Cerebral Cortical Cultures Research Articles

2-APV and DGAMS are superior to MK-801 in preventing hypoxia-induced injury to developing neurons in vitro

The relative efficacy of competitive and noncompetitive excitatory amino acid antagonists in preventing hypoxic neuronal injury recently was examined in vitro. Immature (26 days post-conception) fetal mouse cerebral cortical cell cultures were exposed 10 days after plating to 5% oxygen for 24 hrs and returned to normoxia. After hypoxic insult, cultures were either not treated or the medium was supplemented with the competitive excitatory amino acid antagonists 2-amino-5-phosphonovalerate (2-APV), γ- d-glutamylaminomethylsulphonate (DGAMS), or the noncompetitive antagonist methyl-10,11-dihydro-5-H-dibenzocyclohepten-5,10-imine maleate (MK-801). By 48 hrs after restitution of normoxia, untreated hypoxic cultures evidenced sereve neuronal deterioration, elevated LDH concentrations in the medium, depressed benzodiazepine receptor binding, and reduced GABA and glutamate uptake. Enhanced glial cell activity was reflected by modestly elevated glutamine synthetase activity. In hypoxic cultures treated with 2-APV (10 μM) or DGAMS (30 μM), neuronal morphology and biochemical profiles were both improved significantly when compared to both untreated hypoxic cultures and also to those treated with MK-801; 2-APV provided greater, although incomplete, protection. MK-801, at the highest nonneurotoxic concentration (25 nM), did not improve neuronal viability when compared to untreated controls. These results suggest that competitive excitatory amino acid antagonists are superior to noncompetitive antagonists in preventing hypoxic neuronal injury to developing neurons in vitro. MK-801, at low concentrations, produced significant neurotoxicity without improving cell survival.

Comparison of rates of neuronal development and survival in human and rat cerebral cortical cell cultures

A problem that has not been addressed directly at the cellular and molecular levels concerns the basic mechanisms governing rates of development and aging in the nervous system. We have begun to examine this problem by employing parallel cell cultures of cerebral cortical neurons taken from rat and man, two species with markedly different developmental periods and life spans. Cortical cultures were established from rat and human fetuses and were maintained under identical conditions. Long-term neuronal survival in human cortical cultures was strikingly greater than in rat cortical cultures, and axonal outgrowth was significantly slower in the human neurons. These differences were consistently observed in a variety of culture media. Exchanges of culture medium between cultures of the two species did not alter the differences in neuronal survival and axon outgrowth suggesting that the differences between human and rat neurons were probably not due to factors released into the culture medium. Furthermore, the differences were not related to developmental stage at the time of culture initiation since similar results were obtained in cultures established from fetuses of different gestational ages. These initial data indicate that differences in the developmental time courses and life spans of human and rat cerebral cortex are reflected at the level of the individual neuron, and provide a system in which to explore the mechanisms responsible for these differences.

Cell culture of cryopreserved human fetal cerebral cortical and hippocampal neurons: neuronal development and responses to trophic factors

Past knowledge of the human brain at the cellular and molecular levels has come largely from studies of postmortem fixed tissue or by way of extrapolation from studies of lower mammalian species. The ability to study living human brain neurons would provide a new avenue for further insight into mechanisms operative in human brain development, function, and disease. The present study established procedures for the cryopreservation and culture of human fetal cerebral cortical and hippocampal neurons, and characterized the development of the cells in culture. The predominant cell type in both cortical and hippocampal cultures was pyramidal-like neurons that extended one long axon-like process and a few minor dendrite-like processes. Bipolar and stellate-like glia were also present in cultures from both brain regions. Fibroblast growth factor (FGF), but not nerve growth factor (NGF), enhanced long-term neuronal survival in both cerebral cortical and hippocampal cultures. Immunocytochemistry demonstrated the presence of both FGF- and NGF-like immunoreactivities in neurons and glia, from both cerebral cortex and hippocampus, suggesting that these endogenous growth factors may play roles in human fetal brain development. The ability to cryopreserve large numbers of viable dissociated human fetal brain neurons, and subsequently study them in cell cultures, provides new opportunities to understand dynamic aspects of the human brain at the cellular and molecular levels.

