Effects of flurazepam on kindled amygdala convulsions in catecholamine-depleted rats

The benzodiazepines (BZs) are potent antiepileptic agents with diverse biochemical actions. One of their effects is to suppress the catecholamines, which are themselves important inhibitors of seizures. Surprisingly, it was reported that the antiepileptic action of one of the BZs, flurazepam (FZ), for pentylenetetrazol seizures was potentiated after depletion of the catecholamines. This led to the suggestion that the catecholamines interfere with the general antiepileptic action of FZ and other BZs. We examined the antiepileptic action of FZ against a kindled amygdala seizure in four groups: (i) rats depleted of norepinephrine with the neurotoxin 6-hydroxydopamine, ( (ii) rats partially depleted of dopamine, with norepinephrine protected by an injection of desmethylimipramine shortly before administration of the neurotoxin, (iii) a desmethylimipramine control, and (iv) a saline vehicle control. At the highest dose of FZ (20 mg/kg), a substantial antiepileptic effect against the generalized seizure was observed in all groups, whereas only partial suppression of the focal seizure occurred. There were no differences between the four treatment groups. Clearly, with the kindling model of epilepsy, the antiepileptic effect of FZ is not altered by depletion of catecholamines.     

Primary and "transfer" seizure development in the kindled rat.

An investigation was made of both primary and "transfer" kindling as they occur in ipsilateral limbic sites. Primary kindling was found to involve progressive growth of afterdischarge (AD), propagation and convulsive behavior. It was noted that AD growth did not take place gradually but occurred in sudden, large increments. "Transfer" (a significant acceleration of secondary kindling) was found at every secondary limbic site. It was associated with the early appearance of full-blown AD's, super-normal propagation, and well-developed seizures. The post-transfer interference of primary site function previously reported by Goddard et al was also found, but it occurred in significant amounts only after transfer kindling of the amygdala. It is believed that the data offer some support for both of the hypothetical mechanisms of transfer which have been proposed.     

Endogenous dopaminergic asymmetry affects development of seizures kindled in the rat amygdala

Cerebral dominance in 56 rats was determined by observing the direction of their turning behavior in response to injection of d-amphetamine (1.5 mg/kg, i.p.). Rats subsequently subjected to kindling of the amygdala developed full epileptic seizures after significantly fewer kindling sessions if the kindling electrodes were in the amygdala of the nondominant, rather than the dominant hemisphere. Kindling rate also showed a significant negative correlation with the total amount of turning after amphetamine. Rats kindled through electrodes in the ventral mesencephalon generally failed to develop full convulsive seizures, and did not show interhemispheric differences in the rate at which they developed preictal behaviors. As the dominant hemisphere of the rat, identified by rotational preference, contains a higher concentration of dopamine, we conclude that even small differences in dopaminergic activity, within the normal physiologic range, can affect the relative susceptibility of the two hemispheres to the development of kindled epilepsy.     

Inter- and intraobserver agreement of seizure behavior scoring in the amygdala kindled rat

IntroductionThe Racine scale is a 5-point seizure behavior scoring paradigm used in the amygdala kindled rat. Though this scale has been applied widely in experimental epilepsy research, studies of reproducibility are rare. The aim of the current study was, therefore, to assess its interobserver variability and intraobserver variability.Material and methodsA video database set was acquired in the course of amygdala kindling of 67 Wistar rats. Six blinded observers received scoring instructions and then viewed a set of 15 random videos (session #1). Next, each observer scored 379 to 1048 additional videos (session #2) and finally scored the same set of 15 videos again (session #3). Scores included the occurrence of seizures (yes or no), the total seizure time (start of stimulus until the absence of seizure behavior), and the highest Racine stage. Interobserver variability and intraobserver variability were assessed in and between sessions #1 and #3 using a 2-way mixed intraclass correlation or Cohen's kappa depending on the variable.ResultsInterobserver agreement in session #1 was 0.664 for seizure occurrence, 0.861 for total seizure time, and 0.797 for the highest Racine stage. In session #3, interobserver agreement on seizure occurrence declined to 0.492, total seizure time declined to 0.625, and agreement for the highest Racine stage was 0.725. Interobserver agreement was scored insufficiently on focal R2 seizures in both sessions (0.287 and 0.182). Intraobserver agreement reached > 0.80 agreement for seizure occurrence, highest seizure score, and total seizure time in 3 out of 4 observers. Racine's scale stage 2 seizure scores were only 0.135 in one observer but 0.650, 0.810, and 0.635 in the other observers.Discussion and conclusionOverall, interobserver agreement and intraobserver agreement in scoring with Racine's scale were adequate. However, because interobserver agreement declined after a period of individually scoring videos, we suggest periodic repetition of the standardized instruction in the course of evaluating videos in order to ensure reproducible results.     

