The phenomenon of lowering a seizure readiness level after frequent audiogenic seizures in Krushinskiĭ-Molodkina rats

The spectral analysis of the EEG activity of several brain structures (somatosensory, visual and auditory areas of the cortex, hippocampus, caudate nucleus and central medial thalamic nucleus) in the wakefulness-sleep cycle in Krushinskii--Molodkina strain rats with inherited predisposition to audiogenic seizures using long-lastint reducing of the seizure readiness level revealed in these animals after frequentative audiogenic generalized tonicclonic seizures, was performed. The reducing susceptibility to convulsiogenic sound stimuli in rats correlated with a decrease of the theta-range' waves level and increase of the expression alpha-range waves in the EEG during wakefulness. Probable role of reorganized functions of ascending activating brain systems in origination of the long reduction of susceptibility to the sound after frequentative audiogenic convulsions in rats is discussed.     

Effects of lateralized electric stimulation of the brain on audiogenic convulsive attack in rats

The influence of lateralized and bilateral electrical stimulations of the rat's brain on the course of audiogenic convulsive fit was analyzed. It was found, that after operative application of electrodes to the animal's skull the strength of fit and audiogenic seizure susceptibility decreased in all experimental groups. At the same time the second group, in which the left hemisphere was stimulated, essentially differed from other groups. The occasions of omission of some stages of fit were not so frequent, there, and only in this group after electrostimulation fits increased in the form of arising of new stages. This allows us to propose a special role of the left hemisphere of rats in forming a complete convulsive fit. The described observations resemble those of human asymmetry.     

The influence of neonatal ketamine injection on pain sensitivity and audiogenic seizures in adult rats

The remote effects of neonatal (on the 3d-to-9th postnatal days) ketamine injections (10 and 50 mg/kg in 20 microliters of distilled water, s.c.) were analyzed in adult Wistar, WAG/Rij, and KM (a strain with high audiogenic sensitivity) rats. Both ketamine and water injections increased pain sensitivity in adult rats. Neonatally injected water increased the mean score of seizures in Wistar and WAG/Rij, whereas ketamine water solution injected in the dose of 50 mg/kg did not change the seizure intensity (as compared to the intact control). Consequently, ketamine significantly reduced the mean score of the audiogenic seizure fit without change in its latency. In highly sensitive KM rats the neonatally injected ketamine (50 mg/kg) significantly shortened the mean latency of the fit onset, and fit stages developed faster. Thus, the neonatal ketamine injection increased the audiogenic seizure susceptibility of brain structures in KM rats.     

Interrelations between the contents of transmitter amino acids in the brain structures and the level of convulsion readiness in rats

In rats with high and low levels of the audiogenic excitability (68 and 62 animals, respectivels, we measured the thresholds of convulsive reactions elicited by electrical stimulation of a few limbic and brainstem structures. The obtained figures were compared with the those of the levels of transmitter amino acids (glutamate, aspartate, glycine, and taurine) measured in the same brain structures in corresponding groups of the animals (80 rats in each group. It was found that the thresholds of convulsive reactions evoked by electrical stimulation of most tested brain structures are lower in the audiogenically excitable animals than those in the animals with high thresholds of the audiogenic excitability. These dissimilarities demonstrated certain correlation with increased glutamate concentrations in some structures under study and, probably, to a considerable extent depended on a deficiency of inhibitory amino acids, glycine and taurine, in most tested brain structures.     

Age-Dependent Changes in Brain GABA Turnover Rates in Two Inbred Strains of Mice

gamma-Aminobutyric acid (GABA) steady-state levels and turnover rates have been determined in 15 brain areas of 21-day- and 3-month-old DBA/2J (DBA) and C57B1/6J (C57) mice. These two inbred strains differ by their susceptibility to audiogenic seizures; moreover, the involvement of GABAergic neurotransmission has been suggested in the control of this behavior. Turnover rates are generally higher at 21 days than at 3 months of age. There are few significant differences in the GABA steady-state levels between 21-day-old seizure-prone DBA mice when compared with seizure-resistant C57 mice. In the DBA mice, the steady-state level is higher in the olfactory bulbs and lower in the posterior colliculus and the olfactory tubercles than in the C57 mice. Although there are some significant differences in GABA turnover rates and steady-state levels, intra or inter strains, it is difficult to correlate directly these differences with seizure susceptibility.     