The effects of acidosis on chronically hypoxic neurons in culture

Neuropathologic changes associated with perinatal hypoxic-ischemic events in the human infant most often result from chronic hypoxia rather than from acute asphyxia. To characterize the effects of acidosis associated with chronic hypoxia in developing neurons, cerebral cortical cultures obtained from fetal mice were exposed to 5% O 2 continuously for either 24 or 48 h at 10 days after plating. At the conclusion of the hypoxic insult (HI), neuronal morphology was relatively intact for both conditions even though culture medium reflected significant reductions in pH and bicarbonate with elevation of lactate; cultures exposed to the longer HI manifested statistically greater aberrations from control values. Total benzodiazepine (BDZ) binding and clonazepam (CLO)-displaceable BDZ binding, reflecting the neuronal component of the receptor, were only modestly reduced immediately after HI, but were thereafter significantly and progressively lower over the 72 h normoxic recovery period. Although neuronal integrity was progressively diminished with both insults, morphology was always more normal and CLO higher in cultures subjected to 48-h HI compared to the 24-h HI (34.0 ± 9.8 vs 1.8 ± 1.1% of control values at 72 h, respectively; P < 0.001). In contrast, values obtained for the glial marker Ro5-4864-displaceable BDZ binding were higher than control values for both conditions. Outcome was not influenced by removal of acidotic medium, nor by normalization of lactate. These data suggest that, with time, neurons in vitro adapt to severe hypoxia and that alterations in pH, lactate, and bicarbonate by themselves are probably not neurotoxic since the most acidotic cultures exhibited improved survival.

Chronic hypoxia in neuronal cell culture metabolic consequences

Cerebral cortical cell cultures obtained from fetal mice were subjected to 5% O2 for 24 h at a developmental stage equivalent to that of the human neonate. Immediately after the hypoxic insult (HI), medium lactate was elevated and pH, partial pressure of oxygen, and bicarbonate concentration were depressed compared to controls. At this time, the cultures evidenced a modest reduction in high-affinity GABA uptake but minimal morphologic or other biochemical evidence of cellular dysfunction. Within 24 h of restitution of normoxia, there was prominent disruption of neuronal integrity as well as significant reductions in benzodiazepine (BDZ) receptor binding, clonazepam-displaceable (CLO) BDZ binding, high-affinity uptake of GABA and beta-alanine, choline acetyltransferase (ChAT) activity, and total protein. Except for the neuronal marker CLO, GABA uptake was depressed more than other parameters for the subsequent 72 h. In contrast, the non-neuronal marker, Ro5-4864-displaceable BDZ binding, was always increased. Both morphologic and biochemical changes occurred independent of correction of pH, bicarbonate, and lactate. These data suggest that chronically hypoxic nervous tissue in vitro exhibits considerable delay in the evolution of maximal abnormality but that a population of GABAergic cells may be relatively more vulnerable. Although glial cell stimulation may contribute to neuronal survival, it also is possible that, because of the temporal association with cellular dysfunction, restitution of normoxia may contribute to nervous tissue injury.

Growth hormone-releasing factor immunoreactivity in the hypothalamus and cortex of the rat: in vivo and in vitro studies.

Utilizing a specific RIA for rat (r) GRF, hypothalamus and cerebral cortex from adult rat and long term dissociated fetal rat hypothalamic and cerebral cortical cell cultures were investigated for the presence of rGRF immunoreactivity (IR-GRF). After homogenization in an acidic medium, tissues and cultures were extracted on octadecylsilyl-silica columns, and IR-GRF and somatostatin (IR-SS) were measured by RIA. In extracts from the hypothalamus from the adult rat the content of IR-GRF was 3.02 +/- 0.16 ng ( +/- SE) per hypothalamus. IR-GRF was identical with synthetic rGRF on gel filtration chromatography and by parallel displacement of dilutions of extract in RIA. In extracts from cerebral cortex isolated from the adult rat, no IR-GRF was detected. In extracts from long term dissociated cell cultures from fetal hypothalami, 7.3 +/- 7 pg/10(6) cells of IR-GRF were present and were identical with synthetic rGRF by chromatographic and immunological criteria. In the extracts from cerebral cortical cell cultures cross-reacting material was present which on gel filtration chromatography revealed two peaks of immunoreactivity of higher mol wt than synthetic rGRF. There was nonparallelism on dilution of the extract. The ratio of IR-SS to IR-GRF by weight (IR-SS/IR-GRF) was calculated to compare the relative abundance of IR-GRF in cultured hypothalamic cells. In the hypothalamus isolated from the adult rat the ratio of IR-SS/IR-GRF by weight was 15.8 +/- 1.4 as compared to 48.9 +/- 10.3 in hypothalamic cultures. We conclude that IR-GRF indistinguishable from synthetic rGRF is present in long term dissociated hypothalamic cell cultures, but is relatively less abundant than in the hypothalamus of the adult rat when compared on the basis of IR-SS. No IR-GRF was detectable in cerebral cortex of the adult rat. At least one cross-reacting molecular species is detected in cerebral cortical cultures by the rGRF RIA, but exhibits nonparallelism and has a higher mol wt than synthetic rGRF. The increase of the ratio of IR-SS/IR-GRF in hypothalamic cell cultures in vitro compared to hypothalamus in vivo suggests that the culture conditions change differentially the expression of IR-SS and IR-GRF.

