was to determine the role of glutathione in the neuroprotective effects of lithium and valproate against oxidative damage. We found that chronic treatment with lithium and valproate inhibited reactive oxygen metabolite H 2O 2-induced cell death in primary cultured rat cerebral cortical cells, while buthionine sulfoximine, an inhibitor of glutathione rate-limiting synthesis enzyme glutamate-cysteine ligase, reduced the neuroprotective effect of lithium and valproate against H 2O 2-induced cell death. Further, we found that chronic treatment with lithium and valproate increased glutathione levels in primary cultured rat cerebral cortical cells and that the effects of lithium and valproate on glutathione levels were dose-dependent in human neuroblastoma SH-SY5Y cells. Chronic treatment with lithium and valproate also increased the expression of glutamate-cysteine ligase in both rat cerebral cortical cells and SH-SY5Y cells. In addition, chronic treatment with other mood stabilizing drugs lamotrigine and carbamazepine, but not antidepressants desipramine and fluoxetine, increased both glutathione levels and the expression of glutamate-cysteine ligase in SH-SY5Y cells. These results suggest that glutathione plays an important role in the neuroprotective effects of lithium and valproate, and that glutathione may be a common target for mood stabilizing drugs.

Estradiol stimulates progenitor cell division in the ventricular and subventricular zones of the embryonic neocortex

Two distinct populations of cerebral cortical progenitor cells that generate neurons during embryogenesis have been identified: radial glial cells and intermediate progenitor cells. Despite advances in our understanding of progenitor cell populations, we know relatively little about factors that regulate their proliferative behaviour. 17-beta-Estradiol (E2) is present in the adult and developing mammalian brain, and plays an important role in central nervous system processes such as neuronal differentiation, survival and plasticity. E2 also stimulates neurogenesis in the adult dentate gyrus. We examined the role of E2 during embryonic cortical neurogenesis through immunohistochemistry, in situ hybridization, functional enzyme assay, organotypic culture and in utero administration of estradiol-blocking agents in mice. We show that aromatase, the E2 synthesizing enzyme, is present in the embryonic neocortex, that estrogen receptor-alpha is present in progenitor cells during cortical neurogenesis, that in vitro E2 administration rapidly promotes proliferation, and that in utero blockade of estrogen receptors decreases proliferation of embryonic cortical progenitor cells. Furthermore, the E2 inhibitor alpha-fetoprotein is expressed at high levels by radial glial cells but at lower levels by intermediate progenitor cells, suggesting that E2 differentially influences the proliferation of these cortical progenitor cell types. These findings demonstrate a new functional role for E2 as a proliferative agent during critical stages of cerebral cortex development.

Effects of streptokinase on the development of rat cerebral cortical cells in vitro

The aim of the present work was to study the effects of streptokinase (SK) on the ultrastructure of the cellular elements of the cerebral cortex of neonatal rats in vitro. Three series of cultures were used: cultures maintained in DMEM enriched with 15% fetal calf serum (control 1), some transferred to minimal nutritive medium containing only 0.5% serum (control 2), and some supplemented with SK (2000 MU/ml) (experimental). Addition of SK to the nutritive medium prevented a decrease in the viability of cells in a mature (14 days) dissociated culture from the neocortex of rat pups aged 1-2 days induced by transfer of the cultures to medium lacking serum proteins. The action of SK on 7-day cultures enhanced the loss of cell viability. Electron microscopy studies of organotypic cultures showed that at this concentration, SK prevented the development of destructive changes of astrocytes, oligodendrocytes, and neurons induced in explants by serum protein deficiency. Neurons showed an abundance of mitochondria, some of which had few cristae. Signs of destruction affected only the nuclei of neurons. After exposure to SK for 48 h, oligodendrocytes were activated (they contained an abundance of myelin-like bodies), with degradation of most astrocytes (surviving examples showing nuclear hyperchromicity). Neurons were resistant to these effects.

