Regional changes in gene expression after limbic kindling.

Repeated electrical stimulation results in development of seizures and a permanent increase in seizure susceptibility (kindling). The permanence of kindling suggests that chronic changes in gene expression are involved. Kindling at different sites produces specific effects on interictal behaviors such as spatial cognition and anxiety, suggesting that causal changes in gene expression might be restricted to the stimulated site. We employed focused microarray analysis to characterize changes in gene expression associated with amygdaloid and hippocampal kindling. Male Long-Evans rats received 1 s trains of electrical stimulation to either the amygdala or hippocampus once daily until five generalized seizures had been kindled. Yoked control rats carried electrodes but were not stimulated. Rats were euthanized 14 days after the last seizures, both amygdala and hippocampus dissected, and transcriptome profiles compared. Of the 1,200 rat brain-associated genes evaluated, 39 genes exhibited statistically significant expression differences between the kindled and non-kindled amygdala and 106 genes exhibited statistically significant differences between the kindled and non-kindled hippocampus. In the amygdala, subsequent ontological analyses using the GOMiner algorithm demonstrated significant enrichment in categories related to cytoskeletal reorganization and cation transport, as well as in gene families related to synaptic transmission and neurogenesis. In the hippocampus, significant enrichment in gene expression within categories related to cytoskeletal reorganization and cation transport was similarly observed. Furthermore, unique to the hippocampus, enrichment in transcription factor activity and GTPase-mediated signal transduction was identified. Overall, these data identify specific and unique neurochemical pathways chronically altered following kindling in the two sites, and provide a platform for defining the molecular basis for the differential behaviors observed in the interictal period.

Amygdaloid kindling is anxiogenic but fails to alter object recognition or spatial working memory in rats.

Kindling in rats produces enduring behavioral changes that parallel the psychobehavioral disturbances frequently accompanying temporal lobe epilepsy. Some evidence suggests that the site of kindling is an important determinant of the type of behavioral changes observed following kindling, although this variable has not been systematically investigated. In the present experiments, the effects of amygdaloid kindling were assessed on a battery of behavioral tests we used previously to assess the effects of kindling in dorsal hippocampus or perirhinal cortex. Three generalized seizures were kindled with stimulation in or near the basolateral amygdala. One week later, rats were tested successively on measures of anxiety, activity, object recognition memory, and spatial working memory over a period of 3 weeks. Amygdaloid kindling produced increased anxiety, but spared all other behaviors assessed. This pattern of results is partially distinct from the previously described effects of perirhinal cortical kindling, which increases anxiety but also impairs object recognition memory, and is completely distinct from dorsal hippocampal kindling, which selectively increases activity and impairs spatial working memory. The observations suggest that kindling of distinct highly interconnected temporal lobe sites produces distinct patterns of behavioral comorbidity. The underlying mechanisms are thus most likely localized to intrinsic circuits at the site of seizure origination.

Susceptibility to kindling and neuronal connections of the anterior claustrum.

The claustrum has been implicated in the kindling of generalized seizures from limbic sites. We examined the susceptibility of the anterior claustrum itself to kindling and correlated this with an anatomical investigation of its afferent and efferent connections. Electrical stimulation of the anterior claustrum resulted in a pattern of rapid kindling with two distinct phases. Early kindling involved extremely rapid progression to bilaterally generalized seizures of short duration. With repeated daily kindling stimulations, early-phase generalized seizures abruptly became more elaborate and prolonged, resembling limbic-type seizures as triggered from the amygdala. We suggest that the rapid rate of kindling from the anterior claustrum is an indication that the claustrum is functionally close to the mechanisms of seizure generalization. In support of our hypothesis, we found significant afferent, efferent, and often reciprocal connections between the anterior claustrum and areas that have been implicated in the generation of generalized seizures, including frontal and motor cortex, limbic cortex, amygdala, and endopiriform nucleus. Additional connections were found with various other structures, including olfactory areas, nucleus accumbens, midline thalamus, and brainstem nuclei including the substantia nigra and the dorsal raphe nucleus. The anatomical connections of the anterior claustrum are consistent with its very high susceptibility to kindling and support the view that the claustrum is part of a forebrain network of structures participating in the generalization of seizures.

Dorsal hippocampal kindling produces a selective and enduring disruption of hippocampally mediated behavior.