Benzodiazepine receptor affinity alterations at physiologic temperature after chronic clonazepam exposure

Cerebral cortical cell cultures obtained from fetal mice were exposed to 200 nM clonazepam (CZP) for 14 days and benzodiazepine (BDZ) receptor binding was measured on intact cells in situ at 37 degrees C. Total, specific, and CZP-displaceable BDZ binding were significantly reduced from control values immediately after drug removal (77.7 +/- 1.4%, 75.1 +/- 3.0%, and 40.9 +/- 6.0% of control, respectively) but Ro5-4864-displaceable binding was not affected (87.6 +/- 5.1%). Binding returned to control values within 48 hours. Saturation analysis of the binding data indicated that a high-affinity binding site could not be detected in CZP-exposed cultures immediately after drug removal (162 nM versus 49 nM in controls, p less than .001), but was present 24 hours later.

Transferrin binding by mammalian cortical cells

Predominantly neuronal (neuronal) or non-neuronal (glial) cerebral cortical cell cultures were employed to study the kinetics and changes with maturation of 125I-diferric-transferrin uptake. The diferric-transferrin association curve of neuronal cultures at 37 degrees C was nonphasic and indicated equilibrium at 90 minutes. Dissociation was completed by 70 minutes. Diferric-transferrin specific uptake (80% of total) in neuronal cells (evaluated at days 6, 9, 13, 16, and 23 in culture) increased with maturation. Scatchard transformation of the data revealed increasing Bmax from day 6 to day 16 in culture (1626 to 2740 fmoles/mg protein). However, the K uptake was statistically unchanged over time and equaled 48.7 +/- 13.9 nM (mean +/- SD). In contrast, association studies of glial cultures documented equilibrium by 45 minutes and dissociation by 40 minutes. The concentration curves for diferric-transferrin uptake in glial cells, evaluated at days 11, 15, and 18 in culture, revealed virtually identical uptake at the three ages studied, but the percent specific uptake (58%) was less than for neurons (88%). Scatchard transformation of the data revealed no statistical alteration of Bmax or K uptake from days 11 to 18 in culture. Bmax ranged from 595 to 751 fmol/mg protein; overall K uptake was 48.3 +/- 13.2 nM (mean +/- SD).

Long-term exposure of cortical cell cultures to clonazepam reduces benzodiazepine receptor binding

To determine if reduced drug efficacy after long-term exposure to clonazepam may be a consequence of benzodiazepine receptor alterations, cerebral cortical cell cultures were exposed to the drug (200 n M) for 14 days. Receptor binding was assayed on living cells in situ. After drug exposure, binding in experimental cultures differed markedly from controls with respect to total, specific, and clonazepam-displaceable (neuronal) benzodiazepine binding (60%, 53%, and 6% of control values, respectively) but recovered within 96 h of drug removal. R05-4864-displaceable (nonneuronal) binding was modestly reduced at 0 time (72% of control), but returned to control values in 24 h. The differences in binding could be attributable to a relatively reduced affinity of the high-affinity binding site ( K d ∼ 18 n M for controls and ∼ 30 n M for drug-exposed cultures) but not to changes in the low-affinity binding site or to reduced numbers of receptors.

30] Somatostatin release from dissociated cerebral cortical cell cultures

Primary monolayer cultures of dispersed fetal cerebral cortical cells can be used to measure the release of the neuropeptide, somatostatin. Three to five percent of cellular IRS is released basally into KRB in 10 min. Basal release is stable for at least 60 min and stimulated levels of release can be induced by introducing ionophores, neurotransmitters, or peptides. The peptide content of the incubation samples is readily measured by a well-characterized, sensitive RIA. Table II summarizes the major factors that must be taken into consideration when developing this system for measuring peptide release.