Increased expression of HIF-1α, nNOS, and VEGF in the cerebral cortex of anemic rats

This study tested the hypothesis that specific hypoxic molecules, including hypoxia-inducible factor-1alpha (HIF-1alpha), neuronal nitric oxide synthase (nNOS), and vascular endothelial growth factor (VEGF), are upregulated within the cerebral cortex of acutely anemic rats. Isoflurane-anesthetized rats underwent acute hemodilution by exchanging 50% of their blood volume with pentastarch. Following hemodilution, mean arterial pressure and arterial Pa(O(2)) values did not differ between control and anemic rats while the hemoglobin concentration decreased to 57 +/- 2 g/l. In anemic rats, cerebral cortical HIF-1alpha protein levels were increased, relative to controls (1.7 +/- 0.5-fold, P < 0.05). This increase was associated with an increase in mRNA levels for VEGF, erythropoietin, CXCR4, iNOS, and nNOS (P < 0.05 for all), but not endothelial NOS. Cerebral cortical nNOS and VEGF protein levels were increased in anemic rats, relative to controls (2.0 +/- 0.2- and 1.5 +/- 0.4-fold, respectively, P < 0.05 for both). Immunohistochemistry demonstrated increased HIF-1alpha and VEGF staining in perivascular regions of the anemic cerebral cortex and an increase in the number of nNOS-positive cerebral cortical cells (3.2 +/- 1.0-fold, P < 0.001). The nNOS-positive cells costained with the neuronal marker, Neu-N, but not with the astrocytic marker glial fibrillary acidic protein (GFAP). These nNOS-positive neurons frequently sent axonal projections toward cerebral blood vessels. Conversely, VEGF immunostaining colocalized with both neuronal (NeuN) and astrocytic markers (GFAP). In conclusion, acute normotensive, normoxemic hemodilution increased the levels of HIF-1alpha protein and mRNA for HIF-1-responsive molecules. nNOS and VEGF protein levels were also increased within the cerebral cortex of anemic rats at clinically relevant hemoglobin concentrations.

Differences in the effect on neural stem cells of fetal bovine serum in substrate-coated and soluble form

The influence of fetal bovine serum (FBS) adsorbed to poly(ethylene-co-vinyl alcohol) (EVAL) and polyvinyl alcohol (PVA) substrates (coated FBS) and FBS present in the culture medium (soluble FBS) on the behavior of embryonic rat cerebral cortical neural stem cells was studied at neurosphere level. When both coated FBS and soluble FBS were not present in the culture system, the fate and behavior of neurospheres were mediated mainly by the substrates used. When neurospheres were cultured either on FBS-coated EVAL or FBS-coated PVA substrates in the serum-free medium, the most striking morphological characteristic of neurospheres was that these neurosphere-forming cells attached and were induced to differentiate into process-bearing cell phenotypes predominantly; however, the differentiated cell phenotypes were dissimilar on these two substrates. On the contrary, when neurospheres were cultured in the medium containing 10% FBS, the neurosphere-forming cells were induced into protoplasmic cells typically but no difference in differentiated cell phenotypes on EVAL and PVA substrates was observed. Interestingly, instead of promoting process outgrowth under serum-free medium condition, coated FBS enhanced migration of differentiated protoplasmic cells when soluble FBS were present. These results inform that the substrates, coated serum, and soluble serum within the culture environment together can significantly alter cell behavior and morphological differentiation and will therefore be an important clue for the development of biomaterials to regulate the potential of the CNS neural stem cells.

The effect of chitosan and PVDF substrates on the behavior of embryonic rat cerebral cortical stem cells

In this study, the behavior of neural stem cells from embryonic rat cerebral cortex were compared on the chitosan and poly(vinylidene fluoride) (PVDF) substrates at single-cell and neurosphere level. It was found that chitosan and PVDF substrates inhibited the proliferation and differentiation of single neural stem cells. It seemed that single-cell cultures on both substrates show cells remained dormant. However, neurospheres could exhibit different or similar behavior on these two substrates, which is dependent on the presence or absence of serum. More cells migrated outside from the neurospheres and longer processes extended from differentiated cells on chitosan than on PVDF when neurospheres were cultured in the serum-free medium. On the contrary, when serum was added to the culture system, chitosan and PVDF could induce the neurosphere-forming cells into an extensive cellular substratum of protoplasmic cells upon which process-bearing cells spread. In addition, based on the immunocytochemical analysis, the percentages of differentiated cell phenotypes of neurospheres cultured on chitosan and PVDF substrates became similar in the presence of serum. Therefore, it is reasonable to suggest that biomaterials may stimulate or inhibit the proliferation and differentiation of neural stem cells according to the complex environmental conditions. The information presented here should be useful for the development of biomaterials to regulate the preservation, proliferation, and differentiation of neural stem cells.

