Repeated brief epileptic seizures by pentylenetetrazole cause neurodegeneration and promote neurogenesis in discrete brain regions of freely moving adult rats

Recurrent epileptic seizures are known to provoke various forms of cellular reorganization in the brains of humans and experimental animals. However, little is known about the mechanism of neuronal cell death resulting from epileptic seizures elicited by GABA antagonists. In the present study, we explored the effect on the central nervous systems of freely moving adult rats, of repeated brief epileptic seizures induced by systemic injection of pentylenetetrazole, a GABA-A receptor antagonist. Starting with minor convulsions, repeated epileptic seizures elicited a progressive increase in seizure severity, culminating in the fully kindled state. Histological examination showed that the epileptic seizures caused overt neuronal cell death in the limbic system, including the hippocampus and amygdala, and its adjoining cortex. During the recurrent epileptic seizures, neurogenesis occurred in the subgranular zone of the hippocampus, the subventricular zone of the lateral ventricle, and the amygdala. This type of pentylenetetrazole-induced neurogenesis was seen at an early stage of epileptogenesis in some regions in which massive cell loss was not evident. This suggests that neurogenesis is not a secondary consequence of neuronal cell death, but rather an independent effect of recurrent epileptic seizures.     

Hippocampal cell proliferation and epileptogenesis after audiogenic kindling are not accompanied by mossy fiber sprouting or Fluoro-Jade staining

Repetitive sound-induced seizures, known as audiogenic kindling (AK), gradually induce the transference of epileptic activity from brainstem to forebrain structures along with behavioral changes. The aim of our work was to correlate the behavioral changes observed during the AK with possible alterations in neuronal proliferation, cell death, hippocampal mossy fiber sprouting and in the EEG pattern of Wistar audiogenic rats, a genetically susceptible strain from our laboratory.Susceptible and non-susceptible animals were submitted to repeated sound stimulations for 14-16 days and hippocampal mitotic activity was studied through the incorporation of bromodeoxyuridine (BrdU). Cell death and mossy fiber sprouting were assessed, respectively, by using Fluoro-Jade and Timm staining, 2 and 32 days after the last kindling stimulation. In addition, we used immunofluorescent double labeling for a glial and a mitotic marker to evaluate newly born cell identity. Some animals had hippocampus and amygdala electrodes for EEG recordings.Our results show that kindled animals with 6-11 generalized limbic seizures (class IV-V) had increased cell proliferation in the dentate gyrus when compared with animals with zero or one to three seizures. BrdU-positive cells labeled on day 2 and on day 32 were both GFAP negative. In the later group, rounded and well-defined BrdU-positive/GFAP-negative nuclei were seen in different portions of the granule cell layer. We did not observe any Fluoro-Jade or differential Timm staining in kindled animals at both killing times. However, EEG recordings showed intense epileptic activity in the hippocampus and amygdala of all animals with limbic seizures.Therefore, our data indicate that AK-induced limbic epileptogenicity is able to increase the hippocampal mitotic rate, even though it does not seem to promote neuronal death or mossy fiber sprouting in the supragranular layer of the dentate gyrus.     

Extinction deficit and fear reinstatement after electrical stimulation of the amygdala: implications for kindling-associated fear and anxiety