Cerebral, cerebellar, and brain stem gangliosides in mice susceptible to audiogenic seizures

Abstract— The quantitative and qualitative distribution of gangliosides was investigated in the cerebrum, cerebellum and brain stem of audiogenic seizure resistant (C57BL/6J) and susceptible (DBA/2J) mice at 21 days of age. The concentration of gangliosides (μg/unit weight) was higher in the DBA cerebrum and brain stem, but lower in the DBA cerebellum compared to the concentration in C57 mice. In general, the brain water content was lower in DBA mice than in C57 mice. The distributions of a number of gangliosides were found to be different between the two strains and the differences were often in the same direction across the three brain regions. The most consistant and significant difference in ganglioside pattern observed between the strains was the higher concentration of G_M1 in all three regions of the DBA brain. These results suggest that DBA mice have a more heavily myelinated CNS than C57 mice. The relationship of these observations to inherent audiogenic seizure susceptibility is discussed.     

听源性遗传癫痫易感大鼠大脑皮层胆囊收缩素mRNA的原位杂交检测

本实验用原位杂交法,对听源性遗传癫痫易感大鼠（P77PMC)癫痫发作前,单次癫痫发作和多次发作时大脑颞皮层CCK mRNA阳性信号进行了检测,结果显示：（1）单次和多次癫痫发作后颞皮层CCK mRNA阳性神经元数量明显增加（P＜0．01）;（2）多次癫痫发作者上述脑区CCK mRNA阳性神经元数量较单次癫痫发作有明显的下降（P＜0．01）,大脑颞皮层CCK mRNA增高表明,CCK mRNA在癫痫发作过程中起了某种作用,多次癫痫发作大鼠CCK mRNA表达降低提示,单次和多次癫痫发作时大脑皮层CCK mRNAl转录的调控可能存在不同的机制。     

Determination of Amino Acids and Monoamine Neurotransmitters in Caudate Nucleus of Seizure-Resistant and Seizure-Prone BALB/c Mice

Abstract: Amino acid and monoamine concentrations were examined in tissue extracts of caudate nucleus of genetic substrains of BALB/c mice susceptible or resistant to audiogenic seizures. Amino acids [aspartate, glutamate, glycine, taurine, serine, γ-aminobutyric acid (GABA)], monoamines, and related metabolites were separated by isocratic reverse-phase chromatography and detected by a coulometric electrode array system. In situ activity of tyrosine hydroxylase and tryptophan hydroxylase were determined by measuring the accumulation of L-DOPA and 5-hydroxytryptophan after administration of the decarboxylase inhibitor NSD-1015. Highly significant decreases in concentrations of both excitatory (glutamate and aspartate) and inhibitory amino acids (GABA and taurine) were observed in extracts of caudate nucleus of seizure-prone mice. Substantial decreases in concentrations of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were also noted. Decreased accumulation of L-DOPA after NSD-1015 administration provided evidence for decreased tyrosine hydroxylase activity and decreased DA synthesis in striatum of seizure-prone mice compared with seizure-resistant mice. Decreased concentrations of the DA metabolite 3-methoxytyramine (after NSD-1015 administration) suggested that DA release was also compromised in seizure-prone mice. No significant difference in 5-hydroxytryptophan accumulation in striatum of seizure-prone and seizure-resistant mice suggested that tryptophan hydroxylase activity and serotonin synthesis were not affected. The data suggest that seizure-prone BALB/c mice have a deficiency in intracellular content of both excitatory and inhibitory amino acids. The data also raise the issue of whether GABAergic interactions with the nigrostriatal DA system are important in the regulation of audiogenic seizure susceptibility.     

Role of brain 5-hydroxytryptamine in audiogenic seizure in the rat

The effects of various treatments on brain 5-hydroxytryptamine (5-HT) levels were compared to their effects on audiogenic seizure (AGS) intensity. Although some of these procedures also altered norepinephrine (NE) and dopamine (DA) concentrations, it was possible to observe differences in the depletion of these 2 catecholamines and 5-HT. The data indicated that depletion of brain 5-HT itself enhances AGS, whereas an increase in the concentration of this amine above physiologic levels diminishes the intensity of seizure. It appears that 5-HT exerts an inhibitory effect in the brain which limits spread of the seizure discharge. Thus, depletion of this amine disrupts inhibitory activity, allows more efficient spread of seizure activity in the brain and increases severity of AGS.     