Proliferation and differentiation of neural stem cells on lysine–alanine sequential polymer substrates

The purpose of this study was to explore the phenotypic potential of embryonic rat cerebral cortical stem cells by inducing differentiation on lysine–alanine sequential (LAS) polymer substrates at neurosphere level. LAS polymer is a heterologous polymer of lysine and alanine and has been demonstrated to enhance axon growth of neurons in a serum-free medium in vitro. It was found that very few cells migrated outside of the neurospheres but extremely long processes extended from differentiated cells could form a network between remote neurospheres when cells were cultured on LAS substrates at a low density of 120 neurospheres/cm 2 in the serum-free medium. On the contrary, when the neurosphere density was increased to 360 neurospheres/cm 2, many neurosphere-forming cells migrated out from their original aggregate and exhibited short processes morphology. Furthermore, when serum was added to the culture system, the neurosphere-forming cells could be induced into an extensive cellular substratum of protoplasmic cells upon which process-bearing cells spread. Clearly, neurospheres could exhibit different behaviors on LAS substrates according to the complex environmental conditions. Here, we proposed that neurospheres would change their social communication and adopt different strategies to communicate with other neurospheres when they detected each other's presence. Therefore, the mediation of cell behavior on LAS substrates by communication between neurospheres should be taken into account.

Nicotine blocks ethanol-induced apoptosis in primary cultures of rat cerebral cortical and cerebellar granule cells.

Nicotine's counteraction of adverse effects of ethanol on cognitive function and motor coordination may play a major role in the observed high incidence of smoking among alcoholics. Previously, we have observed protective effects of nicotine against ethanol-induced neurotoxicity in cultured cortical and cerebellar granule cells as determined by lactate dehydrogenase assay. Ethanol-induced apoptosis may be a contributory mechanism to its neuronal toxicity. In this study we sought to determine whether ethanol induces formation of caspase 3 (reflective of apoptosis) in these cells and whether these effects may be blocked by nicotine pretreatment. Primary cultures of cerebral cortical and cerebellar granule cells were prepared from the brains of 20 day old Sprague-Dawley fetuses. Exposure of cells to ethanol (10-100 mM) for 3 days resulted in a dose-dependent increase in caspase 3 activity and cytotoxicity. Pretreatment with nicotine (5-20 microM) dose dependently attenuated these effects of ethanol. Complete block of ethanol effects was achieved by the highest dose of nicotine (20 microM). Nicotine, at concentrations administered, did not affect caspase activity or neuronal viability. These results suggest that at least some of the neurotoxic effects of ethanol may be mediated by apoptosis and that pretreatment with nicotine can prevent these effects of ethanol. Anti-apoptotic effects of nicotine in this model may be suggestive of potential use of nicotinic agonists in neurotoxic insults and/or neurodegenerative disorders.

Mood stabilizing drug lithium increases expression of endoplasmic reticulum stress proteins in primary cultured rat cerebral cortical cells

The mood stabilizing drug lithium is a highly effective treatment for bipolar disorder. Previous studies in our laboratory found that chronic treatment with the mood stabilizing drug valproate in rat brain increased the expression of endoplasmic reticulum (ER) stress proteins GRP78, GRP94 and calreticulin. We report here that in primary cultured rat cerebral cortical cells, expression of GRP78, GRP94 and calreticulin are increased not only by valproate, but also by lithium after chronic treatment for 1 week at therapeutically relevant concentrations. However, two other mood stabilizing drugs carbamazepine and lamotrigine had no effect on expression of GRP78, GRP94 or calreticulin. Chronic treatment with lithium for 1 week increased both mRNA and protein levels of ER stress proteins. In contrast to a classic GRP78 inducer thapsigargin, an inhibitor of the ER Ca 2+-ATPase, chronic treatment with lithium or valproate for 1 week modestly increased GRP78 expression in neuronal cells, had no effect on basal intracellular free Ca 2+ concentration and does not induce cell death. These results indicate that lithium and valproate may increase expression of GRP78, GRP94 and calreticulin in primary cultured rat cerebral cortical cells without causing cell damage. These results also suggest that the mechanism of GRP78 increase induced by lithium and valproate may be different from that of thapsigargin.