Generalized seizures produced by electrical kindling of the amygdala in laboratory rats are a widely used animal model of temporal lobe epilepsy. In addition to seizure evolution amygdala kindling enhances emotionality. The relative roles of electrical stimulation and seizure induction in fear responding are unclear. Here we investigate this issue using extinction and reinstatement of fear-potentiated startle. After classical conditioning (light+footshock pairings) laboratory rats were fear extinguished with each light presentation followed by nonepileptogenic amygdala stimulation. In contrast to the normal extinction learning of control subjects, amygdala stimulated animals exhibited conditioned fear after 120 presentations of the nonreinforced conditioned stimulus (CS). In a second experiment electrical stimulation of the amygdala restored extinguished fear responding and the fear reinstatement was specific to extinction context. The reinstatement effect did not involve sensitized fear to the CS produced by amygdala stimulation. The possibility that electrical activation of the amygdala produces unconditioned fear was considered. Animals uniformly failed to demonstrate fear-potentiated startle using electrical stimulation of the amygdala as the unconditioned stimulus. This was the case with a subthreshold afterdischarge stimulus and a stimulation schedule that produced kindled seizures. The extinction deficit and fear reinstatement results were interpreted to suggest that amygdala stimulation activates acquired excitatory stimulus-affect neural connections formed during Pavlovian fear conditioning. Our data supports a model in which excitation of an amygdala-based memory-retrieval system reinforces the expression of learned fear behaviors.     

Decreased levels of disrupted-in-schizophrenia 1 (DISC1) are associated with expansion of the dentate granule cell layer in normal and kindled rats

Disrupted-in-schizophrenia 1 (DISC1) is a candidate gene involved in the pathogenesis of schizophrenia. DISC1 expression is particularly abundant in the adult dentate gyrus, in which decreased levels lead to aberrant growth, impaired migration, and accelerated integration of adult generated neurons. Because seizures can also result in similar changes, we tested the hypothesis that DISC1 expression may be altered in an animal model of epilepsy. We found that extended amygdala kindling (i.e., 99-electrical stimulations) significantly decreased DISC1 labeling in the dentate granule cell layer and subgranular zone. Extended kindling also led to an increase in the number of ectopic granule cells in the hilus. In addition, although the width of the granule cell layer was not generally affected by kindling, decreased levels of DISC1 in the subgranular zone and granule cell layer were associated with an expansion of the upper blade and crest of the dentate gyrus in both normal and kindled rats. These novel findings suggest that seizure activity affects DISC1 signaling in the dentate gyrus and that DISC1 expression may regulate the cytoarchitectural organization of the granule cell layer.     

Independence of cocaine sensitization and amygdaloid kindling in the rat

The reported sensitization to repeated administration of cocaine has been described as a type of pharmacological kindling resembling the seizure development seen with repeated electrical stimulation of certain brain sites. If these two phenomena share a common neural mechanism, prior chronic administration of cocaine would be expected to facilitate the process of subsequent electrical kindling. Rats were pretreated with saline or cocaine (40 mg/kg IP) daily for 21 days, followed by electrical stimulation of the basolateral amygdala. Progressive sensitization to the behavioural effects of cocaine administration occurred. However, no significant differences in the subsequent development of electrically kindled seizures were observed between the cocaine and saline-treated groups. These results suggest that cocaine sensitization and electrical kindling of the amygdala are subserved by independent neural mechanisms.     

