Glutathione levels in specific brain regions of genetically epileptic (tg/tg) mice

The tottering (tg/tg) mouse is a genetic model of human generalized epilepsy; these mice exhibit spontaneous absence seizures accompanied by bilaterally synchronous spike-wave discharges (6). The mechanism(s) for seizure activity are unknown in these mice. Several recent studies have suggested that membrane lipid peroxidation may be causally involved in some forms of experimentally induced epilepsies (18). Since reduced glutathione (GSH) is the most important free radical scavenging compound in vivo that can prevent membrane lipid peroxidation, the objective of this study was to investigate GSH concentrations in specific central nervous system regions of genetically epileptic, tg/tg, mice as compared to age-matched controls. Three brain regions, cerebellum, hippocampus, and occipital cortex, were dissected, weighed and the concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) were measured in each of these tissues. GSH content was significantly lower in the occipital cortex of tg/tg mice compared to controls; no differences were observed in the other two brain regions examined. Total GSH content (GSH plus 2 × GSSG) paralleled GSH concentration differences. GSSG content from tg/tg mice was lower in the hippocampus and occipital cortex, compared to controls. This is the first report of an association between decreased central nervous system glutathione concentrations and seizure activity in animals exhibiting generalized seizures.     

Increased methionine-enkephalin levels in genetically epileptic (tg/tg) mice.

Recent experimental data indicate that endogenous brain ligands for the opioid receptors such as enkephalins, beta-endorphin (beta-End) and dynorphin (Dyn) may be involved in both generalized and partial seizures. The "tottering" (tg/tg) mouse provides an electrophysiological representation of generalized spontaneous human epilepsy. These mice exhibit behavioral absence seizures with accompanying spike-wave discharges. Methionine-enkephalin (M-Enk), beta-End and Dyn levels in various regions of brain were measured by radioimmunoassay (RIA) in 15-18-week-old tg/tg and control (+/+) mice to elucidate the relation between seizures and the opioid system. beta-End and Dyn levels were similar in tg/tg and +/+ mice. However, M-Enk levels were significantly increased in the striatum, cortex, pons and medulla of the tg/tg mice. Our data suggest that in the tottering mouse model of generalized epilepsy there is an alteration of enkephalinergic pathways and not of the endorphinergic or dynorphinergic pathways.     

Glutathione levels in specific brain regions of genetically epileptic (tg/tg) mice

The tottering (tg/tg) mouse is a genetic model of human generalized epilepsy; these mice exhibit spontaneous absence seizures accompanied by bilaterally synchronous spike-wave discharges (6). The mechanism(s) for seizure activity are unknown in these mice. Several recent studies have suggested that membrane lipid peroxidation may be causally involved in some forms of experimentally induced epilepsies (18). Since reduced glutathione (GSH) is the most important free radical scavenging compound in vivo that can prevent membrane lipid peroxidation, the objective of this study was to investigate GSH concentrations in specific central nervous system regions of genetically epileptic, tg/tg, mice as compared to age-matched controls. Three brain regions, cerebellum, hippocampus, and occipital cortex, were dissected, weighed and the concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) were measured in each of these tissues. GSH content was significantly lower in the occipital cortex of tg/tg mice compared to controls; no differences were observed in the other two brain regions examined. Total GSH content (GSH plus 2 x GSSG) paralleled GSH concentration differences. GSSG content from tg/tg mice was lower in the hippocampus and occipital cortex, compared to controls. This is the first report of an association between decreased central nervous system glutathione concentrations and seizure activity in animals exhibiting generalized seizures.     

Brain catecholamine metabolism in catechol-O-methyltransferase (COMT)-deficient mice