Chronic Treatment with Mood Stabilizers Lithium and Valproate Prevents Excitotoxicity by Inhibiting Oxidative Stress in Rat Cerebral Cortical Cells

Recent studies indicate that chronic treatment with the mood-stabilizing drugs lithium and valproate produces a neuroprotective effect against excitotoxicity. In this study, we aimed to determine whether inhibiting oxidative damage plays a role in a neuroprotective effect of lithium and valproate against excitotoxicity. Intracellular free calcium concentration was measured with the fluorescent calcium ion indicator fluo-3. Malondialdehyde, an end product derived from peroxidation of polyunsaturated fatty acid, and protein carbonyls were used to assess oxidative damage to lipid and protein. Excitotoxicity was assayed by measuring cell viability with the MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] method and by measuring deoxyribonucleic acid (DNA) fragmentation with TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) staining. We found that chronic treatment with lithium and valproate at their therapeutically relevant concentrations significantly inhibited the glutamate-induced increase of intracellular free calcium concentration, lipid peroxidation, protein oxidation, DNA fragmentation, and cell death in primary cultured rat cerebral cortical cells. This treatment had no effect on basal intracellular free calcium concentration, lipid peroxidation, protein oxidation, DNA fragmentation, and cell death. Our results suggest that chronic treatment with lithium and valproate inhibits oxidative damage to lipid and protein and in turn produces a neuroprotective effect against excitotoxicity.

Moderate lead exposure elicits neurotrophic effects in cerebral cortical precursor cells in culture

Lead (Pb) persists as an environmental toxicant despite aggressive environmental and occupational regulation. Neurotoxicological effects of acute Pb poisoning range from subtle cognitive deficits, to clumsiness and ataxia, to coma and seizures. In adult neurotoxicity, reductions of blood Pb levels are often associated with reversal of clinical signs. In children, however, the effects are more likely to endure, with even low levels of chronic Pb exposure correlating with decreasing IQ. These persistent effects likely result from neurodevelopmental insults, such as altered cell survival or maturation, although the mechanisms have not been fully defined. In the present study we define the effects of moderate-level Pb exposure on mammalian neurogenesis using a well-characterized cortical precursor model. Gestational day 14.5 rat cerebral cortical precursor cells were cultured in defined media and cell number, precursor proliferation, apoptosis, and neuritic process outgrowth were assessed following exposure to a range of Pb acetate concentrations. Surprisingly, whereas a concentration of 30 microg/ml Pb acetate was acutely toxic to neurons, concentrations between 1 and 10 microg/ml Pb acetate (approximately 3 microM and 30 microM Pb, respectively) increased cell number: 10 times as many cells exposed to 10 microg/ml Pb were present on day 4 as compared to control. The increase in cell number was not a result of increased proliferation, however, as DNA synthesis did not increase. Rather, Pb exposure maintained the survival of cortical precursors, as the progressive apoptosis occurring under control conditions was markedly reduced by the metal. Additionally, neuritic process initiation and outgrowth increased in a concentration-dependent manner, with processes four times as abundant on day 1 and twice as long on day 2. These results suggest that brief exposure to lead during neurogenesis directly affects cell survival and process development, potentially altering cortical arrangement. Consequently, alterations in neural circuitry may underlie some of the neurological effects of Pb exposure during brain development.