Regional expression of c-fos mRNA in rat brain during the evolution of amygdala kindled seizures.

The biochemical alterations eliciting the growth and spread of afterdischarge and accompanying the evolution of behavioral seizure stages in electrical kindling are not known. In situ hybridization for c-fos mRNA was used to map potential brain structures recruited during the evolution of major seizures from electrical kindling of the amygdala in rats. Two different patterns of c-fos induction were observed in the earliest stages of kindling (stages 1 and 2). A unilateral cortical distribution included the insular, temporal, perirhinal and parietal cortices and the amygdala. No changes in the hippocampus were noted in this group. The second distribution pattern was limited to the hippocampus (either unilateral or bilateral) and amygdala (unilateral) with no changes in the cortical areas. The afterdischarge durations were significantly (2 fold) longer in the 'hippocampal' group as compared to the 'cortical' group. In the later stages of kindling (stages 4 and 5) the distribution of c-fos mRNA was uniformly bilateral and involved a combination of the hippocampal and cortical distributions observed in the earlier stages and including the amygdala bilaterally as well. The induction of c-fos mRNA appears to provide a map of two different routes in the sequential pathways involved in the evolution of kindled seizures; it may also ultimately prove to be an important component of the kindling process itself. Additionally, c-fos mRNA was elevated bilaterally in the inferior colliculus of animals exhibiting running fits with their seizures. The inferior colliculus was previously shown by others to be involved in running fits accompanying convulsions.     

Neocortical and thalamic spread of amygdala kindled seizures

PURPOSE: Amygdala kindling is an epilepsy model involving long-term network plasticity in the nervous system. In this model, repeated weak stimulation of the amygdala eventually leads to severe motor seizures. The mechanisms for worsening behavioral seizures, and the possible role of enhanced connectivity between the amygdala and other structures have not been thoroughly investigated. METHODS: We performed simultaneous field potential recordings from the amygdala, frontal cortex, and medial thalamus during kindling in rats. Seizures were analyzed for signal power compared with baseline and for correlation between recording sites. Interictal signals were analyzed for changes in coherence between electrode contacts in kindled animals compared with sham kindled controls. RESULTS: We found that increased behavioral severity of seizures was related to increased seizure duration and to increased signal power in the frontal cortex and medial thalamus. Kindling was associated with increased connectivity between the amygdala and frontal cortex, based on increased amygdala-frontal signal correlation during seizures. In addition, during the interictal period, increased coherence was noted between amygdala and frontal contacts in kindled animals compared with controls. CONCLUSIONS: We found evidence for increased connectivity between the amygdala and frontal cortex both during seizures and in the interictal period, as a result of kindling. Enhanced connections between limbic and neocortical circuits may be important for the development of epilepsy, as well as for normal long-range network plasticity in the nervous system.     

