Hugh E. Laird

Neurotransmitter abnormalities as determinants of seizure susceptibility and intensity in the genetic models of epilepsy

Normal subjects are not considered to be epileptic merely because they exhibit a seizure in response to an appropriate stimulus of sufficient intensity. In epileptics, a relatively weak stimulus will elicit an explosive neuronal response, whereas in a normal individual it will cause only trivial responses. Fortunately, only a few subjects in any given animal or human population are epileptic; however, most if not all epileptics are genetically predisposed to seizures. The importance of genetic determinants in human epilepsy has become established only recently [l]. Genetic factors probably control seizure susceptibility and, therefore, the likelihood of developing epilepsy [l-8]. The genetic determinant is not solely responsible for the appearance of seizures but is an important contributing component. Seizures probably do not occur in the absence of other factors such as stress, biochemical and hormonal imbalances, or other environmental or physical initiators [l]. Thus, epilepsy is a composite of the individual’s genetically controlled seizure propensity plus the presence of one or more seizure triggering factors. We believe that neurochemical studies in subjects with a genetically determined predisposition to seizures are helping establish the molecular basis of epileptogenicity. However, heretofore most investigations of epilepsy have used animal models with very high degrees of inherited seizure resistance. These are normal animals which are made “epileptic” by the use of various chemical or electrical methods. Except in the kindling model of epilepsy, these normal animals retain their relatively high innate seizure resistance. Although valuable information has been obtained on the initiation of seizures in normal animals, this approach has revealed very little about genetically determined seizure susceptibility. Genetic models of epilepsy. At least one genetic model of epilepsy is available within each of several animal species, including the rat [9, 101, mouse

Effect of Midbrain and Pontine Tegmental Lesions on Audiogenic Seizures in Genetically Epilepsy-Prone Rats

Summary: A bilateral mechanical lesion of the midbrain and pontine tegrnentum was found to abolish completely the tonic components of sound‐induced seizures in genetically epilepsy‐prone rats (GEPR) that display tonicclonic seizures. Correlations between varied lesion placements and effects on maximal audiogenic seizures provided evidence that damage to the nucleus reticularis pontis oralis (RPO) of the midbrain and pontine reticular formation (RF) was responsible for the seizure‐attenuating effects. Moreover, electrolytic lesions of the pontine RF involving the RPO nucleus were found to abolish the tonic components of the maximal audiogenic seizure. Additionally, bilateral mechanical lesions involving the RPO nucleus were found to attenuate the clonic components of sound‐induced seizures in GEPR that display only running seizures and clonus. These findings are consistent with previous studies showing that pontine tegmental lesions attenuate the tonic components of maximal electroshock‐ and pentylenetetrazol‐induced seizures, and lend further support to the hypothesis that all generalized tonic seizures share a common neural substrate. The role of the brainstem RF in tonic versus clonic convulsions is discussed in light of the present findings.

Abnormalities in Monoamine Levels in the Central Nervous System of the Genetically Epilepsy-Prone Rat

Summary: Norepinephrine, dopamine, and 5‐hydroxytryptamine concentrations were determined in the central nervous systems of genetically epilepsy‐prone rats (GEPR) and in control rats. Norepinephrine concentrations were abnormal in all major areas of the central nervous system of the GEPR, with decrements existing in the telencephalon, hypothalamus‐thalamus, midbrain, pons‐medulla and spinal cord. An increment in the concentration of this neurotransmitter existed in the cerebellum. Dopamine concentrations were normal in all areas of the GEPR brain. Abnormalities in 5‐hydroxytryptamine concentrations were also present in the GEPR. They were exclusively decrements and occurred in the telencephalon, hypothalamus‐thalamus, midbrain, and pons medulla. Concentrations of this neurotransmitter were normal in the cerebellum and spinal cord. Coupled with our earlier pharmacologic data, these observations support our concept that noradrenergic and/or 5‐hydroxytryptaminergic decrements are etiologically important in seizure susceptibility in the GEPR. The lack of abnormalities in brain dopamine concentrations strengthens our hypothesis that dopaminergic transmission does not regulate seizure susceptibility in this model.