Behavior of embryonic rat cerebral cortical stem cells on the PVA and EVAL substrates

Cell behavior is determined by intrinsic programs and complex interactions among cells, medium components and substrates. Several previous reports have demonstrated the usefulness of extrinsic signals coming from soluble growth factors and cell–cell contact for regulating the proliferation and differentiation of neural stem cells. At present, the effects of substrate on neural stem cells are not known. In this study, the behavior of neural stem cells, isolated from embryonic rat cerebral cortex, was observed and compared on the polyvinyl alcohol (PVA) and poly (ethylene- co-vinyl alcohol) (EVAL) substrates in the presence of the mitogenic effect of basic fibroblast growth factor (bFGF) in the serum-free medium. It was found that PVA and EVAL exerted different influences on the fate of neural stem cells. The behavior of neural stem cells on the EVAL was independent of cell density at the single-cell level. Single neural stem cells seemed to remain dormant on the EVAL. Conversely, the development of cell clusters, termed neurospheres, was in a density-dependent manner on the EVAL. Neurospheres continuously proliferated under high-density culture condition, but differentiated into neurons and astrocytes under low-density culture condition. However, regardless of single cells or neurospheres, cultured cells could not survive on the PVA. Therefore, it is reasonable to assume that biomaterials may stimulate or inhibit the proliferation and differentiation of neural stem cells. These in vitro results are very encouraging since this information should be useful for the development of strategies for regulating the preservation, proliferation and differentiation of neural stem cells.

Fabrication and biocompatibility of polypyrrole implants suitable for neural prosthetics

Finding a conductive substrate that promotes neural interactions is an essential step for advancing neural interfaces. The biocompatibility and conductive properties of polypyrrole (PPy) make it an attractive substrate for neural scaffolds, electrodes, and devices. Stand-alone polymer implants also provide the additional advantages of flexibility and biodegradability. To examine PPy biocompatibility, dissociated primary cerebral cortical cells were cultured on PPy samples that had been doped with polystyrene-sulfonate (PSS) or sodium dodecylbenzenesulfonate (NaDBS). Various conditions were used for electrodeposition to produce different surface properties. Neural networks grew on all of the PPy surfaces. PPy implants, consisting of the same dopants and conditions, were surgically implanted in the cerebral cortex of the rat. The results were compared to stab wounds and Teflon implants of the same size. Quantification of the intensity and extent of gliosis at 3- and 6-week time points demonstrated that all versions of PPy were at least as biocompatible as Teflon and in fact performed better in most cases. In all of the PPy implant cases, neurons and glial cells enveloped the implant. In several cases, neural tissue was present in the lumen of the implants, allowing contact of the brain parenchyma through the implants.

D1 dopamine receptor regulation of the levels of the cell-cycle-controlling proteins, cyclin D, P27 and Raf-1, in cerebral cortical precursor cells is mediated through cAMP-independent pathways.

Previously, we demonstrated that dopamine D1 receptor (D1R) agonists inhibit epidermal growth factor (EGF)-induced passage of mouse fetal cerebral cortical precursor cells from the G1 phase to the S phase of the cell cycle. Here, we report that this action of D1R agonists may involve regulation of cyclin D, and P27, which respectively promote and suppress the G1 to S transition. Furthermore, regulation of Raf-1, a component of the receptor tyrosine kinase mitogen-activated protein kinase pathway engaged in the mitogenic activity of EGF, may also be involved. Specifically, levels of cyclin D and Raf-1 decrease, whereas those of P27 first increase and then decrease in a dose-dependent fashion in response to the D1R agonist, SKF38393. This agonist also promotes Raf-1 phosphorylation on serine 338 residue, suggesting increased activation of this protein. Only the latter effect can be blocked by adenylyl cyclase (AC) and cAMP-dependent protein kinase A (PKA) inhibitors, and mimicked by agonists of the cAMP signaling pathway. Another D1R agonist, SKF83959, which stimulates phospholipase Cbeta (PLCbeta) but not AC, reduces levels of Raf-1 and cyclin D similar to SKF38393. However, we detected only down-regulation of P27 by this agonist. Additionally, the concentration-dependent patterns of both SKF38393- and SKF83959-induced alterations in the levels of P27 closely resemble the effects of these ligands on the levels of the D1R-PLCbeta-associated second-messenger cascades linker, calcyon. These findings suggest that D1R-induced suppression of the cell cycle progression in EGF-supported fetal cortical precursor cells represents a net effect of competing cell cycle promoting and inhibiting molecular changes, which involve cyclin D, P27 and Raf-1. The data also show that cAMP second messenger cascade is not engaged in the D1R-induced regulation of the levels of these three proteins. Such regulation probably involves PLCbeta-associated pathways.