THE ROLE OF THE PIRIFORM CORTEX IN KINDLING

In epilepsy research, there is growing interest in the role of the piriform cortex (PC) in the development and maintenance of limbic kindling and other types of limbic epileptogenesis leading to complex partial seizures, i.e. the most common type of seizures in human epilepsy. The PC (“primary olfactory cortex”) is the largest area of the mammalian olfactory cortex and receives direct projections from the olfactory bulb via the lateral olfactory tract (LOT). Beside the obvious involvement in olfactory perception and discrimination, the PC, because of its unique intrinsic associative fiber system and its various connections to and from other limbic nuclei, has been implicated in the study of memory processing, spread of excitatory waves, and in the study of brain disorders such as epilepsy with particular emphasis on the kindling model of temporal lobe epilepsy with complex partial seizures. The interest in the kindling model is based primarily on the following observations. (1) the PC contains the most susceptible neural circuits of all forebrain regions for electrical (or chemical) induction of limbic seizures. (2) During electrical stimulation of other limbic brain regions, broad and large afterdischarges can be observed in the ipsilateral PC, indicating that the PC is activated early during the kindling process. (3) The interictal discharge, which many consider to be the hallmark of epilepsy, originates in the PC, independent of which structure serves as the kindled focus. (4) Autoradiographic studies of cerebral metabolism in rat amygdala kindling show that, during focal seizures, the area which exhibits the most consistent increase in glucose utilization is the ipsilateral paleocortex, particularly the PC. (5) During the commonly short initial afterdischarges induced by stimulation of the amygdala at the early stages of kindling, the PC is the first region that exhibits induction of immediate-early genes, such as c-fos. (6) The PC is the most sensitive brain structure to brain damage by continuous or frequent stimulation of the amygdala or hippocampus. (7) Amygdala kindling leads to a circumscribed loss of GABAergic neurons in the ipsilateral PC, which is likely to explain the increase in excitability of PC pyramidal neurons during kindling. (8) Kindling of the amygdala or hippocampus induces astrogliosis in the PC, indicating neuronal death in this brain region. Furthermore, activation of microglia is seen in the PC after amygdala kindling. (9) Complete bilateral lesions of the PC block the generalization of seizures upon kindling from the hippocampus or olfactory bulb. Incomplete or unilateral lesions are less effective in this regard, but large unilateral lesions of the PC and adjacent endopiriform nucleus markedly increase the threshold for induction of focal seizures from stimulation of the basolateral amygdala (BLA) prior to and after kindling, indicating that the PC critically contributes to regulation of excitability in the amygdala. (10) Potentiation of GABAergic neurotransmission in the PC markedly increases the threshold for induction of kindled seizures via stimulation of the BLA, again indicating a critical role of the PC in regulation of seizure susceptibility of the amygdala. Microinjections of NMDA antagonists or sodium channel blockers into the PC block seizure generalization during kindling development. (11) Neurophysiological studies on the amygdala-PC slice preparation from kindled rats showed that kindling of the amygdala induces long-lasting changes in synaptic efficacy in the ipsilateral PC, including spontaneous discharges and enhanced susceptibility to evoked burst responses. The epileptiform potentials in PC slice preparations from kindled rats seem to originate in neurons at the deep boundary of PC. Spontaneous firing and enhanced excitability of PC neurons in response to kindling from other sites is also seen in vivo, substantiating the fact that kindling induces long-lasting changes in the PC comparable to abnormalities seen in primary foci. Taken together, these observations indicate that the PC might be part of an epileptic network which is pivotal in the genesis of kindling, facilitating and intensifying the spread of seizures from a focus in amygdala or hippocampus to cortical and subcortical regions along pathways that also are utilized in normal movements. Although direct evidence implicating the PC in the pathogenesis of human epilepsy is not yet available, the experimental data reviewed in this paper should initiate clinical studies on the potential role of this brain structure as a pacemaker or secondary focus in TLE and other types of epilepsy. Copyright © 1996 Elsevier Science Ltd.     

Unilateral low-frequency stimulation of central piriform cortex inhibits amygdaloid-kindled seizures in Sprague-Dawley rats

The central piriform cortex (cPC) is considered to be critically involved in the generation and propagation of kindled seizures. Our previous study found that low-frequency stimulation (LFS) of the cPC inhibits the development process of amygdala kindling. In this study, we determined whether unilateral LFS of the cPC had an inhibitory effect on amygdaloid-kindled seizures in Sprague-Dawley rats. When fully-kindled seizures were achieved by daily amygdala electrical stimulation (2 s train of 1 ms pulses at 60 Hz and 150-300 microA), LFS (15 min train of 0.1 ms pulses at 1 Hz and 50-150 microA) was applied to the ipsilateral or contralateral cPC 1 s after cessation of kindling stimulation for 10 days. LFS of the ipsilateral cPC significantly decreased the incidence of generalized seizures and seizure stage, and shortened cumulative afterdischarge duration and cumulative generalized seizure duration. LFS of the contralateral cPC also significantly decreased the expression of seizure stage, but had no appreciable effect on the generalized seizure incidence, cumulative afterdischarge duration and cumulative generalized seizure duration. On the other hand, LFS of the ipsilateral cPC significantly increased the afterdischarge threshold and further increased the differences of current intensity between afterdischarge threshold and generalized seizure threshold. Our data suggest that LFS of the cPC may be an effective method of inhibiting kindled seizures by preventing both afterdischarge generation and propagation. It provide further evidence that brain regions like the cPC, other than the seizure focus, can serve as targets for deep brain stimulation treatment of epilepsy.     

