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We evaluated the neuroprotective effect of MK-801, a noncompetitive, selective N-methyl-D-aspartate receptor antagonist, in a neonatal hypoxic-ischemic animal model. Seven-day-old rats underwent bilateral ligation of the carotid arteries followed by exposure to an 8 oxygen atmosphere for 1 hr. We sacrificed the animals 72 hrs later and assessed the hypoxic-ischemic brain damage histologically. MK-801 (10 mg/kg), administered IP 0.5 hr before the hypoxia, completely prevented hypoxic-ischemic infarction in cerebral cortex, while treatment immediately and 1 hr after the end of the hypoxia resulted in 76 and 52 reduction in the infarcted area, respectively. MK-801, given 0.5 hr before and immediately after the insult, reduced striatal damage and, given 0.5 hr before, attenuated neuronal necrosis in hippocampal regions. These results show that in neonates MK-801 is neuroprotective even when administered up to 1 hr after the end of a hypoxic-ischemic insult.

Septal kindling was associated with an inhibition of the post hoc phosphorylation of several synaptic plasma membrane proteins of rat hippocampus. In control rats, the 32P incorporation into proteins of molecular weights 50,000, 58,000, and 60,000 was markedly stimulated by combined calcium/calmodulin, whereas in kindled animals, the response to combined calcium/calmodulin was reduced. Calcium alone, cAMP, or cGMP modulated 32P incorporation into several synaptic plasma membrane proteins but did not differentiate control from kindled tissues. Both control and kindled rats showed nonspecific inhibition of calcium/calmodulin-stimulated phosphorylation in the post hoc assay by corticotropin and by [Leu]enkephalin. The differences between control and kindled animals were most striking in hippocampus and in the amygdaloid-entorhinal area; less pronounced in cortex, basal ganglia, and brain stem; and not significant in cerebellum, a region where kindling cannot be elicited. An 8-wk period of rest after kindling did not reduce these changes, suggesting that they may be a persistent as the kindling behavior itself.

When stimulated by the cholinergic agonist carbachol, PC12 cells rapidly secrete a large fraction of the intracellular catecholamines by exocytotic release from the large dense-core secretory vesicles in a Ca(2+)-dependent manner. To investigate whether Ca2+/calmodulin kinase II plays a role in the regulated secretion of catecholamines, we examined the effect of the specific Ca2+/calmodulin kinase II inhibitor KN-62 on the carbachol-induced release of norepinephrine from PC12 cells. Approximately 50% of the regulated release of norepinephrine, stimulated either by carbachol or direct depolarization, was inhibited by pretreatment with KN-62, while the remaining 50% was resistant to KN-62 and therefore independent of Ca2+/calmodulin kinase II. In contrast, H7, an inhibitor of protein kinase C, had no effect on any of the stimulated release. FURA 2 imaging experiments demonstrated that KN-62 does not act by blocking the stimulation-induced increase in intracellular [Ca2+]. The most likely model consistent with these data is that all the dense-core vesicles fuse with the plasma membrane in a Ca(2+)-dependent process, but that approximately 50% of the vesicles require an additional step that is dependent on the action of Ca2+/calmodulin kinase II. This step occurs between the influx of Ca2+ and the fusion of vesicle membranes with the plasma membrane, and may be analogous to the Ca2+/calmodulin kinase II phosphorylation of synapsin which mobilizes small, clear synaptic vesicles for exocytosis at the synapse.

The effect of bicuculline-induced status epilepticus (SE) on local cerebral metabolic rates for glucose (LCMRglc) was studied in 2-wk-old ketamine-anesthetized marmoset monkeys, using the 2-[14C]-deoxy-D-glucose autoradiographical technique. To estimate LCMRglc in cerebral cortex and thalamus during SE, the lumped constant (LC) for 2-deoxy-D-glucose (2-DG) and the rate constants for 2-DG and glucose were calculated for these regions. The control LC was 0.43 in frontoparietal cortex, 0.51 in temporal cortex, and 0.50 in thalamus; it increased to 1.07 in frontoparietal cortex, 1.13 in temporal cortex, and 1.25 in thalamus after 30 min of seizures. With control LC values, LCMRglc in frontoparietal cortex, temporal cortex, and dorsomedial thalamus appeared to increase four to sixfold. With seizure LC values, LCMRglc increased 1.5- to 2-fold and only in cortex. During 45-min seizures, LCMRglc in cortex and thalamus probably increases 4- to 6-fold initially and later falls to the 1.5- to 2-fold level as tissue glucose concentrations decrease. Together with our previous results demonstrating depletion of high-energy phosphates and glucose in these regions, the data suggest that energy demands exceed glucose supply. The long-term effects of these metabolic changes on the developing brain remain to be determined.

Felbamate (2-phenyl-1,3-propanediol dicarbamate) is a novel agent effective against maximal electroshock, pentylenetetrazol and other chemically induced seizures in mice and rats. Felbamate has been proposed as a novel anticonvulsant for the treatment of generalized tonic-clonic and complex partial seizures. In addition, felbamate has been shown to have neuroprotectant effects (in vitro and in vivo) in neonate models of cerebral ischemia. However, few existing studies have contributed to the elucidation of the mechanism of anticonvulsant and neuroprotectant action of felbamate. Because glycinergic mechanisms have been demonstrated to be involved with seizure disorders and neuroprotection, we investigated the binding interaction of felbamate with strychnine-insensitive glycine receptors and compared these findings with brain and plasma levels of felbamate after drug treatment. Inhibition of [3H]5,7-dichlorokynurenic acid (a high-affinity glycine receptor antagonist) binding by felbamate (IC50 = 374 microM) corresponded well with peak felbamate concentrations found in brain (683 and 759 microM) and plasma (679 and 807 microM) 8 hr after 300 (i.p.) or 500 mg/kg (p.o.) doses, respectively. Chemically diverse anticonvulsants tested and MK 801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine maleate] did not modulate [3H]5,7-dichlorokynurenic acid binding. Additional studies have shown that felbamate does not interact with other sites associated with the N-methyl-D-aspartate receptor complex. Thus, the data presented in this report strongly indicate a mechanism of action for felbamate through strychnine-insensitive glycine receptor interaction.