Catechol-O-methyltransferase (COMT) catalyses the O-methylation of compounds having a catechol structure and its main function involves the elimination of biologically active or toxic catechols and their metabolites. By means of homologous recombination in embryonic stem cells, a strain of mice has been produced in which the gene encoding the COMT enzyme is disrupted. We report here the levels of catecholamines and their metabolites in striatal extracellular fluid in these mice as well as in homogenates from different parts of the brain, under normal conditions and after acute levodopa administration. In immunoblotting studies, COMT-knockout mice had no COMT protein in brain or kidney tissues but the amounts of catecholamine synthesizing and other metabolizing enzyme proteins were normal. Under normal conditions, COMT deficiency does not appear to affect significantly brain dopamine and noradrenaline levels in spite of relevant changes in their metabolites. This finding is consistent with previous pharmacological studies with COMT inhibitors and confirms the pivotal role of synaptic reuptake processes and monoamine oxidase-dependent metabolism in terminating the actions of catecholamines at nerve terminals. In contrast, when COMT-deficient mice are challenged with l-dihydroxyphenylalanine, they show an extensive accumulation of 3,4-dihydroxyphenylacetic acid and dihydroxyphenylglycol and even dopamine, revealing an important role for COMT under such situations. Notably, in some cases these changes appear to be Comt gene dosage-dependent, brain-region specific and sexually dimorphic. Our results may have implications for improving the treatment of Parkinson's disease and for understanding the contribution of the natural variation in COMT activity to psychiatric phenotypes.     

The significance of increase in striatal D(2) receptors in epileptic EL mice

The present study systematically and quantitatively analyzed the immunohistochemical distribution of various substances involved in synthesis, binding, and transport of dopamine in the forebrain of epileptic mice (EL mouse strain) using a brain mapping analyzer. A reduction in serum calcium levels decreases calcium/calmodulin-dependent-dopamine synthesis in the brain and subsequently increases susceptibility to epileptic convulsions and induces abnormal behavior in EL mice. The immunohistochemical levels of D(2) receptors in the medial area of the neostriatum were significantly higher in EL mice than in ddY mice (mother strain of EL mice), while there were no differences in the levels of tyrosine hydroxylase, calcium/calmodulin-dependent protein kinase II, calmodulin, D(1) receptors, and dopamine transporters. Together with our previous findings, the results suggest that the decrease in serum calcium levels and subsequent decrease in brain dopamine synthesis comprise the primary physiologic disorder in EL mice, and convulsions or increased D(2) receptors are secondarily-induced phenomena to improve or compensate for the principal disorder.     

Repeated electroconvulsive shock normalizes blood glucose levels in genetically obese mice (C57BL/6J ob/ob) but not in genetically diabetic mice (C57BL/KsJ db/db)

There are several conflicting reports on the effect of electroconvulsive shock (ECS) on diabetes in humans. The present study investigated the effect of repeated ECS on blood glucose levels in genetically obese mice, which are considered an animal model for non-insulin dependent (maturity onset) diabetes. These mice were compared with genetically diabetic mice which are thought to be an animal model for insulin-dependent (juvenile-type) diabetes. A marked decrease in blood glucose concentrations was observed in obese mice after the first ECS which lasted for 14 days after the last ECS. No effect was seen in genetically diabetic mice. The neural mechanisms by which ECS normalizes blood glucose in genetically obese mice are discussed.     

Brain mapping of epileptic activity in a case of idiopathic occipital lobe epilepsy (Panayiotopoulos syndrome)

The Panayiotopoulos type of occipital lobe epilepsy has generated great interest, but the particular brain areas involved in the peculiar seizure manifestations have not been established. We studied a patient with the syndrome, using high-resolution EEG and simultaneous EEG and functional magnetic resonance imaging (fMRI). Resolution of the scalp EEG was improved using a realistic spline Laplacian algorithm, and produced a complex distribution of current sinks and sources over the occipital lobe. The spike-related blood oxygen level dependent (BOLD) effect was multifocal, with clusters in lateral and inferior occipital lobe and lateral and anterior temporal lobe. We also performed regional dipole seeding in BOLD clusters to determine their relative contribution to generation of scalp spikes. The integrated model of the neurophysiologic and vascular data strongly suggests that the epileptic activity originates in the lateral occipital area, spreading to the occipital pole and lateral temporal lobe.     