Further evidence for a role of the anterior claustrum in epileptogenesis

The anatomy of the claustrum (CLA) has been well characterized, but its functional role remains uncertain. The results of recent research suggest that the CLA may be part of a network of structures involved in seizure generalization, and we set out to test this idea. To test persistence, seizures were kindled in the anterior CLA. Following a 14-day suspension of kindling, all rats required only one stimulation to evoke a stage 5 seizure. In another experiment, groups of rats received bilateral lesions of the anterior CLA before and after amygdaloid kindling. We found that small lesions of the anterior CLA retard amygdaloid kindling, but do not block the expression of generalized seizures. Lesions produced after amygdaloid kindling resulted in a shorter seizure duration, but had no marked effect on seizure expression. Another group of rats was tested for transfer of kindling between the anterior CLA and contralateral amygdala. We found an asymmetrical transfer of kindling to the CLA from the amygdala wherein amygdaloid kindling facilitated subsequent kindling of the CLA but kindling of the anterior CLA failed to facilitate kindling of the amygdala. The results add support to the notion that the CLA contributes to the development of generalized limbic seizures.     

Partial kindling induces neurogenesis,activates astrocytes and alters synaptic morphology in the dentate gyrus of freely moving adult rats

A partial kindling procedure was used to investigate the correlation between focal seizure development and changes in dendritic spine morphology, ongoing neurogenesis and reactive astrogliosis in the adult rat dentate gyrus (DG). The processes of neurogenesis and astrogliosis were investigated using markers for doublecortin (DCX), 5-bromo-2-deoxyuridine (BrdU) and glial fibrillary acidic protein (GFAP). Our data demonstrate that mild focal seizures induce a complex series of cellular events in the DG one day after cessation of partial rapid kindling stimulation consisting (in comparison to control animals that were electrode implanted but unkindled), firstly, of an increase in the number of postmitotic BrdU labeled cells, and secondly, an increase in the number of DCX labeled cells, mainly in subgranular zone. Ultrastructural changes were examined using qualitative electron microscope analysis and 3-D reconstructions of both dendritic spines and postsynaptic densities. Typical features of kindling in comparison to control tissue included translocation of mitochondria to the base of the dendritic spine stalks; a migration of multivesicular bodies into mushroom dendritic spines, and most notably formation of “giant” spinules originating from the head of the spines of DG neurons. These morphological alterations arise at seizure stages 2–3 (focal seizures) in the absence of signs of the severe generalized seizures that are generally recognized as potentially harmful for neuronal cells. We suggest that an increase in ongoing neurogenesis, reactive astrogliosis and dendritic spine reorganization in the DG is the crucial step in the chain of events leading to the progressive development of seizure susceptibility in hippocampal circuits.     

Expression of vesl-1S/homer-1a, a gene associated with long-term potentiation, in the brain of the epileptic EI mouse.

The El mouse is an animal model for hereditary epilepsy. It undergoes a convulsive seizure lasting 10-30 s after vestibular stimulation, such as "tossing-up". To better characterize the effects of seizure activity in the El mouse, we examined the expression profile of vesl-1S/homer-1a mRNA in the brain. It has been shown that this mRNA is expressed after long-term potentiation or seizures induced by electrical or pharmacological stimulations. The basal level of vesl-1S mRNA was very low even in seizure-susceptible El mice. The El mouse hippocampus, but not that of ddY mice, showed a remarkable increase in vesl-1S mRNA expression in the dentate granule cell layer after seizure induced by "tossing-up". The mRNA remained for about 3 h after the seizure and disappeared after 8 h.     