Kindling produces enduring neural changes that are subsequently manifest in enhanced susceptibility to seizure-evoking stimuli and alterations in some types of behavior. The present study investigated the effects of dorsal hippocampal (dHPC) kindling on a variety of behaviors to clarify the nature of previously reported effects on spatial task performance. Rats were kindled twice daily with dHPC stimulation until three fully generalized seizures were evoked. Beginning 7 d later and on successive days, rats were tested in an elevated plus maze, a large circular open field, an open field object exploration task, and a delayed-match-to-place (DMTP) task in a water maze to assess anxiety-related and activity-related behavior (tasks 1 and 2), object recognition memory (task 3), and spatial cognition (task 4). Kindling disrupted performance on the DMTP task in a manner that was not delay dependent and produced a mild enhancement of activity-related behaviors in the open field task but not the elevated plus maze. All other aspects of testing were spared. These findings indicate that dHPC kindling produces enduring and selective effects on behavior that are consistent with a restricted disruption of hippocampally mediated functions. Possible bases for these effects are changes in local NMDA receptor function and/or changes in local inhibition, which might alter the optimal conditions for experience-dependent induction of intrahippocampal plasticity. This preparation may be useful for studying the mechanisms of mnemonic dysfunction associated with temporal lobe epilepsy and may offer unique insights into the mechanisms underlying normal hippocampal function.

The mnemonic effects of kindling.

Kindling produces enduring changes in the brain that are evident in not only enhanced susceptibility to seizure-evoking stimuli but also alterations in non-epileptic behaviors or functions. The present review examines the effects of kindling on one class of non-epileptic functions, learning and memory, and explores the dependence of these effects on variables such as the site of kindling, extent of kindling, and interval between kindling and testing. Current research shows that kindling is capable of altering performance on a variety of tasks including those that require spatial cognition, aversive conditioning, and object-related cognition and that non-mnemonic effects are unlikely, in at least some cases, to underlie these effects. Consideration of the conditions under which these effects are observed indicates a distinct relation between specific mnemonic effects and both the site and extent of kindling. Continued characterization of the mnemonic effects of kindling should provide a theoretical framework to guide discovery of their underlying mechanisms, which, in turn, may lead to rational therapy for mnemonic dysfunction associated with epilepsy and insights into the mechanisms of learning and memory.

Characteristics of accelerated kindling after depletion of noradrenaline in adult rats.

The characteristics and pattern of the acceleration of kindling produced by 6-hydroxydopamine-induced depletion of noradrenaline (NA) were investigated in adult rats receiving electrical stimulation of the amygdala once daily. NA-depleted rats developed generalized seizures very rapidly by spending significantly less time than controls in the early stages of nonconvulsive or partial seizures, and they required significantly less cumulative total time in afterdischarge than controls to develop generalized seizures. The results suggest that attempts to identify noradrenergic correlates of kindling should be directed to the early stages of seizure development.

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.

Time course of mossy fiber sprouting following bilateral transection of the fimbria/fornix.

Lesions of the fimbria/fornix (FF) lead to a variety of epileptiform changes in hippocampal activity. In epilepsy, as well as in several experimental preparations of epilepsy, aberrant sprouting of the dentate gyrus mossy fibers (MF) is observed and has been hypothesized to play a critical role in the generation of seizure activity. We therefore sought to determine whether MF sprouting also follows FF transections. FF transections did indeed lead to MF sprouting, which became apparent at 14 days and reached asymptotic levels at 28 days post-lesion. These results indicate a possible mechanism for the epileptiform activity seen following FF lesions and provide an additional example of the diverse epileptogenic treatments that are accompanied by MF sprouting.

Mossy fiber sprouting is dissociated from kindling of generalized seizures in the guinea-pig.

Controversy surrounds whether aberrant mossy fiber sprouting in the hippocampus is necessary for the establishment of seizure states. We investigated the association between mossy fiber sprouting and kindling in guinea-pigs, using either single-site or alternate-site stimulation. Kindling with single-site amygdaloid stimulation did not induce significant sprouting, despite the development of partial seizures. In contrast, single-site septal and alternating amygdaloid-septal stimulation produced moderate but significant sprouting in about 60% of animals that failed to develop stage 5 generalized seizures. Since the magnitude of sprouting was similar despite striking differences in the intensity of seizures that developed, we conclude that mossy sprouting is not causally associated with seizure development.

Forebrain noradrenaline and oral self-administration of ethanol by rats.