Noradrenergic and serotonergic determinants of seizure susceptibility and severity in genetically epilepsy-prone rats

Pharmacological studies demonstrate a reciprocal relationship between both noradrenergic and serotonergic transmission and audiogenic seizure severity and susceptibility in the genetically epilepsy-prone rat (GEPR). In contrast, drug-induced changes in the neurochemical indices of dopaminergic activity do not result in alterations in seizure severity. These pharmacological investigations led to the hypothesis that both noradrenergic and serotonergic neurons are capable of regulating seizure severity in the GEPR. Pharmacological investigations also provided evidence that monoaminergic neurons serve as determinants of seizure susceptibility in these epileptic animals. The GEPR is susceptible to environmentally-induced seizures which cannot be precipitated in neurologically normal subjects. Drug studies suggest that monoaminergic decrements serve as one set of susceptibility determinants. However, non-monoaminergic abnormalities also play important roles in the seizure predisposition which characterizes the GEPR. Pathophysiological studies have confirmed and extended the concepts generated by the pharmacological investigations. Noradrenergic and serotonergic deficits do indeed characterize the seizure naive state of the GEPR. These studies have provided a basis for tentative identification of areas of the brain in which monoaminergic abnormalities regulate seizure severity and susceptibility. Monoaminergic defects in some areas such as the thalamus may regulate both susceptibility and severity. In other areas, defects may regulate only severity or susceptibility. In the striatum, noradrenergic defects do not appear to be present and probably are not determinants of the epileptic state of the GEPR.     

Effects of thermal preconditioning on convulsive activity in rats with inheritable form of audiogenic epilepsy

Effects of thermal preconditioning universal recognized method of increase in concentration of inducible Heat shock protein 70 kDa (Hsp70i) on characteristics of convulsive activity in Krushinskii-Molodkina (KM) rats with inheritable audiogenic epilepsy were studied. For the first time, it was found that short-term thermal preconditioning (41 degrees C during 5 minutes) increased duration of the latency of audiogenic seizure onset. Thermal preconditioning resulted in an increase in concentration of Hsp70i in amygdale, hypothalamus, midbrain; the uttermost increase was observed in hippocampus and inferior colliculus: the brain areas responsible for initiation of audiogenic seizures. A coincidence was found in the term of increase in concentration of Hsp70i and the latency of seizure onset (on day 4 after thermal preconditioning). Results of this research confirm the proposition that inducible Hsp70i is capable of taking part in the processes of seizure development in rats with inheritable form of audiogenic epilepsy.     

The effect of audiogenic seizures on regional CNS energy reserves, glycolysis and citric acid cycle flux

Selected energy reserves, glycolytic intermediates and citric acid cycle intermediates were measured in the cerebral cortex, thalamus, brain stem, cerebellum and spinal cord of susceptible mice during audiogenic seizures. Changes in energy reserves (ATP, phosphocreatine and glucose) differed strikingly in extent and temporal pattern from region to region. The audiogenic seizure produced a transient, large decrease in thalamic energy reserves during the early, pretonic phase of the seizure. Less extensive decreases were observed in brain stem and spinal cord; but in these latter regions the changes persisted throughout the pretonic and tonic phases of the seizures. In cerebellum there was a biphasic decrease in energy reserves; a small decrease was observed immediately after the sound stimulus and a second much greater decrease was observed during the tonic phase of the seizure. No change in energy reserves was observed in cerebral cortex. Changes in glycolytic intermediates (glucose 6-phosphate, fructose diphosphate, pyruvate and lactate) also varied from region to region in response to the decreases in energy reserves. In contrast, changes in the two citric acid cycle intermediates, α-oxoglutarate and malate, were essentially the same in all regions studied. α-Oxoglutarate decreased during the tonic phase of the seizure and rose during recovery. Malate remained at control levels throughout the seizure and then slowly increased. These findings are interpreted as indicating regional variations in nueronal activity during audiogenic seizures. During the period when clinical seizure activity is apparent neuronal activity increases in the subcortical regions. This is reflected by an increase in energy utilization and an increase in glycolytic flux in these areas. However, a concomitant increase in citric acid cycle flux does not seem to occur during this period. Citric acid cycle flux does appear to increase after the seizure is over.     

Role of the brain-stem reticular formation in tonic-clonic seizures: lesion and pharmacological studies

Bilateral lesions of the pontine tegmentum involving the superior cerebellar peduncles and the nucleus reticularis pontis oralis have been shown to attenuate the tonic components of maximal seizures induced by electroshock, sound stimulation (audiogenic), or pentylenetetrazol, although having no effect on clonus in three separate seizure models. The pontine tegmental lesion also abolishes the clonus of minimal audiogenic seizures that have a motor pattern different from that of other clonic models, and are believed to originate in the brain stem. The preponderant suppression of tonus by the pontine tegmental lesion as well as the inhibition of clonus in audiogenic seizures is strikingly similar to the effects of phenytoin in these same seizure models. The findings presented are consistent with the hypothesis that the pontine reticular formation (RF) plays a key role in the generation and/or expression of tonic convulsions. Additional findings are presented that suggest that serotonin may attenuate the tonic components of maximal electroshock seizures by an action on the brain stem. Thus, it seems likely that pontine tegmental lesions as well as antiepileptic drugs and neurotransmitters with preferential effects on tonic seizures act on a common neural substrate that appears to include the brain-stem RF.     