Receptor-Mediated Activation of G-Proteins by κ Opioid Agonists in Frog (Rana esculenta) Brain Membranes

This study delineates the heterotrimeric guanine nucleotide binding regulatory protein (G-protein) types in frog (Rana esculenta) brain membranes and their activation by κ opioid agonists. Ethylketocyclazocine (EKC), trans-(±)-3,4-dichloro-N-methy-N-(2-[1-pyrrolidinyl]cyclohexyl)benzene-acetamide (U-50,488) and bremazocine displayed dose-dependent, norbinaltorphimine-reversible stimulation of guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding in crude membrane preparations. G-proteins were identified by Western-blotting using previously characterized specific antisera that were generated against mammalian G-protein α-subunits and β-subunits. A photoreactive guanosine 5′-triphosphate (GTP) analog, [α-³²P]GTP azidoanilide ([α-³²P]AA-GTP) irreversibly labeled four proteins in the molecular weight range of 39–43 kDa. Ethylketocyclazocine and U-50,488 stimulated photolabelling of these proteins among which the 39 kDa band comigrated with the protein specifically labelled with the α_i2 antibody and the 40 kDa band was identified as α_o1. The other two bands were also stained with the α_common antibody, but were not further identified. These results suggest that the endogenously expressed κ opioid receptors that are present in frog brain interact with multiple G-proteins in situ. Furthermore, the structure of most G-proteins seems to be well preserved during phylogenesis.     

Vibrator (vb): a spinocerebellar system degeneration with autosomal recessive inheritance in mice

Vibrator (gene symbol vb), an autosomal recessive mutation, occurred spontaneously in the DBA/2J strain of mice, was rescued by a single cross to C57BL/6J and subsequent brother × sister mating, and has been mapped near shaker-2 (sh-2) and vestigial tail (vt) on chromosome 11. The name emphasizes the unusually rapid (18–20 Hz) postural action tremor expressed in juvenile homozygotes. Selected neurons in spinal cord, and later in brainstem and cerebellum, show progressive degenerative changes featuring dilated cisternae of endoplasmic reticulum in cell bodies, dendrites and axons, with eventual severe intracellular vacuolation and some cell death.     

A calcium-activated, nonselective cationic conductance in Aplysia silent neurons

We have previously reported the activation of a triphasic current response by calcium injection in voltage-clamped, nonbursting neurons of Aplysia californica. Present evidence indicates that the second phase, a delayed inward current that peaks 10–20 seconds after the end of the injection, is a calcium-activated, nonselective cationic conductance. It can be carried by both sodium and calcium, is not sensitive to chloride concentration changes, but is voltage sensitive, decreasing in amplitude with hyperpolarization.     

Interhemispheric connections in neonatal owl monkeys (Aotus trivirgatus) and galagos (Galago crassicaudatus)

Interhemispheric connections were studied by injecting a mixture of horseradish peroxidase (HRP) and wheatgerm agglutinin conjugated with horseradish peroxidase (WGA-HRP) into multiple sites in dorsolateral occipital and parietal cortex of one cerebral hemisphere of three galagos (Galago crassicaudatus) and two owl monkeys (Aotus trivirgatus) within seven days of birth. Cortex was either separated from the rest of the brain, flattened and cut parallel to the surface to aid reconstructing surface-view patterns of labeled neurons and processes, or cut in standard coronal or parasagittal planes to better reveal laminar patterns of connections. In both primate species, the surface-view pattern of callosal connections in infants was remarkably adult-like. In infant owl monkeys, callosal connections were concentrated along the margin of area 18 with area 17, and only a few labeled cells were found within area 17. Other visual areas including the second visual area, V-II, and the middle temporal visual area, MT, had patchy distributions of labeled neurons that extended over large parts of the visual field representations. Primary motor, auditory, and somatosensory fields also had patchy distributions of labeled neurons, with regions of areas 3b and adjoining somatosensory fields having few callosal connections in portions that appeared to correspond with representations of the hand and foot. Results were very similar in galagos, except that newborn galagos, as in adults, had a patchy distribution of callosally projecting neurons that extended well within area 17. Furthermore, the labeled neurons were concentrated in patches that aligned with the cytochrome oxidase blobs of area 17. Finally, callosal connections were concentrated in cytochrome oxidase poor regions of area 3b.     

Excessive intra- and supragranular mossy fibers in the dentate gyrus of tottering (tg/tg) mice

In Timm's sulfide silver preparations, intragranular and supragranular mossy fiber staining is found to be much more prevalent in the temporal dentate gyrus of the spontaneous epileptic mouse, tottering, than at matching levels in unaffected littermate controls. This abberant distribution of mossy fibers may be due to the spontaneous seizures affecting this mutant.