Limbic epileptogenicity, cell loss and axonal reorganization induced by audiogenic and amygdala kindling in wistar audiogenic rats (WAR strain)

Audiogenic seizures are a model of generalized tonic-clonic brainstem-generated seizures. Repeated induction of audiogenic seizures, in audiogenic kindling (AuK) protocols, generates limbic epileptogenic activity. The present work evaluated associations between permanence of AuK-induced limbic epileptogenicity and changes in cell number/gluzinergic terminal reorganization in limbic structures in Wistar audiogenic rats (WARs). Additionally, we evaluated histological changes after only amygdala kindling (AmK) and only AuK, and longevity of permanence of AuK-induced limbic epileptogenicity, up to 160 days. WARs and Wistar non-susceptible rats were submitted to AuK (80 stimuli) followed by both 50 days without acoustic stimulation and AmK (16 stimuli), only AmK and only AuK. Cell counting and gluzinergic terminal reorganization were assessed, respectively, by using Nissl and neo-Timm histochemistries, 24 h after the last AmK stimulus. Evaluation of behavioral response to a single acoustic stimulus after AuK and up to 160 days without acoustic stimulation was done in another group. AuK-induced limbic epileptogenicity developed in parallel with a decrease in brainstem-type seizure severity during AuK. AmK was facilitated after AuK. Permanence of AuK-induced limbic epileptogenicity was associated with cell loss only in the rostral lateral nucleus of amygdala. Roughly 20 generalized limbic seizures induced by AuK were neither associated with hippocampal cell loss nor mossy fiber sprouting (MFS). AmK developed with cell loss in hippocampal and amygdala nuclei but not MFS. Main changes of gluzinergic terminals after kindling protocols were observed in amygdala, perirhinal and piriform cortices. AuK and AuK-AmK induced a similar number and type of seizures, higher than in AmK. AmK and AuK-AmK were associated with broader cell loss than AuK. Data indicate that permanent AuK-induced limbic epileptogenicity is mainly associated to gluzinergic terminal reorganization in amygdala but not in the hippocampus and with no hippocampal cell loss. Few AmK-induced seizures are associated to broader and higher cell loss than a higher number of AuK-induced seizures.     

Electroshock seizures protect against apoptotic hippocampal cell death induced by adrenalectomy.

Seizures evoked by electroshock induce rapid changes in the expression of several genes in the adult brain, including those encoding for neurotrophic factors. Some of the neurotrophic factors induced by brief seizures such as basic fibroblast growth factor and nerve growth factor have been shown to have neuroprotective action. We reasoned therefore that these seizures may protect against neural injury. To test this hypothesis, we examined the effect of electroshock-induced seizures on the vulnerability to cell death in the hippocampus. Cell death was induced by adrenalectomy, which results in a highly selective apoptotic neuronal death in the dentate granule cell layer of the hippocampus. Daily electroshock seizures were administered for seven days to sham-operated and adrenalectomized rats. Neuronal degeneration was evaluated by the highly sensitive and reliable cupric-silver impregnation method. Animals experiencing electroshock seizures were completely protected against adrenalectomy-induced cell death, whereas adrenalectomized animals not exposed to electroshock seizures exhibited substantial neuronal cell degeneration in the dentate granule cell layer. Daily restraint stress did not prevent the adrenalectomy-induced neuronal death, indicating that the neuroprotective effect of the seizure treatment is not accounted for by stress. We conclude that brief controlled seizure-evoked neural activation may allow the sparing of otherwise vulnerable neuronal populations in the injured adult brain. This prompts a need to explore the possibility that controlled administration of electroshock seizures may have therapeutic potential in treating neurodegenerative disorders.     