Effect of Ro 4-1284 on audiogenic seizure susceptibility and intensity in epilepsy-prone rats

The effects of CNS monoamine depletion on audiogenic seizure (AGS) susceptibility and intensity were studied in two types of Sprague-Dawley derived rats: (1) the progeny of a nonsusceptible strain (controls); and (2) the nonsusceptible progeny of epilepsy-prone (audiogenic seizure susceptible) parents (NSPSP). Forty-five minutes after injection of the benzoquinolizine Ro 4-1284, a significant fraction of the NSPSP developed AGS susceptibility, whereas the incidence in controls was not significant. AGS intensity was also significantly elevated 45 minutes and 19 days following Ro 4-1284 in NSPSP. In controls, there was a smaller, but significant, elevation of seizure intensity only at the earlier time period. Both types of rats exhibited a marked depletion of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in all of the six different areas of the CNS which were examined. In the NSPSP, a significant incidence of seizure susceptibility was retained as long as 19 days after Ro 4-1284 administration, despite the complete repletion of monoamine stores. These observations suggest that CNS monoaminergic neurons function as determinants of AGS susceptibility and intensity in animals which also carry some other genetically determined susceptibility factor(s). A deficiency in monoaminergic transmission is insufficient to cause susceptibility in animals not carrying the other trait(s). Also, although a monoaminergic deficit may initially cause the appearance of susceptibility, the presence of the deficit may not be necessary for the continuation of susceptibility once an animal has actually sustained an AGS in the presence of the monoaminergic deficit.     

INCREASED CEREBRAL EXCITABILITY CAUSED BY p-CHLOROPHENYLALANINE IN YOUNG RATS

—Single intraperitoneal doses of p-chlorophenylalanine (p-CP; 100 mg/kg or more), lowered the threshold of flurothyl-induced seizures in 1- and 2-week old rats after 24 h, but not after 4 or 72 h. In older rats there was no change in cerebral excitability after a single dose, but the seizure threshold was lowered after several daily doses. Rats given p-CP in lower doses daily from birth exhibited lowered seizure thresholds only until 3-4 weeks of age. Depletion of brain serotonin and inhibition of liver phenylalanine hydroxylase by p-CP exhibited time courses and dose responses similar to those reported for older animals. Activity of phenylalanine hydroxylase returned to normal more rapidly than the content of serotonin after a dose of p-CP, and recovery of normal seizure threshold seemed to be more closely associated with recovery of the hepatic enzyme than with restoration of the brain amine. It appears, therefore, that hyperphenylalanemia or some consequence of it may be an important factor contributing to increased cerebral excitability in p-CP intoxication and possibly in clinical phenylketonuria and that depletion of brain serotonin may not be the only or even the predominant cause of the changes in cerebral excitability in these conditions.     

Involvement of synaptosomal neurotransmitter amino acids in audiogenic seizure-susceptibility and-severity of Rb mice

The involvement of synaptosomal neurotransmitter amino-acids in seizure susceptibility and seizure severity was explored. The amino-acid contents of brain synaptosomes were determined in three sublines of Rb mice differing in their response to an acoustic stimulus: Rb1, clonic-tonic seizure-prone, Rb2, clonic seizure-prone, and Rb3, seizure-resistant. Synaptosomes were prepared from 6 brain areas considered to be involved in seizure activity: olfactory bulbs, amygdala, inferior colliculus, hippocampus, cerebellum, pons-medulla. The steady-state levels of GABA and glycine (Gly), inhibitory amino-acids, of taurine (Tau), an inhibitory neurotransmitter of neuromodulator, of aspartate (Asp) and glutamate (Glu), excitatory amino-acids, as well as of serine (Ser) and glutamine (Gln), two precursors of neurotransmitter amino-acids, were determined by HPLC. Low levels of Tau, GABA, and Ser in hippocampus, Gly in amygdala, Glu in hippocampus, inferior colliculus and pons, Gln and Asp in inferior colliculus appeared to correlate with seizure-susceptibility. GABA and Asp in olfactory bulb, Gln in amygdala, hippocampus and pons, ser in olfactory bulb and pons, appeared to be associated either with seizure-severity or-diversity. A strong involvement of hippocampus (Tau, GABA, Ser, Glu, and Gln) and inferior colliculus (Asp, Glu, Gln) in audiogenic seizure-susceptibility, and of olfactory bulb (GABA, Asp) in seizure-severity and/or-diversity is suggested.Special issue dedicated to Dr. Alan N. Davison.     