The effects of 6-hydroxydopamine-induced depletion of forebrain noradrenaline (NA) on oral intake of ethanol were studied in male Wistar rats. Prior depletion of NA produced a smaller and significantly less variable intake of a concentrated solution of ethanol than that of control rats, and this effect was not accompanied by hyperreactivity to aversive solutions of quinine. NA-depleted rats also displayed rejection 'thresholds' for ethanol solutions that were significantly lower than those of controls. Depletion of forebrain NA did not, however, affect the punishing effects of ethanol injections measured in the conditioned taste aversion paradigm. In contrast to these effects of NA depletion on initiation of ethanol intake, depletion of forebrain NA after a preference for ethanol had been established failed to affect subsequent intake of ethanol. These results suggest that forebrain NA is involved in the initiation of ethanol intake by naive rats but not in the maintenance of established patterns of intake by experienced rats. Possible mechanisms for this differential involvement of NA are discussed.

Kindling of hippocampal field CA1 impairs spatial learning and retention in the Morris water maze.

We used two procedures to assess the spatial learning and memory of rats in the Morris water maze task subsequent to kindling of hippocampal field CA1: (1) seizures were kindled with stimulation of CA1 prior to training in the water maze (acquisition); and (2) maze training was imposed until performance stabilized, seizures were kindled with stimulation of CA1, and then performance in the maze was reassessed (retention). In both conditions, behavioral testing occurred 24 h after the last kindled seizure. When the effects of CA1 kindling on acquisition were tested, we found that kindling of generalized seizures with stimulation of field CA1 (kindling), but not kindling of non-convulsive or partial seizures (partial kindling), produced deficits in the water maze. When the effects of CA1 kindling on retention were tested, however, we found that kindling of either partial or generalized seizures produced deficits in the water maze. The results suggest that the processing of spatial information is vulnerable to the long-lasting changes in neural excitability associated with kindling.

Limbic seizures, but not kindling, reversibly impair place learning in the Morris water maze.

We investigated the effects of kindling and kindled seizures in different limbic structures on place and cue learning in the Morris water maze. The triggering of seizures by stimulation of the perforant path, septum, or amygdala prior to daily training impaired place learning, but had little effect on visible platform training or swim speed. Seizures triggered by stimulation of the medial perforant path after daily training also impaired place learning. Conversely, place learning proceeded normally in rats tested 24 h after kindling triggered by stimulation of the perforant path, septum, or amygdala, indicating that kindling per se does not affect place learning. Each group was able to learn the location of a reversed platform when pretraining seizures were discontinued; and perforant path and septal kindled rats, but not amygdaloid kindled rats, were impaired at learning the location of a reversed platform when seizures were triggered before training. The results confirm previous reports that limbic seizures produce amnesia, but they contradict the finding that hippocampal kindling impairs learning on tasks sensitive to hippocampal lesions.

Epileptiform effects of met-enkephalin, beta-endorphin and morphine: kindling of generalized seizures and potentiation of epileptiform effects by handling.

Repeated spaced injection of small amounts of beta-endorphin or Met-enkephalin into the hippocampus or posterior amygdala of the rat led to the development of kindled generalized convulsions. Similar injection of morphine into the hippocampus or anterior amygdala resulted in epileptiform spiking followed by tolerance. The epileptiform spiking and convulsive behavior varied in a dose-related manner. Naloxone blocked or greatly attenuated the electrographic seizure and convulsive behavior. Prior kindling with beta-endorphin or Met-enkephalin significantly facilitated electrical kindling of the amygdala. Handling or conspecific threat potentiated the epileptiform spiking and convulsive behavior in some cases. The results indicate that the epileptogenic response to intracerebrally applied opioid peptides is site-specific within the rat brain, and they support the idea that endogenous opioid mechanisms may play a role in convulsive seizures. They also suggest a possible opiate-based mechanism for the stress-induced exacerbation of seizures.

Relations between amygdaloid and anterior neocortical kindling.

Primary kindling of generalized seizures with amygdaloid stimulation facilitated the subsequent kindling of generalized seizures with neocortical stimulation. Similarly, initial kindling of generalized seizures with anterior neocortical stimulation facilitated the kindling of generalized amygdaloid seizures. Unexpectedly, there were striking differences in the reliability with which seizures could be triggered by amygdaloid and neocortical stimulation. The results support the idea that there is a single mechanism that is responsible for seizure generalization.