Glutathione S‐transferase is a novel target for mood stabilizing drugs in primary cultured neurons

Oligonucleotide microarray technology was used to analyze gene expression profiles after chronic treatment with the mood stabilizing drug valproate at a therapeutically relevant concentration in primary cultured rat cerebral cortical cells. We discovered that valproate regulates expression of 28 genes, including three isoenzymes (M1, A3 and A4) of glutathione S-transferase (GST), an important protective factor against oxidative stress. Because previous studies in our laboratory found that chronic valproate treatment protected cultured neurons against oxidative stress, further experiments on the regulation of GST were performed. Regulation of GST M1, GST A3 and GST A4 was verified using northern blotting hybridization. Chronic valproate treatment increased mRNA levels of M1 and A4, but decreased the A3 mRNA level dose-dependently, indicating further complexities in the regulation of GST by valproate. The level of GST M1 protein and GST activity were also increased by chronic valproate treatment. In addition, chronic treatment with lithium, another commonly prescribed mood stabilizer, also increased levels of GST M1 mRNA and protein. The present findings suggest that regulation of GST M1, and possibly GST A4, may mediate the anti-oxidative effects of valproate treatment, and regulation of GST may be involved in the mood stabilizing effect of valproate and lithium.

Primary culture of cortical neurons, type-1 astrocytes, and microglial cells from cynomolgus monkey ( Macaca fascicularis) fetuses

We established selective primary cultures of neurons, astrocytes, and microglial cells from cryopreserved fetal cerebral cortex of cynomolgus monkeys ( Macaca fascicularis). At 14 days in serum-containing medium, the cell cultures of the fetal cerebral cortex consisted primarily of neurons, astrocytes, and floating microglial cells. At 21 days, we observed a small number of myelin basic protein (MBP)-positive oligodendrocytes. The addition of cytosine arabinoside (a selective DNA synthesis inhibitor) at 2 days in culture eliminated proliferative glial cells, allowing adequate numbers of neurons to survive selectively. A chemically defined serum-free medium successfully supported neuronal survival at a level equivalent to that supported by the serum-containing medium. Brain-derived neurotrophic factor (BDNF) significantly affected the survival of primate neurons. Glutamate induced a significant degree of neuronal cell death against primate neurons and MK-801, a selective N-methyl- d-aspartate receptor (NMDAR) antagonist, blocked cell death, which suggests that primate cortical neurons have NMDAR and the glutamate-induced cell toxicity is mediated by NMDAR. In the serum-free medium, type-1 astrocytes responded to dibutyryl cyclic AMP and showed a process-bearing morphology. The growth of type-1 astrocytes in the serum-free medium was stimulated by epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and hydrocortisone, which are known growth factors in rat type-1 astrocytes. Cultured microglial cells expressed CD68, a monocyte marker. Macrophage–colony stimulating factor (M–CSF) stimulated microglial cell growth in the serum-free medium. These selective primary culture systems of primate cerebral cortical cells will be useful in issues involving species specificity in neuroscience.

P2Y receptor regulation of cultured rat cerebral cortical cells: calcium responses and mRNA expression in neurons and glia.

1 We have investigated increases in cytosolic Ca(2+) in response to nucleotides in mixed rat cerebrocortical cultures (neurons and glia in similar numbers) and in essentially neuron-free glial cultures. 2 In both cultures, the agonist-response profile was 2-methylthioADP(2MeSADP)>2-methylthioATP(2MeSATP)>ADP>ATP>adenosine 5'-O-(3-thiotriphosphate), consistent with a P2Y(1) receptor. The maximal responses to 2MeSADP, 2MeSATP and ADP were identical, but that to ATP was higher. 3 Suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, reactive blue 2 (RB2), and adenosine biphosphate (A3P5P) were antagonists with apparent pA(2) values of 5.5 for suramin, 6.4 for RB2, and 4.7 for A3P5P. 4 Single cell imaging divided the cells from the mixed neuronal-glial cultures into two populations: responsive (neurons) and unresponsive (glial cells) to high [K(+)]. The response of cells to nucleotides was almost exclusively limited to those not responsive to high K(+). 5 In the presence of extracellular Mn(2+), the response of the mixed cultures to 30 mM K(+) and 20 micro M Bay K 8644 was attenuated. However, when 2MeSADP was added there was no reduction in response in cultures previously loaded with Mn(2+). This further indicated that the 2MeSADP response was not in the neurons. 6 Reverse transcriptase-polymerase chain reaction studies detected transcripts for P2Y(1), P2Y(4) and P2Y(6) in RNA preparations from embryonic rat cortex, and from both mixed and glial cultures. P2Y(2) transcripts were not detected in the embryonic cortex. 7 Based on this and previous work, it is proposed that the principal P2Y influences in the brain are on cytosolic Ca(2+) in glial cells and presynaptic sites on neurons.