Microinjections of GABA agonists into the amygdala complex attenuates kindled seizure expression in the rat

The effects of intraamygdala injections of either gamma-vinyl GABA or muscimol on the behavioral and electrographic expression of stable, fully generalized, kindled seizures were assessed. Results suggest that intraamygdala administration of GABA agonists preferentially attenuates the behavioral, but not the focal, electrographic expression of kindled seizures elicited from either the insular or entorhinal cortex. These results, in conjunction with those of others, suggest that the amygdala becomes an integral and necessary structure for the expression of seizures kindled from a variety of forebrain areas.     

Modulation of audiogenically kindled seizures by gamma-aminobutyric acid-related mechanisms in the amygdala.

Repetitive induction of audiogenic seizures (AGSs) ("AGS kindling") results in expansion of the AGS neuronal network from the brainstem to forebrain structures. AGSs in kindled genetically epilepsy-prone rats (GEPR-9s) exhibit a significant increase in the duration of posttonic clonus (PTC). The amygdala (AMG) does not appear to be a required network component before AGS kindling, but this structure is implicated in the seizure network after AGS kindling. gamma-Aminobutyric acid (GABA) is a major neurotransmitter in AMG, and histamine receptor activation is also reported to stimulate GABA release. The present study examined the effect on audiogenically kindled seizures of focal microinjections into the AMG of GEPR-9s. AGS kindling involved induction of 14 AGSs in GEPR-9s. Bilateral microinjection of a GABA(A) agonist, muscimol (0.3 nmol/side), into the AMG significantly reduced the duration of PTC, starting 0.5 h after drug infusion, with recovery by 24 h. Microinjection of histamine (60 nmol/side) suppressed PTC at 0.5 h, with total blockade at 24 h, but the seizure pattern did not revert to that observed before kindling until 120 h. This long duration suggests that mechanisms in addition to modulation of GABA function may be involved in the effect of histamine. The wild running and tonic components of AGS were never affected by microinjection of these agents into the AMG. These findings confirm previous work suggesting that the AMG is not a required nucleus in the AGS neuronal network before kindling. However, the AMG becomes critical in expansion of the seizure network during AGS kindling, and audiogenically kindled seizures are negatively modulated by increased GABA function.     

The kindled amygdala model of epilepsy: anticonvulsant action of amino acid antagonists

The amygdalas of rats were stimulated daily to produce kindled epilepsy. Superfusion of the ipsilateral ventricle allowed collection of amino acids before, during and after stimulation. At the stage where stimulation evoked full seizures there was a correlated increase in the extent of glutamate release. Other amino acids, including aspartate, showed no significant changes at this time. Aspartate, threonine and serine showed smaller responses not significantly different from those seen at the pre-kindled stage. Antagonists of excitatory amino acids (omega-phosphono-alpha-amino dicarboxylic acids) effectively antagonized both the behavioral and electrical components of the kindled seizures.     

The effect of amygdala kindled seizures on locus coeruleus activity

Locus coeruleus (LC) neuronal activity was recorded in anesthetized and in awake behaving rats during the production of amygdala afterdischarges (AD's) using kindling protocol. Both LC multiple and single unit discharges were temporally correlated with the appearance of AD's in the amygdala. Seizures were manifested in single unit activity as significant increases in firing rate. In awake animals, a bursting pattern of discharges was observed. We postulate that the observed changes in the LC activity pattern may have a modulatory role in the development of kindled seizures.     

Effects of seizures and carbamazepine on interictal spiking in amygdala kindled cats

We examined the influence of seizures and carbamazepine (CBZ) on spiking rates in kindled cats. In the first experiment, spiking rates were measured before and after seizures, with and without CBZ. CBZ was administered immediately after seizures in order not to affect them. Spiking rates were measured over 9 h during the different sleep stages. In a second experiment, CBZ was administered before and after seizures so as to affect seizure strength and thus measure its effect on spiking. Results confirmed earlier findings of a large increase in spiking following a stage 6 seizure in fully kindled animals. We also established that: (1) repeated daily seizures caused a further increase in spiking until a ceiling was reached; (2) increased spiking was not a direct effect of postictal alterations in sleep stages; (3) CBZ, despite its effectiveness as an anticonvulsant, did not reduce spiking but rather increased it; (4) postictal increases in spiking were related to seizure 'strength'. These findings support the hypothesis that spiking rates are primarily influenced by seizure occurrence, as was found in patients with temporal lobe seizures, and that anticonvulsants act differently on seizures and spikes. This emphasizes the possibility of distinct pathophysiological mechanisms for interictal spikes and seizures.     

