Attenuation of neurotoxicity following anoxia or glutamate receptor activation in EGF- and hippocampal extract-treated neuronal cultures.

Neurotoxicity following anoxia or glutamate receptor activation was studied in primary neuronal cultures grown in serum-free, chemically defined CDM R12 medium. Exposure to 1 mM KCN, 0.5 mM kainic acid and 0.5 mM N-methyl-D-aspartate led to progressive neuronal degeneration. This damage was quantified by measuring lactate dehydrogenase released in the culture medium. The toxic effects were observed early during the development of the neuronal culture (from 4 days in vitro on) and seemed to be neuron-specific since astrocyte cultures were not affected. Chronic treatment of the neuronal cultures with epidermal growth factor at 10 ng/ml and hippocampal extract at dil. 1/833 (w/v) induced morphological alterations, increased beta-adrenergic receptor coupled adenylate cyclase activity, increased level of total lactate dehydrogenase activity in the case of epidermal growth factor-treated cultures, and attenuation of lactate dehydrogenase release following exposure to KCN or glutamate receptor agonists. The alterations observed are probably due to the proliferation and differentiation of glial cells in these treated cultures. This suggests that glial cells protect neurons in vitro from degeneration induced by anoxia or glutamate receptor activation.

Towards an improved survival of rat brain neurons in culture by cerebrospinal fluid of patients with senile dementia of Alzheimer's type.

Neuronal cell survival was investigated in rat brain cortical cultures in the presence of increasing concentrations of human brain extracts or cerebrospinal fluid (CSF) from control and Senile Dementia of Alzheimer's type (SDAT) patients. Using hippocampal brain extracts, converted 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was compared to the content of the neuronal marker MAP2 in foetal rat brain neuronal cultures in order to test converted MTT as a quantitative parameter for neuronal cell survival. A significant correlation was found between both parameters. SDAT frontal cortex brain extracts induced a two four-fold increase in neuronal cell survival at 25 to 125 micrograms protein extract, whereas control brain extracts induced at similar protein concentrations a decline in neuronal cell survival. The enhanced survival yielded by SDAT brain extracts was fully abolished in the presence of control brain extract. Control CSF concentration-dependently increased neuronal cell survival in postnatal rat brain neuronal cultures independent of the difference in the protein content of CSF samples and age of the patients. SDAT CSF also concentration-dependently enhanced neuronal cell survival, however, the effect was more pronounced compared to control CSF. These observations are in favour of the hypothesis that there might be a higher neurotrophic activity in SDAT brain tissue.

Ca2+-mediated neuronal death in rat brain neuronal cultures by veratridine: protection by flunarizine.

Neuronal cell degeneration was studied in vitro in primary rat brain neuronal cultures grown in serum-free, chemically defined, CDM R12 medium, by measuring lactate dehydrogenase (LDH) released in the culture medium. A Ca2+-dependent neuronal cell degeneration was observed after prolonged and transient exposure 30 microM veratridine. The release of LDH occurred gradually and could be completely prevented by 2 mM ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.1 microM tetrodotoxin, and 1 microM flunarizine. Flunarizine was without effect on neuronal cell loss induced by 1 mM glutamate, 1 mM kainic acid, and 5 mM KCN. The lack of effect on neurotoxicity induced by 1 mM glutamate differentiates flunarizine from N-methyl-D-aspartate antagonists such as MK-801. The latter protected at nanomolar concentrations against glutamate-induced neuronal cell death but had a maximal effect only at 0.1 mM on the veratridine-induced released LDH. It is suggested that, besides the excitatory amino acid receptor pathway, prolonged opening of the veratridine-sensitive Na+ channel can be neurotoxic. The latter can be prevented by flunarizine. The role of Na+ channel blockers as therapeutic agents in cerebral ischemia is discussed.

Neurotoxic action of veratridine in rat brain neuronal cultures: mechanism of neuroprotection by Ca++ antagonists nonselective for slow Ca++ channels.