The seizure-related phenotype of brain-derived neurotrophic factor knockdown mice

The present investigation focused on the seizure-related phenotype of mice lacking one allele of brain-derived neurotrophic factor. Thresholds for producing seizures in brain-derived neurotrophic factor wild-type and brain-derived neurotrophic factor heterozygous mice were compared in several seizure models, including thresholds for electrically-induced clonic, tonic-clonic and 6 Hz limbic seizures, as well as seizures induced chemically by kainate, pilocarpine and pentylenetetrazol. In addition, the rate of amygdala kindling, as well as pre- and post-kindling seizure thresholds was determined. Seizure thresholds for clonic and tonic-clonic electrically induced seizures did not differ between brain-derived neurotrophic factor wild-type and heterozygous mice. However, heterozygous mice had higher thresholds for 6 Hz limbic seizures compared with wild-type mice. Heterozygous mice also required larger doses of kainate to produce limbic seizures. Somewhat surprisingly, heterozygous mice required significantly lower doses of pilocarpine to produce limbic seizures. However, heterozygous mice required a higher dose of pentylenetetrazol to induce twitches, but not clonic seizures, compared with wild-type mice. In addition, heterozygous mice required more current to elicit focal afterdischarges in the amygdala both pre- and post-kindling than did wild-type mice, and, heterozygous mice kindled more slowly than wild-type mice. The present findings provide additional support for the hypothesis that brain-derived neurotrophic factor is involved not only in normal excitability, but may also be involved in abnormal excitability such as occurs in seizure disorders and epileptogenesis.     

Bilateral lesions of the central but not anterior or posterior parts of the piriform cortex retard amygdala kindling in rats

The piriform cortex is thought to be involved in temporal lobe seizure propagation, such as that occurring during kindling of the amygdala or hippocampus. A number of observations suggested that the circuits of the piriform cortex might act as a critical pathway for limbic seizure discharges to assess motor systems, but direct evidence for this suggestion is scarce. Furthermore, the piriform cortex is not a homogeneous structure, which complicates studies on its role in limbic epileptogenesis. We have previously reported data indicating that the central part of the piriform cortex might be particularly involved during amygdala kindling. In order to further evaluate the role of different parts of the piriform cortex during kindling development, we bilaterally destroyed either the central, anterior or posterior piriform cortex by microinjections of ibotenate two weeks before onset of amygdala kindling. Lesions of the anterior piriform cortex hardly affected kindling acquisition, except that fewer animals exhibited stage 3 (unilateral forelimb) seizures compared to sham controls. Lesions of the central piriform cortex significantly retarded kindling, which was due to a decreased progression from stage 3 to stage 4/5 seizures, i.e. the lesioned rats needed significantly longer for the acquisition of generalized clonic seizures in the late stages of kindling development. Lesions of the posterior piriform cortex did not significantly affect kindling development.The data demonstrate that different parts of the piriform cortex mediate qualitatively different effects on amygdala kindling. The central piriform cortex seems to be a neural substrate involved in the continuous development of kindling from stage 3 to stages 4/5, indicating that this part of the piriform cortex may have preferred access, either directly or indirectly, to structures capable of supporting generalized kindled seizure expression.     

Oxidative stress is involved in seizure-induced neurodegeneration in the kindling model of epilepsy

Reactive oxygen species have been implicated in the development of seizures under pathological conditions and linked to seizure-induced neurodegeneration. There has been little direct evidence, however, of free radical production resulting from seizures. Using amygdala-kindled rats, we have examined the generation of reactive oxygen species following seizures, and their possible contribution to seizure development and seizure-induced neuronal loss. The concentrations of two products of free radical-induced lipid peroxidation, malonaldehyde and 4-hydroxy-2(E)-nonenal, were measured using colorimetric assays. Lipid peroxidation was increased in both hemispheres of kindled rats as compared to sham-operated controls. Cell death was also significantly increased in all hippocampal areas. Antioxidants (vitamin E and glutathione) prevented the rise in lipid peroxides and hippocampal neuronal death during kindling, but did not arrest the development of seizures.Thus, epileptiform activity can result in free radical production which may be one of the factors leading to cell death.     