Growth hormone action on proliferation and differentiation of cerebral cortical cells from fetal rat.

To define the role of GH during central nervous system development, we performed studies in cultured rat cerebral cortical cells from 14- (E14) and 17-d-old embryos (E17). The expression of GH receptor, IGF-I receptor, and IGF-I mRNAs was confirmed. In E17, GH increased total cell number (3.9-fold), [(3)H]-thymidine incorporation (3.5-fold), proliferating cell nuclear antigen levels (2.5-fold), and bromodeoxyuridine (BrdU)-positive cells (2.5-fold). GH action on nestin/BrdU-positive cells was increased in E14 cells at 3 d in vitro (80-fold) but not at 7 d in vitro. In E14 cells, GH increased (9.5-fold) beta-tubulin/BrdU cells. In E17 cells, GH induced neuronal differentiation, as indicated by the absence of beta-tubulin/BrdU-positive cells and the 5.9-fold increment of beta-tubulin protein, and increased glial fibrillary acidic protein/BrdU-positive cells (2.5-fold) and glial fibrillary acidic protein expression (4.5-fold). GH-induced proliferation and differentiation was blocked by IGF-I antiserum. GH increased IGF-binding protein-3 (IGFBP-3), IGF-I receptor protein and its phosphorylation. This study shows that GH promotes proliferation of neural precursors, neurogenesis, and gliogenesis during brain development. These responses are mediated by locally produced IGF-I. GH-induced IGFBP-3 may also have a role in these responses. Therefore, GH is able to activate the IGF-I/IGFBP-3 system in these cerebral cells and induce a physiological action of IGF-I.

3P-0640 Examination of smooth muscle cell origin in human intimal hyperplasia: A study on bone marrow-transplanted patients

To examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells.Neuronal cells (primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin (Rap) or N-acetyl-l-cysteine (NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3′-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays.Cd-induced activation of Akt/mTOR signaling, including Akt, mTOR, p70 S6 kinase (p70 S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Rap, an mTOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/mTOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G).Cd-induced ROS generation activates Akt/mTOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that mTOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.

Valproate inhibits oxidative damage to lipid and protein in primary cultured rat cerebrocortical cells

Valproate is often prescribed as a long-term therapeutic mood stabilizing agent for individuals with bipolar disorder. Although research suggests that this drug may produce a neuroprotective effect, its neuroprotective mechanism is not yet clear. The purpose of this study was to determine if valproate provides a neuroprotective effect against damage caused by oxidative stress in primary cultured rat cerebral cortical cells. We found that chronic treatment with valproate at therapeutically relevant concentrations for 7 days inhibited lipid peroxidation and protein oxidation induced by treatment with 0.25 mM oxidant FeCl 3 for 90 min, indicating that valproate inhibits oxidative damage to lipid and protein. Our results suggest that chronic treatment with valproate may protect neuronal cells from damage caused by oxidative stress and that neuroprotection from oxidative damages may be involved in the mechanism of action of valproate. Supporting this possibility are recent findings that chronic treatment with valproate increased the expression of endoplasmic reticulum stress protein GRP78 (Mol Pharmacol 55 (1999) 521) and antiapoptotic factor bcl-2 (J Neurochem 72 (1999) 379) in rat cerebral cortex. Since GRP78 binds Ca 2+ and folds damaged protein, bcl-2 stabilizes mitochondrial transmembrane potential and inhibits cytochrome C release, and both GRP78 and bcl-2 have been shown to inhibit oxyradical accumulation, together these findings indicate that valproate may target one or more of these processes in order to produce neuroprotective effects.