Neurotoxicity Screening in a Multipotent Neural Stem Cell Line Established from the Mouse Brain

Neural stem cells (NSCs) have mainly been applied to neurodegeneration in some medically intractable neurologic diseases. In this study, we established a novel NSC line and investigated the cytotoxic responses of NSCs to exogenous neurotoxicants, glutamates and reactive oxygen species (ROS). A multipotent NSC line, B2A1 cells, was established from long-term primary cultures of oligodendrocyte-enriched cells from an adult BALB/c mouse brain. B2A1 cells could be differentiated into neuronal, astrocytic and oligodendroglial lineages. The cells also expressed genotypic mRNA messages for both neural progenitor cells and differentiated neuronoglial cells. B2A1 cells treated with hydrogen peroxide and L-buthionine-(S,R)-sulfoximine underwent 30-40% cell death, while B2A1 cells treated with glutamate and kainate showed 25-35% cell death. Cytopathologic changes consisting of swollen cell bodies, loss of cytoplasmic processes, and nuclear chromatin disintegration, developed after exposure to both ROS and excitotoxic chemicals. These results suggest that B2A1 cells may be useful in the study of NSC biology and may constitute an effective neurotoxicity screening system for ROS and excitotoxic chemicals.

Neuroprotective Effect of Citicoline on Retinal Cell Damage Induced by Kainic Acid in Rats

PURPOSE: To examine whether citicoline has a neuroprotective effect on kainic acid (KA) -induced retinal damage. METHODS: KA (6 nmol) was injected into the vitreous of rat eyes. Citicoline (500mg/kg, i.p.) was administered to the rats once before and twice a day after KA-injection for 3- and 7-day intervals. The neuroprotective effects of citicoline were estimated by measuring the thickness of the various retinal layers using hematoxylin-eosin (H and E) staining. In addition, immunohistochemistry was conducted to elucidate the expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS). RESULTS: Morphometric analysis of retinal damage in KA-injected eyes showed significant cell loss in the inner nuclear layer (INL) and inner plexiform layer (IPL) of the retinas at 3 and 7 days after KA injection, but not in the outer nuclear layer (ONL). At 3 days after citicoline treatment, no significant changes were detected in the retinal thickness and immunoreactivities of eNOS and nNOS. The immunoreactivities of eNOS and nNOS increased in the retina at 7 days after the KA injection. However, prolonged treatment for 7 days significantly attenuated the immunoreactivities and the reduction of thickness. CONCLUSIONS: The results indicate that citicoline has a neuroprotective effect on KA-induced neurotoxicity in the retina.

Identification of hemolytic and neuroactive fractions in the venom of the sea anemone Bunodosoma cangicum

Sea anemones are a rich source of biologically active substances. In crayfish muscle fibers, Bunodosoma cangicum whole venom selectively blocks the I K(Ca) currents. In the present study, we report for the first time powerful hemolytic and neuroactive effects present in two different fractions obtained by gel-filtration chromatography from whole venom of B. cangicum. A cytolytic fraction (Bcg-2) with components of molecular mass ranging from 8 to 18 kDa elicited hemolysis of mouse erythrocytes with an EC50 = 14 æg/ml and a maximum dose of 22 æg/ml. The effects of the neuroactive fraction, Bcg-3 (2 to 5 kDa), were studied on isolated crab nerves. This fraction prolonged the compound action potentials by increasing their duration and rise time in a dose-dependent manner. This effect was evident after the washout of the preparation, suggesting the existence of a reversible substance that was initially masking the effects of an irreversible one. In order to elucidate the target of Bcg-3 action, the fraction was applied to a tetraethylammonium-pretreated preparation. An additional increase in action potential duration was observed, suggesting a blockade of a different population of K+ channels or of tetraethylammonium-insensitive channels. Also, tetrodotoxin could not block the action potentials in a Bcg-3-pretreated preparation, suggesting a possible interaction of Bcg-3 with Na+ channels. The present data suggest that B. cangicum venom contains at least two bioactive fractions whose activity on cell membranes seems to differ from the I K(Ca) blockade described previously

Neuroprotection, excitotoxicicity and NMDA antagonists

PURPOSE: To analyze the main aspects of neuroprotection and excitotoxicity. DISCUSSION: This is a significant theory on the pathophysiology of cerebral ischemia; it is based on the release of excitatory aminoacid (EAA), mainly glutamate. The sequence starts with a decrease of the blood flow and ends in neuronal death. The main stages of this reaction are herein presented and discussed. An in depth study of the effects of the excessive intracellular calcium is undertaken. Neuroprotectors (NP) are a group of drugs that reduce the excitotoxicity, opposing the excessive release of EAA and its intracellular effects. Neuroprotectors represent a rational approach to stroke treatment and offer a number of potential advantages. They prevent or limit ischemia-induced damage. CONCLUSION: There are many experimental and clinical NP trials. A minimum of 800 trials are currently under study worldwide. The most important NP subgroups are: N-methyl D-aspartate (NMDA) antagonists, gamma-amino butyric acid (GABA) agonists, amino-hydroxy-methyl-isoxalone propionic acid (AMPA) antagonists, reducers of intracellular Ca++ inhibitors of nitric oxide modulation pathway free radicals scavengers, sodium channel antagonists, glutamate release inhibitor, growth factors, hypothermia and potassium channel activators.

Electroencephalographic study of the effect of neurotoxin DSP-4 in iron model of chronic focal epilepsy.