Spike densities of the amygdala and neocortex reflect progression of kindled motor seizures

Amygdala kindling is a common temporal lobe-like seizure model. In the present study, temporal and spectral analyses of the ictal period were investigated throughout amygdala kindling in response to different behavioral seizures. Right-side amygdala was kindled to induce epileptiform afterdischarges (ADs). ADs of both the frontal cortex and amygdala were analyzed. Powers of the low (0–9 Hz)- and high (12–30 Hz)-frequency bands in response to different behavioral seizures were calculated. Densities of upward and downward peaks of spikes, which reflected information of spike count and spike pattern, throughout kindle-induced ADs were calculated. Progression was seen in the temporal and spectral characteristics of amygdala-kindled ADs in response to behaviors. Numbers of significant differences of all 1-s AD segments between two Racine’s seizure stages were significantly higher in upward and downward indexes of the temporal spike than those using the spectral method in both the amygdala and neocortex. Ability for distinguishing seizure stages was significantly higher in temporal spike density of amygdala ADs compared to those of frontal ADs. Our results showed that amygdala kindling caused spectrotemporal changes of activities in the amygdala and frontal cortex. The density of spike-related peaks had better distinguishability in response to behavioral seizures, particularly in a seizure zone of amygdala. The present study provides a new temporal index of spike’s peak density to understand progression of motor seizures in the kindling process.     

Unilateral low-frequency stimulation of central piriform cortex delays seizure development induced by amygdaloid kindling in rats

Low-frequency stimulation of the kindling site interferes with the course of kindling epileptogenesis. The present study examined the effect of unilateral low-frequency stimulation of the central piriform cortex on seizure development induced by amygdaloid kindling in rats. The ipsilateral or contralateral central piriform cortex received low-frequency stimulation (15 min train of 0.1 ms pulses at 1 Hz and 50-150 muA) immediately after termination of once daily kindling stimulation (2 s train of 1 ms pulses at 60 Hz and 150-300 microA) in the right amygdala for 30 days. Low-frequency stimulation of either the ipsilateral or contralateral central piriform cortex significantly suppressed the progression of seizure stages and reduced afterdischarge duration throughout the course of amygdaloid kindling. The marked suppression induced by low-frequency stimulation of the central piriform cortex on either side was predominantly due to the significant retardation of progression from stage 0 to stage 1 and stage 3 to stage 4 seizures. In addition, the suppressive effect of low-frequency stimulation did not disappear when the stimulation was stopped; it could persist for at least 10 days. These findings indicate that brain areas other than the kindling focus, such as the central piriform cortex on both sides, can also be used as reasonable targets for low-frequency stimulation to retard seizure development induced by amygdaloid kindling. Secondly, like the ipsilateral central piriform cortex, the contralateral central piriform cortex may also participate in the progression and secondary generalization of focal seizures. The study suggests that unilateral low-frequency stimulation of the central piriform cortex may have a significant antiepileptogenic effect, and may be helpful for exploring effective and long-lasting therapies for human temporal lobe epilepsy.     

Conventional anticonvulsant drugs in the guinea pig kindling model of partial seizures: effects of acute phenobarbital,valproate, and ethosuximide