The effect of various Ca++ antagonists and local anesthetics on neuronal cell degeneration induced by veratridine was studied in primary rat brain neuronal cultures. Cell death was quantified by measuring lactate dehydrogenase (LDH) released in the culture medium. The neuronal cell degeneration was Ca+(+)-dependent because, in the absence of extracellular Ca++, 16 hr of exposure to 30 microM veratridine failed to produce release of LDH. Ca++ antagonists, nonselective for slow Ca++ channels (flunarizine, cinnarizine, lidoflazine, prenylamine and bepridil) inhibited veratridine-induced release of LDH with IC50 values between 0.11 and 0.47 microM. Ca++ antagonists selective for slow Ca++ channels were less potent and inhibited veratridine-induced release of LDH at concentrations in the following order of potency: nicardipine greater than gallopamil and verapamil greater than niludipine greater than nitrendipine greater than nifedipine greater than nimodipine greater than diltiazem. Tested local anesthetics were incomplete inhibitors of veratridine-induced release of LDH. A good correlation was found between the potency of the drugs to inhibit released LDH induced by 30 microM veratridine in neuronal cultures and their binding affinity for the batrachotoxin binding site of Na+ channels in rat cortex synaptosomal preparation. It is concluded that protection against veratridine-induced neurotoxicity can be mediated by blocking a veratridine-sensitive Na+ channel. It is a property of certain nonselective Ca++ antagonists. There is apparently no direct relationship with Ca++ antagonistic activity. The effect is unrelated to local anesthetic activity.

Depolarization of chick myotubes triggers the appearance of (+)-[3H]PN 200-110-binding sites.

Regulation of the appearance of dihydropyridine-binding sites was studied in primary cultures of chick myotubes. Labeling of surface dihydropyridine-binding sites on intact myotubes at 37 degrees was achieved with (+)-[3H]PN 200-110. The appearance of the sites was prevented in a calcium-free medium using 1.8 mM EGTA, in accordance with the presumed Ca2+ dependency of the appearance of dihydropyridine-binding sites in skeletal muscle cells. Chronic treatment of myotubes with isoproterenol or various other drugs did not modulate either the Bmax or Kd value of the (+)-[3H]PN 200-110 binding to the membrane preparation of treated myotubes (control values were: Kd = 0.16 nm, Bmax = 556 fmol/mg of protein), suggesting a lack of heterologous regulation via beta-adrenergic receptor stimulation. However, depolarization of intact myotubes, either by 47 mM K+ or 10(-5) M veratridine, provoked a 3-fold increase in the Bmax value of (+)-[3H]PN 200-110 binding measured on the intact muscle cells without affecting the Kd value. The effect was reversed upon repolarization of the cells. Depolarizing conditions did not affect the binding on a membrane preparation of the myotubes. Hence, depolarization appeared to specifically trigger the appearance of (+)-[3H]PN 200-110 binding sites on intact myotubes; several hypotheses which could explain the involved mechanism are discussed.

Chronic treatment with sabeluzole protects cultured rat brain neurons from the neurotoxic effects of excitatory amino acids.

The neuroprotective properties of the cognitive enhancer sabeluzole were investigated in rat brain neuronal cultures derived from the hippocampal formation of 17-day-old rat embryos. Measurement of the neuronal cytoskeletal microtubule-associated protein, MAP2, was used to assess survival of neurons after exposure of neuronal cultures to glutamate. MAP2 was quantified in neuronal cell homogenates by means of an enzyme-linked immunosorbent assay (ELISA) using a mouse monoclonal MAP2 antibody, peroxidase-labeled goat anti-mouse Ig antiserum, and 2,2'-azido-di-[3-ethylbenz-thiazoline] sulphonate (ABTS) as substrate. Exposure of 7-day-old neuronal cultures to 1 mM glutamate for 16 hours led to a three-fold increase in released lactate dehydrogenase (LDH) and a 40% decrease in cellular MAP2 content. Acute treatment of neuronal cultures with 10 microM sabeluzole yielded a 40% drop in released LDH induced by glutamate. Cultures treated chronically with 0.1 microM sabeluzole on days 1 and 4 in culture showed, after 1 week in culture, a MAP2 content and total LDH activity that was not different from control cultures. A 16-hour exposure to 1 mM glutamate did not induce LDH release or changes in MAP2 levels in sabeluzole-treated cultures. A single treatment with 0.1 microM sabeluzole between day 1 to 5 induced a 70-80% drop in glutamate-induced released LDH in 7-day-old neuronal cultures. Full and partial neuronal protection after chronic sabeluzole treatment at 0.1 microM was also observed for neurotoxicity induced by 5 mM N-methyl-D-aspartate (NMDA) and 1 mM kainic acid or 30 microM veratridine, respectively. Within a series of compounds such as Ca++ and Na+ channel antagonists, glutamate receptor antagonists and various neurotransmitter receptor antagonists, sabeluzole, chronically given, were the most potent for inhibition of released LDH induced by 1 mM glutamate (IC50-value: 34 +/- 13 nM). In conclusion, chronic sabeluzole treatment protects cultured rat brain neurons from excitotoxic aggression.