The effect of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on electroencephalographic activity (EEG) was studied in the model of chronic focal epilepsy induced by intracortical injection of FeCl3 in the rat. EEG activity was recorded from the epileptogenic focus (ipsilateral and contralateral) in chronic experiments before and after DSP-4 treatment. In some experiments EEG activity was also simultaneously recorded from the cortical epileptogenic focus and locus coeruleus before and after DSP-4 treatment to study the effect of iron-induced seizure activity and of DSP-4 on the locus coeruleus electrical activity. The results showed that DSP-4 aggravated the iron-induced epileptiform activity as well as the locus-coeruleus electrical activity. The data also showed that, induction of epilepsy by FeCl3 is accompanied by enhancement of the locus coeruleus electrical activity. Our study demonstrates that DSP-4 intensifies and modifies the epileptic activity in the iron-induced chronic epilepsy model and that the effects of toxin persist for a longer duration.

Use of neurotoxins to study Ca2+ channel functions

The article contains a brief review on the properties and classification of voltage-dependent Ca2+ channels and on the organic blockers of the different channel types. The effects of peptide toxins from the venoms of Conus sp and of the spider Agelenopsis aperta on high voltage-activated Ca2+ channels are discussed. In addition, we present preliminary data on a novel peptide toxin purified from the venom of the spider Phoneutria nigriventer, which is a powerful blocker of L-and N-type Ca2+ channels.

Noncompetitive inhibitors of the acetylcholine receptor help paint a picture of its ion channel

This review describes and analyzes the evidence from studies using noncompetitive inhibitors of the nicotinic acetylcholine receptor that the receptor's ion channel is formed by the second transmembrane segment of all five receptor subunits. Inconsistencies in this generally accepted model are also presented and discussed

Effects of DSP-4-induced depletion of brain norepinephrine on appetitive and aversive memory retrieval.

This study examined whether depletion of central norepinephrine produces an improved retrieval of aversive memories in the same way as pre-exposure to inescapable footshocks, in rats. Animals conditioned in a T-maze with appetitive (10% sucrose) and aversive (2.0 mA footshock) events were given a single dose of DSP-4 (100, 200 or 400 micrograms/rat) or drug vehicle ICV 24 hr later. The retention performance and activity were assessed 48 hr after the treatment with this neurotoxin. DSP-4 had no effect on open field activities but enhanced latencies to enter both, previously shocked and appetitively reinforced, goalboxes. The data thus, suggest that central administration of DSP-4 does not result in selective enhanced aversive memories. On the contrary, post-trial NE depletion with this neurotoxin might interfere with the retrieval of previously learned association with appetitive stimuli. DSP-4 significantly reduced monoamines, depending upon the brain regions assayed and the doses studied. However, only decreased NE in striatum coincided with the memory changes suggesting that NE innervation to striatum may participate in the retrieval process.

Coral snake venoms: mode of action and pathophysiology of experimental envenomation

As cobras corais säo representantes da familia Elapidae nas Américas. Classificam-se em dois gêneros Micruroides e Micrurus. O gênero Micrurus compreende a quase totalidade das espécies de cobra coral e todas as que causam acidentes no homem. Podem-se fazer as seguintes generalizaçöes quanto aos efeitos produzidos por suas peçonhas e a algumas propriedades destas. As peçonhas das cobras corais säo neurotóxicas, causando perda da força muscular e morte por paralisia respiratória. Näo provocam edema local e necrose assim como näo produzem coagulaçäo sanguínea ou hemorragias. A atividade proteolítica das peçonhas de cobras corais é pequena ou nula. Exercem atividade fosfolipase A**2. Näo induzem efeitos nefrotóxicos. Os componentes tóxicos da peçonha das Elapidae säo as neurotoxinas pré-sinápticas, as neurotoxinas pós-sinápticas, as cardiotoxinas e fosfolipases A**2 com atividade mionecrótica ou semelhante à das cardiotoxinas. O modo de açäo das peçonhas de Micrurus frontalis, M. lemniscatus, M. corallinus e M. fulvius foi investigado em preparaçöes neuromusculares isoladas e é aqui exposto. Mostra-se que enquanto as peçonhas de M. frontalis e M. lemniscatus devem conter apenas toxinas que atuam através de ligaçäo com os receptores colinérgicos da placa terminal (neurotoxinas pós-sinápticas), a de M. corallinus atua também na junçäo neurovascular inibindo a liberaçäo de acetilcolina pelos impulsos nervosos e a de M. fulvius induz despolarizaçäo da membrana das fibras musculares. Relatam-se também os efeitos produzidos pelas peçonhas de M. corallinus e M. fulvius in vivo em cäes e os provocados pela peçonha de M. frontalis em cäes e macacos

Eliciting conditioned taste aversion by cobra venom neurotoxin in rats.

An attempt is made to study conditioned taste aversion (CTA) using cobra venom antivenom or lithium chloride as the Unconditioned Stimulus (US). Twenty-four hour water deprived rats were habituated for two consecutive days to drinking tap water in the drinking box for 15 minutes daily. On 3rd day they were allowed to drink 0.1% sodium saccharin. Thirty minutes later, they were injected with cobra venom (45 micrograms), antivenom (0.022 microliter), antivenom followed by venom, lithium chloride (0.15 M, 4% body weight) or physiological saline. After two days of recovery the animals were water deprived for twenty four hours and water intake was measured on the 7th and 8th day. Retention test on the 9th day shows reduced saccharin consumption in the lithium chloride and venom groups. CTA was significantly reduced in the antivenom-venom group and absent in the antivenom and control group. It is concluded cobra venom can induce clear-cut CTA in rats.