Genetic Association Between Ca2+-ATPase Activity and Audiogenic Seizures in Mice

Ca2+-ATPase activity was studied in fresh brain stem homogenates of the audiogenic seizure (AGS)-resistant C57BL/6 and AGS-susceptible DBA/2 inbred strains and in 21 B6 X D2 recombinant inbred strains. A highly significant negative correlation was found between Ca2+-ATPase activity and AGS susceptibility among these strains. In general, strains with low Ca2+-ATPase activities were more AGS-susceptible than strains with high activities. Further, Ca2+-ATPase activity appears to be influenced by a major gene associated with the Ah locus. This gene is designated Caa for Ca2+-ATPase activity and is different from Ias, which is closely linked to the Ah locus. Ias influences AGS spread by a yet unknown biochemical mechanism, whereas Caa may influence AGS susceptibility by regulating Ca2+-ATPase activity in brain tissue.     

Effect of seizure activity on subunit composition of Ca2+/calmodulin-dependent protein kinase II in hippocampus of Krushinskii-Molodkina rats

Using a Western blot method it was been shown that a aontent of beta-CaMKII subunit was decreased on 40 % in hippocampus of Krushinskii-Molodkina rats (rats, genetically prone to audiogenic seizures) in comparison with normal Wistar rats. Additionally, we have investigate the temporal modifications of alpha/beta-CaMKII subunits in dependent from number audiogenic fits (n = 1 or n = 5). The level of beta-CaMKII in hippocampus of naive KM rats and KM rats in 24 h after single audiogenic seizure was not different, but it was increased on 56 % through 72 h after convulsion fit. In contrast, both 5-fold seizures and 20-fold seizures (audiogenic kindling) reduced the beta-CaMKII levels in comparison with naive group. The levels of alpha-subunit protein kinase in hippocampus these experimental groups of animals were not different. We suggest a hypothesis, that modifications alpha/beta ratio can be viewed as specific "homeostatic mechanism", which provide for neuronal function in hippocampus, thereby changing the rules governing synaptic plasticity in dependence from preliminary neuronal activity.     

Neuroanatomical localization of structures responsible for seizures in the GEPR: lesion studies

Identification of the neural substrates subserving audiogenic convulsions in the GEPR is an important task and while it is not yet complete, many laboratories employing various techniques have contributed importantly to our current understanding. The present review focuses on the use of lesions to identify the neural substrates of audiogenic convulsions. Lesions in brain stem nuclei appear to have a much greater ability to attenuate audiogenic convulsions than do forebrain lesions. In fact, some forebrain lesions (dorsal hippocampus, caudate, intralaminar thalamic nuclei) appear to enhance the severity of audiogenic seizures. On the other hand, bilateral lesions in the inferior colliculus (IC) have been shown to completely abolish audiogenic convulsions, while lesions in the pontine reticular formation (PRF nucleus) abolish all aspects except the running episode suggesting that these two brain stem structures are important neural substrates involved in the expression of audiogenic convulsions. Large bilateral lesions of the substantia nigra also appear to attenuate audiogenic convulsions. The effect of lesions on audiogenic convulsions is basically similar to their effect on other generalized seizure models and the data appear to support the hypothesis that there are two anatomical systems involved in the expression of all generalized convulsions: a forebrain system responsible for the expression of face and forelimb clonus; and a brain stem system responsible in the expression of running-bouncing clonus and tonus.     

Glutamate receptor GluR1 expression is altered selectively by chronic audiogenic seizures in the Frings mouse brain

The audiogenic seizure-susceptible mouse, Frings, is genetically susceptible to sound-induced seizures and provides a reliable model of reflex epilepsy that lasts throughout the life span of the animal. We used immunohistochemistry to examine if the expression of the non-N-methyl-D -aspartate glutamate receptor (GluR) subunits GluR1, GluR2, or GluR3 were altered subsequent to multiple seizures. Following a regimen of one seizure per day for 3 weeks, GluR1 immunoreactivity, but not GluR2 or GluR3, was substantially elevated in the outer shell of the nucleus accumbens in 21 of 31 chronically seized Frings mice. No other brain regions such as the hippocampus exhibited any qualitative changes in expression of these subunits. In 9 of the 21 Frings mice exhibiting increased GluR1, but in none of the controls, bilateral structural lesions were observed in the lateral hypothalamus. These results support a model where highly localized changes in the expression of GluR1 occur in response to repeated audiogenic seizure. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 209–216, 1998