Peripheral-type benzodiazepine receptors.

Since their first description as anomalous high affinity diazepam binding sites in rat peripheral tissues, the peripheral-type benzodiazepine receptor (PBR) has been increasingly studied to better understand nonneural effects of the benzodiazepines. The mammalian PBR is ubiquitously distributed with high concentrations in the outer mitochondrial membrane of secretory tissues. In regions of the brain, the density of PBR can equal or exceed the density of central-type benzodiazepine receptors. High affinity PK 11195 binding is diagnostic for the receptor while the affinity for benzodiazepines is species dependent. Recent cDNA cloning of a PBR component, the isoquinoline binding protein (IBP), shows no apparent sequence homology with any GABAA receptor subunits known to comprise central benzodiazepine receptor subtypes. The PBR seems at best only distantly related to CBRs. Recent advances in the pharmacology, biochemistry and molecular biology of the PBR are reviewed.

Optimal antidotal dose of activated charcoal.

Abstract Activated charcoal is an effective antidote for reducing the gastrointestinal absorption of numerous ingested chemicals. However, the dose of activated charcoal for clinical use has often been recommended on the basis of in vitro data. Use of such data tends to overestimate the antidotal capacity of activated charcoal and may result in undertreatment with the adsorbent, whereas in vivo data are believed to be more reliable. The present study quantifies the capacity of activated charcoal to bind 3 drugs, pentobarbital sodium, chloroquine phosphate, and isoniazid in vivo. A fixed dose of each drug was administered to rats and mean control tissue concentrations of the drugs were determined at peak time. The identical dose of each drug was then administered to groups of rats which were immediately antidoted with activated charcoal in a dose of 1, 2,4 or 8 times the weight of the drug. Tissue concentrations of the drugs were then determined and the results were calculated and expressed as percent reduction of tissue drug concentrations, on the basis of the control tissue drug concentration values. Charcoal-drug ratios of 1:1, 2:1, 4:1 and 8:1 reduced absorption as follows: pentobarbital sodium, 7, 38, 62 and 89%; chloroquin phosphate, 20, 30, 70 and 96%; and isoniazid, 1.2, 7.2, 35 and 80%. Binding of drugs by activated charcoal at the 3 lower charcoal-drug ratios was highly variable between drugs. Only the highest charcoal-drug ratio provided uniform and nearly complete binding of the 3 drugs. The results suggest that the optimal activated charcoal-drug ratio for the treatment of pentobarbital, chloroquine, and isoniazid is at least 8:1.

Hepatic protein kinase C: Translocation stimulated by prolactin and partial hepatectomy

Prolactin stimulates a hepatotrophic response similar to that caused by phorbol esters or partial hepatectomy in rats. Since phorbol esters, which activate protein kinase C, mimic prolactin action in liver, the relationship between prolactin administration and subsequent hepatic protein kinase C translocation was assessed. Prolactin administration rapidly stimulated a 4-fold elevation of membrane protein kinase C activity. The effect of prolactin on hepatic protein kinase C was specific for lactogenic hormones but could be duplicated by phorbol esters. Further, an increase in serum prolactin was demonstrated subsequent to partial hepatectomy and preceding hepatic protein kinase C translocation. Therefore, translocation of hepatic protein kinase C appears important for hepatic proliferation in response to prolactin administration and to partial hepatectomy.

Evaluation of diazepam and pyridoxine as antidotes to isoniazid intoxication in rats and dogs.