Intra-amygdaloid infusions of clonidine retard kindling.

It has been reported previously that systemic administration of clonidine, an agonist of alpha-2 noradrenergic receptors, significantly retards amygdaloid kindling by delaying the emergence from partial seizure. We examined the effect of either systemic administration (i.p.) or intra-amygdaloid infusions of clonidine on the kindling of seizures with electrical stimulation of the amygdala. Rats received either low-frequency stimulation of the amygdala, to induce rapid kindling, or conventional high-frequency stimulation. Clonidine and electrical stimulation were administered once every 48 h. We observed a significant retardation of kindling in rats receiving i.p. injections of clonidine (0.1 mg/kg) or infusions of clonidine in concentrations of 10(-7)-10(-4) M, regardless of the stimulation frequency. The prophylactic effect was due to a delay in the progression out of partial seizure. The effect was specific to the amygdala/pyriform region, because infusions of clonidine dorsal to the amygdala were without effect. Intra-amygdaloid clonidine had little effect on established generalized seizures, suggesting that it was producing a genuine prophylactic effect against kindling. We conclude that the subpopulation of alpha-2 adrenoceptors in the amygdala/pyriform region contributes to the antiepileptogenic effect observed after systemic administration of clonidine.

Attenuation of self-stimulation from substantia nigra but not dorsal tegmental noradrenergic bundle by lesions of sulcal prefrontal cortex.

Rats that self-stimulated from electrodes implanted in either the substantia nigra pars compacta (SNC) or the dorsal tegmental noradrenergic bundle (DTB) received bilateral electrolytic lesions of the prefrontal cortex dorsal to the rhinal sulcus. Immediately after the lesions, animals with SNC electrodes (n = 9) showed significant and permanent reductions in self-stimulation rates. The average reduction in the SNC group was 67% of prelesion bar-pressing scores. In contrast, rats with DTB electrodes (n = 11) were not significantly affected by similar sulcal lesions. Subsequent examination of the brains for prograde degeneration using procedure I of Fink and Heimer 11 revealed a descending system of sulcal efferents that was very dense in the region of the SNC but only scattered in the area of the DTB. The close relation between the effects of sulcal lesions on self-stimulation and the resultant density of degeneration as a function of stimulation site is discussed in terms of the contribution of prefrontal cortex to self-stimulation in general, and of the implications for the catecholamine hypothesis of self-stimulation in particular.

Kindling antagonism: interactions of the amygdala with the piriform, perirhinal, and insular cortices.

We employed the kindling antagonism paradigm to characterize the role of the amygdala (AM), piriform cortex (PIR), perirhinal cortex (PRH), and insular cortex (INS) in the propagation and expression of generalized seizures. Alternating kindling stimulation was delivered to the AM and either the PIR, PRH, or INS. We found that kindling from the AM was retarded by stimulation only of the deep layers of both the INS and anterior PRH. Kindling antagonism was specific to the anterior cortical regions, since neither the posterior PRH or PIR were capable of antagonizing AM kindling. The results strongly implicate the deep layers of anterior limbic cortex in establishment of antagonism of AM kindling.

Anterior neocortical kindling in vasopressin-deficient rats.

Kindling of seizures with stimulation of anterior neocortex was examined in control rats and in Brattleboro rats deficient in arginine-vasopressin (AVP). There were no significant differences between control rats, homozygous Brattleboro rats, and heterozygous Brattleboro rats in the rate and pattern of kindling of generalized seizures. Thus AVP is not critically involved in anterior neocortical kindling.

Role of forebrain catecholamines in amygdaloid kindling.

The rate and pattern of seizure development provoked by repeated electrical stimulation of the amygdala (kindling) was assessed in rats that had been pretreated with intracerebral injections of the selective catecholaminergic neurotoxin 6-hydroxy-dopamine. Rats with selective depletion of forebrain noradrenaline displayed a highly significant facilitation of both primary-site and secondary-site kindling, whereas no such effect occurred in rats with selective depletion of forebrain dopamine. The facilitative effects of noradrenaline depletion were apparently related to disinhibition of the spread of seizure discharge from the stimulated site rather than to increased epileptogenicity in the stimulated site itself. These results are consistent with previous evidence that noradrenaline reduces the susceptibility of the central nervous system to epileptiform activity, and they suggest that a lessening of seizure-suppressant noradrenergic function in the forebrain might be part of the mechanism underlying kindling.