大鼠耐苯妥英钠和苯巴比妥钠杏仁核点燃模型及多药耐受基因的表达

目的　探索耐苯妥英钠和苯巴比妥钠（ＰＨＴＰＢ） 杏仁核点燃大鼠模型的建立并评价其可靠性。方法　采用苯妥英钠（ＰＨＴ） 、苯巴比妥钠（ＰＢ） 对４０ 只杏仁核点燃大鼠进行筛选,按照国际通用标准,根据后放电阈值（ａｆｔｅｒ ｄｉｓｃｈａｒｇｅ ｔｈｒｅｓｈｏｌｄ,ＡＤＴ） 的变化判断点燃大鼠对ＰＨＴ和ＰＢ的敏感性,并测定大鼠脑部Ｐ糖蛋白（Ｐｇｌｙｃｏｐｒｏｔｅｉｎ,ＰＧＰ） 的表达,证实耐药的客观性。结果　点燃鼠对ＰＨＴ、ＰＢ具有不同程度的敏感性,６ 只点燃大鼠对ＰＨＴ、ＰＢ不敏感;６ 只点燃大鼠ＰＨＴ、ＰＢ能显著增高其ＡＤＴ。耐药组鼠大脑广泛脑区的ＰＧＰ表达较药物有效组显著增强。结论　用ＰＨＴ、ＰＢ对点燃大鼠进行筛选以制备耐ＰＨＴＰＢ点燃模型的方法是可行的;可利用其建立难治性癫痫模型,进行耐药机制的探讨。     

杏仁核点燃癫痫鼠GAD67mRNA的表达

目的 探讨大鼠杏仁核点燃癫痫后脑组织谷氨酸脱羧酶（GAD）mRNA的表达及其在癫痫发作后表达变化的意义。方法 通过建立与人类癫痫发生,形成具有高度相似性的杏仁核点燃大鼠癫痫模型。采用原位杂交技术检测癫痫鼠颞叶及海马组织不同点燃时程GAD67mRNA表达。结果 点燃后1d,GAD67mRNA表达增多并且表达信号增强,至7d时达高峰,以后表达逐渐下降,但在点燃后49d,表达仍高于正常,与对照组及手术对照组相比有统计学差异。结论 在慢性癫痫发作中,GABA能神经元的活性增强,考虑是由于癫痫过程中兴奋性增强,引起GABA能神经元抑制功能代偿性增加的结果,即癫痫发作后GABA能神经元介入的抑制功能代偿性增加的,这可能是机体内源性抗癫痫机制增强的一种反应。     

Spontaneous interictal spiking in the awake kindled rat.

The spontaneous interictal spike (SIS) noted in EEG recordings is a signal of the abnormally excitable behavior of neurons in an epileptic focus. This study provides a detailed, quantitative, temporal and spatial profile of SIS in the amygdaloid kindled rat. The reproducible nature of the development and decline of SIS in kindling provides a useful model for biochemical study of the mechanisms that initiate and regulate SIS.     