Abstract Because information regarding efficacious treatment of acute isoniazid (INH) toxicity is incomplete and controversial, diazepam and pyridoxine were investigated as iv antidotes in rats and dogs following administration of po lethal doses of INH. There is a marked species variation in the lethality of INH; the lowest consistently lethal dose is 1500 mg/kg for rats and 75 mg/kg for dogs. Of the two species, the dog more closely approximates mans sensitivity to the lethal effect of INH overdose (80–150 mg/kg). Species variation was also observed in the effects of the antidotes. Diazepam exerted dose-related protection against convulsions in rats; paradoxically, survival was increased by the lowest dose (1 mg/kg) but not by higher doses. In dogs, however, diazepam failed to prevent convulsions but provided dose-related protection against death. Pyridoxine, in rats, did not protect against INH toxicity, but in dogs it showed dose-related effectiveness against convulsions, and all doses (75–300 mg/kg) prevented lethality. Significantly, the highest dose of pyridoxine tested in rats (750 mg/kg) was substantially below the optimal pyridoxine-to-INH antidotal ratio recommended for man (a dose that at least equals the amount of INH ingested), but that dose of pyridoxine would be larger than its LD50 for rats. Combined administration of diazepam with pyridoxine protected against convulsions and death in rats and dogs. Used concurrently, the two antidotes are clearly synergistic for controlling the manifestations of experimental INH overdose. These results have important implications for the management of acute INH intoxication in man.

Epilepsy and the concentrations of plasma amino acids in humans

We have examined the correlation between the presence of epilepsy in humans, and plasma amino acid levels. Subjects were divided into those having pure generalized tonic-clonic seizures (grand mal group), those having generalized tonic-clonic seizures plus other types of epilepsy (mixed group), and those suffering from epilepsies other than grand mal (no grand mal group). Compared to non-epileptic controls, the grand mal group had significantly higher fasting plasma levels of aspartate (100% increase) and glutamate (380% increase) but significant decreases were seen with phenylalanine (?23%), lysine (?27%), and tryptophan (?30%). The no grand mal group showed similar changes except for lysine. The mixed group showed elevations in glutamate, but decreases only in cysteine and methionine. In response to a high protein meal, plasma levels of alanine, cysteine and methionine rose significantly less for the no grand mal group compared to the control group. Increases in aspartate and glutamate concentrations strongly correlated with the prescription of phenytoin. However, the concentrations of these amino acids were not significantly correlated with the actual plasma levels of phenytoin.

Prolactin and known modulators of rat splenocytes activate nuclear protein kinase C.

Prolactin (PRL) and other trophic factors rapidly activate a nuclear pool(s) of protein kinase C (nPKC) in purified splenocyte nuclei. The PRL also enhanced [2-3H]glycerol incorporation into nuclear mono- and triacylglycerol. An assay was devised which not only probed the ability of the hormone to activate protein kinase C (PKC) but also demonstrated the presence of nuclear substrates. Using this methodology, a biphasic concentration-response curve to PRL was observed. Heterologous species of PRL and various growth factors also activated nPKC. The PRL-induced nPKC stimulation was antagonized by various immunomodulators, G protein-coupling inhibitors, PKC inhibitors, a calmodulin inhibitor, and a peripheral benzodiazepine agonist and antagonist. A monoclonal antibody to PKC, anti-rat PRL antiserum and a monoclonal anti-rat PRL receptor antibody antagonized PRL-induced PKC-dependent nuclear phosphorylation, further implicating nPKC and a PRL receptor-mediated activation process. Nuclear PKC may be a major target for trophic regulation in response to both positive and negative growth signals.

Correlations between amino acid concentrations in brains of seizure-susceptible and seizure-resistant rats

We have examined the relationship between free amino acid concentrations in the brains of genetically seizure-susceptible and seizure-resistant rats. The concentrations of free amino acids in the two strains do not differ significantly in the inferior colliculus or the cortex. However, animal-to-animal variations in the concentrations of numerous amino acid pairs are highly correlated. Glutamic acid decarboxylase activities did not vary between the two strains. We conclude that the strong correlations reported between glutamate and taurine levels in several species are not unique to this amino acid pair. Furthermore, unlike the situation with some experimentally-induced epilepsies, genetic epilepsy is not associated with major disturbances in free amino acid concentrations. The high correlations between amino acid pairs in some cases may reflect variations in cellular and subcellular compartment sizes that are shared by several amino acids.