This study addressed the anticonvulsant effects of phenobarbital, valproate, and ethosuximide in the amygdala of kindled guinea pigs to further validate this model for the screening of anticonvulsant drugs. Behavioral toxic effects were assessed at 30 min following drug administration using quantitative locomotor tests, as well as scores on a sedation and muscle relaxation rating index. The anticonvulsant efficacy of the drugs were evaluated from measurements of afterdischarge threshold (ADT), afterdischarge duration (ADD), and behavioral seizure severity (SS) during early and late phases of kindling acquisition, and in kindled guinea pigs. ADD and SS were also measured in response to both threshold and suprathreshold kindling stimulation. All drugs exerted slight to moderate sedative effects in guinea pigs on both the behavioral tests and rating index. We found that phenobarbital exhibited effective anticonvulsant properties in guinea pigs by consistently reducing ADD and SS in response to both threshold and suprathreshold kindling stimulation. Valproate exhibited effective anticonvulsant properties at threshold stimulation and less effective properties at suprathreshold stimulation. Lastly, we found that ethosuximide lacked effective anticonvulsant action at either threshold or suprathreshold kindling stimulation. Our results indicate that the guinea pig kindling model correctly predicted the actions of phenobarbital, valproate, and ethosuximide in the treatment of partial seizures. Guinea pig amygdala kindling appears to serve as a useful and valid model for partial epilepsy.     

Kindling increases aversion to saccharin in taste aversion learning

Kindling is a model in which an initially subconvulsive electrical stimulation of certain brain areas eventually develops a generalized seizure that produces behavioral and long term neuronal changes. In the present study we evaluated if kindling can modify conditioning taste aversion (CTA). In this paradigm animals acquire aversion to saccharin when it is presented as the conditioned stimulus (CS) followed by an injection of lithium chloride (LiCl) that induces a gastric irritation as the unconditioned stimulus (US). Male Wistar rats were implanted with bipolar electrodes aimed at the right amygdala (AMG) or at the right insular cortex (IC). The animals were stimulated daily until they reached stages 2-4 (intermediate) or until kindling was fully established (three consecutive stage 5 seizures). At least two weeks after kindling stimulation had ceased the animals were deprived of water for 24 h and given 10-min drinking sessions twice a day for 4 days. On day 5 (morning session) tap water was replaced by saccharin solution (0.1%), 20 min later the animals were injected with LiCl (7.5 ml/kg i.p., 0.2 M) to induce gastric malaise or taste aversion. After three more days of baseline consumption, water was substituted by a fresh 0.1% saccharin solution to test the aversion. AMG-kindling delayed the extinction of CTA. Animals with kindling in the IC had a higher retention than the sham kindling group; that is, they drank significantly less saccharin solution than the other groups. The results of the present experiment show that local modification of brain function induced by kindling stimulation can prolong the aversive effects of CTA.     

Acute and chronic responses to the convulsant pilocarpine in DBA/2J and A/J mice

Characterizing the responses of different mouse strains to experimentally-induced seizures can provide clues to the genes that are responsible for seizure susceptibility, and factors that contribute to epilepsy. This approach is optimal when sequenced mouse strains are available. Therefore, we compared two sequenced strains, DBA/2J (DBA) and A/J. These strains were compared using the chemoconvulsant pilocarpine, because pilocarpine induces status epilepticus, a state of severe, prolonged seizures. In addition, pilocarpine-induced status is followed by changes in the brain that are associated with the pathophysiology of temporal lobe epilepsy (TLE). Therefore, pilocarpine can be used to address susceptibility to severe seizures, as well as genes that could be relevant to TLE. A/J mice had a higher incidence of status, but a longer latency to status than DBA mice. DBA mice exhibited more hippocampal pyramidal cell damage. DBA mice developed more ectopic granule cells in the hilus, a result of aberrant migration of granule cells born after status. DBA mice experienced sudden death in the weeks following status, while A/J mice exhibited the most sudden death in the initial hour after pilocarpine administration. The results support previous studies of strain differences based on responses to convulsants. They suggest caution in studies of seizure susceptibility that are based only on incidence or latency. In addition, the results provide new insight into the strain-specific characteristics of DBA and A/J mice. A/J mice provide a potential resource to examine the progression to status. The DBA mouse may be valuable to clarify genes regulating other seizure-associated phenomena, such as seizure-induced neurogenesis and sudden death.