Testosterone and its metabolites affect afterdischarge thresholds and the development of amygdala kindled seizures

In boys with epilepsy, pubertal increases in seizure frequency may be associated with rising androgen levels. The present study tested the hypothesis that testosterone (T) and/or its metabolites might affect amygdala seizure thresholds and the development of secondary generalization from amygdala foci (kindling). Afterdischarge thresholds and kindling rate were measured in gonadectomized (GDX) male rats, with or without T replacement therapy. Drugs that block either androgen or estradiol (E(2)) receptor-mediated responses were also tested. METHODS: Kindling electrodes were implanted in the basolateral amygdala of adult male Wistar rats. In Experiment 1, subjects were GDX and implanted with a silastic capsule containing either: cholesterol (control); T; 5% E(2) in cholesterol; or 5alpha-dihydrotestosterone (DHT). In Experiment 2, intact subjects were treated with daily injections of vehicle (control); daily injections of flutamide (an androgen receptor antagonist); or Silastic implants containing 1,4,9-androstatriene 3,17-dione (ATD; an aromatase inhibitor). RESULTS: In Experiment 1, initial afterdischarge (AD) thresholds were significantly lowered by E(2) treatment, as compared to cholesterol controls, and remained low throughout the kindling paradigm. In T replaced males, AD threshold significantly decreased over the kindling period, a response that was not observed in DHT treated rats. Rates of kindling were significantly faster as a result of T, E(2) and DHT treatment, as compared to cholesterol controls. E(2) treated males kindled the fastest of all 3 groups. In Experiment 2, initial AD thresholds were significantly lowered by flutamide treatment, as compared to cholesterol controls, and remained low throughout the kindling paradigm. AD threshold significantly decreased over the kindling period in intact males, a response that was blocked by ATD treatment. Both flutamide and ATD significantly slowed the rate of kindling, as compared to intact controls. ATD had the most dramatic inhibitory effect on kindling rate. CONCLUSIONS: In males, T and its two metabolites, E(2) and DHT, all appear to enhance the development of amygdala-kindled seizures. E(2) has the most potent epileptogenic effect. Antagonism of E(2) mediated effects in the brain may have potential therapeutic value for males with epilepsy.     

Activity of locus coeruleus neurons in amygdala kindled rats: role in the suppression of afterdischarge

Kindling is a model of epilepsy. The mechanisms of kindling development are unknown but may involve attenuation of noradrenergic neurotransmission. Single unit recordings, pharmacologic and lesion techniques were used to test the hypothesis that the increased seizure duration of kindled rats is the consequence of an inactivation of noradrenergic neurons in the locus coeruleus (LC). No difference was found between unkindled (naive) and kindled rats in the firing rates of recorded LC neurons either between or during seizures in the paralyzed, ventilated condition. Moreover, in naive rats, frank destruction of the LC did not lengthen seizure duration. We conclude that the lengthened seizure duration of kindled rats, in the paralyzed, ventilated conditions, is not the consequence of inactivation of the LC since the firing rate of recorded LC neurons in kindled rats was not decreased and since destruction of the LC in naive rats did not lengthen seizure duration. If attenuation of noradrenergic neurotransmission does contribute to the kindling phenomenon, then the LC is not likely to be the site at which this attenuation occurs.     

Changes in cholecystokinin mRNA expression after amygdala kindled seizures: an in situ hybridization study

Cholecystokinin (CCK) can be a potent anticonvulsant neuropeptide in certain seizure models. Therefore, we examined whether seizures produced by electrical kindling of the amygdala or electroconvulsive seizures (ECS) would affect the expression of CCK mRNA in rat brain. Following a single kindled seizure, CCK mRNA expression was decreased about 20–58% in the amygdala. In contrast, after multiple consecutive kindled seizures, CCK mRNA expression was increased in the amygdala, cerebral cortex, CA1 pyramidal cell layer of the hippocampus and dentate hilus. A single ECS produced no effect on CCK mRNA expression, but multiple ECS increased expression in the interneurons of the hippocampus 24 h after the last seizure. Since seizures produced by ECS can be anticonvulsant to further ECS or kindled seizures, the CCK increases in the hippocampus may represent a compensatory anticonvulsant adaptation observed in both models. Overall, the kindling-induced alterations in CCK expression appear to be more complex involving multiple brain regions and distinct temporal properties.