Differential toxicity of chronic exposure to phenytoin, phenobarbital, or carbamazepine in cerebral cortical cell cultures

The effects of phenytoin (30 μg/ml), phenobarbital (64 μg/ml), and carbamazepine (24 μg/ml) were assessed in cerebral cortical cell cultures. After antiepileptic drug exposure for eleven days, cultures were assayed for total protein, number of neurons, tetanus toxin fixation, high-affinity uptake of γ-aminobutyric acid and β-alanine, activity of choline acetyltransferase, and benzodiazepine binding. Carbamazepine-exposed cultures demonstrated minimal effects, whereas highly significant deficits related to generalized toxicity were observed in cultures exposed to phenytoin or phenobarbital.

Differential neurochemical effects of chronic exposure of cerebral cortical cell culture to valproic acid, diazepam, or ethosuximide

We have assessed the relative neurochemical effects of valproic acid, ethosuximide, and diazepam on dissociated cultures of mouse cerebral cortex. Cultures were exposed chronically (11 days) to each antiepileptic drug and assayed for number of neurons, total protein, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid and beta-alanine, choline acetyltransferase activity, and specific and clonazepam-displaceable benzodiazepine binding. Ethosuximide-exposed cultures did not evidence neuronal toxicity; exposure to valproic acid and diazepam resulted in modest neuronal toxicity. However, exposure to each of these drugs resulted in a marked reduction in benzodiazepine binding. This effect may relate to a common mechanism of action of drugs used to treat absence seizures.

Combination of immunocytochemistry and radioligand receptor assay to identify beta-adrenergic receptor subtypes on astroglia in vitro

There is an increasing need to assess the distribution of receptors for neuroactive substances on specific neural cell types. This study describes the establishment of methodology that combines the quantification of beta-adrenergic receptor subtypes by radioligand binding assays with immunocytochemical analysis of the contribution of astroglia (identified by the presence of glial fibrillary acidic protein) and fibroblasts (identified by the presence of fibronectin) to cultures prepared from neonatal rat cerebral cortex. The effects of subtle changes in culture methodology on the cellular composition of cerebral cortical cultures and the distribution of beta-adrenergic receptor subtypes were examined. The data indicate that (1) a decrease in the density of the initial plating suspension, (2) an increase in the age of the animals, or (3) supplementation of the cortical cell suspension with meningeal fibroblasts all result in an increase in fibronectin staining and a decrease in glial fibrillary acidic protein antibody staining. This change in the cellular composition of the cortical cultures correlated with an increase in the number of beta 2-adrenergic receptors and a corresponding decrease in the number of beta 1-adrenergic receptors. These observations point out the care which must be exercised when preparing primary astroglial cultures of sufficient purity for large-scale biochemical and pharmacological studies.

Reduced benzodiazepine receptor binding in cerebral cortical cultures chronically exposed to diazepam.

Fetal mouse cerebral cortex in culture was exposed chronically to diazepam, and benzodiazepine (BDZ) receptor activity was assayed on the intact cell. Results have indicated: (1) minimal evidence of generalized drug toxicity, determined by choline acetyltransferase activity, tetanus labeling, high-affinity uptakes of gamma-aminobutyric acid and beta-alanine, protein, and light microscopy; (2) a significant dose-dependent reduction (down-regulation) of BDZ receptor activity that occurs after chronic exposure to clinically relevant concentrations of the drug; (3) a disproportionate suppression of the neuronal portion of the receptor after both acute and chronic exposure; and (4) recovery patterns of the BDZ receptor that differ substantially after acute versus chronic treatment. The latter finding may partially explain the observed clinical manifestations of drug tolerance and reduced anticonvulsant efficacy after prolonged use of this class of drugs.

Influence of glucose on somatostatin synthesis and secretion in isolated cerebral cortical cells.

Somatostatin-producing cerebral cortical cell cultures were grown in either high- (33 mM) or low-glucose (5 mM) medium and then exposed to short repetitive changes of high- or low-glucose Krebs-Ringer's bicarbonate buffer. Equivalent amounts of somatostatin were released in the high-to-high, the low-to-low, and the low-to-high paradigms. The high-to-low experiment produced a rapid rise in somatostatin release, followed by a decline. Cultures exposed to 2-deoxyglucose after high-glucose medium also released much greater amounts of immunoreactive somatostatin. Separate sets of cultures were grown in high- or low-glucose medium for up to 19 days. Cultures grown in high-glucose medium generally contained more somatostatin intracellularly than did those maintained in low glucose, although somatostatin in the medium was only different at day 19. These results identify extracellular glucose as an important determinant of cortical somatostatin production.