Hippocampal Kindling Research Articles

Specific Functions of Grafted Locus Coeruleus Neurons in the Kindling Model of Epilepsy

Grafts of fetal noradrenaline (NA)-rich locus coeruleus (LC) tissue implanted bilaterally into the hippocampus in 6-hydroxydopamine (6-OHDA) treated, hyperexcitable rats retard seizure development in hippocampal kindling. We report that bilateral intrahippocampal LC grafts failed to affect the severity of seizures when implanted in 6-OHDA-treated, already kindled rats. Furthermore, a noradrenergic hyperinnervation of the hippocampus, produced by grafting fetal LC tissue into the intact brain, did not influence hippocampal kindling development. Kindling leads to long-term reduction in basal hippocampal NA release from the intrinsic locus coeruleus system as monitored using intracerebral microdialysis. This deficit could be reversed by intrahippocampal LC grafts implanted into previously kindled rats. However, these unilateral grafts had no effect on the severity of the kindled convulsions despite a noradrenergic hyperinnervation of the hippocampus around the grafts. In conclusion, these data show a distinct functional specificity of intrahippocampal LC grafts in the kindling model of epilepsy. Similar to the intrinsic noradrenergic system, the grafted neurons have a major inhibitory influence on seizure development but are ineffective against established convulsions.

Kindling in the perirhinal cortex

In vitro experiments have indicated that the perirhinal cortex is highly excitable and its relationship to the basolateral amygdala and piriform cortex is altered by previous amygdala or dorsal hippocampal kindling. As a result, we felt it was important to assess the excitability of the perirhinal cortex in vivo by comparing its kindling profile to that of the basal amygdala, piriform cortex or dorsal hippocampus. We observed that the after-discharge (AD) threshold of the perirhinal cortex was higher than the other 3 structures but the AD duration was not different. Subsequently, the perirhinal cortex kindled more rapidly than the other 3 structures, and with extremely short latencies to onset of forelimb clonus. With the view that synchronized discharge in the perirhinal-piriform area provides the critical trigger for limbic kindled convulsions, the relationship of kindling rate and convulsion latencies and durations between the 4 structures was discussed.

Partial hippocampal kindling increases paired-pulse facilitation and burst frequency in hippocampal CA1 neurons

For up to 3 weeks after 15 evoked afterdischarges (partial kindling) in the hippocampus in vivo, paired-pulse facilitation of the CA1 apical dendritic excitatory postsynaptic potentials (EPSPs), recorded from single neurons in vitro, was significantly larger in neurons of kindled than control rats. Partial kindling did not significantly affect the resting membrane potential, the threshold or size of the action potential (AP), the fast afterhyperpolarization, input resistance, time constant or the EPSP threshold. The number of APs induced within the initial 20 ms of a long-duration 0.5-nA depolarizing current was significantly higher in the kindled than control neurons. The increase in paired-pulse facilitation and intrinsic spiking in hippocampal CA1 after partial kindling may contribute to an increase in seizure susceptibility.

Increased production of the TrkB protein tyrosine kinase receptor after brain insults

The protein-tyrosine kinases Trk, TrkB, and TrkC are signal-transducing receptors for a family of neurotrophic factors known as the neurotrophins. Here we show that seizures induced by hippocampal kindling lead to a rapid, transient increase of trkB mRNA and protein in the hippocampus. TrkB is a component of a high affinity receptor for brain-derived neurotrophic factor (BDNF). No change was detected in mRNAs for Trk or TrkC, components of the high affinity nerve growth factor or neurotrophin-3 receptors, respectively. trkB mRNA was also transiently increased in the dentate gyrus following cerebral ischemia and hypoglycemic coma; these treatments had no effect on trk and trkC mRNAs. The increase in trkB m NA and protein showed the same time course and distribution as the increase in BDNF mRNA. These data suggest that BDNF and its receptor may play a local role within the hippocampus in kindling-associated neural plasticity and in neuronal protection following epileptic, ischemic, and hypoglycemic insults.

Effects of long-stimulus intervals and scopolamine administration on hippocampal kindling

Using the low-frequency kindling procedure, we studied the effects of periodic 2-week stimulus-free intervals and chronic scopolamine administration on hippocampal kindling seizure development. In Experiment 1, rats were divided into two groups, interval group and no-interval group. In the interval group a 2-week stimulus interval was set after every five consecutive daily stimulations until the 21st stimulation. The number of stimulating pulses required for the triggering of epileptic afterdischarge, pulse-number threshold (PNT), was used as an indicator of the seizure thershold. PNT, afterdischarge duration (ADD) and behavioral seizure stage (BSS) of each induced seizure in the initial stage of kindling, kindling rate, seizure parameters at the completion of kindling were recorded and compared to the values of the no-interval, control group. Our result showed that PNT at the 6th stimulation, the first stimulation after the first 2-week stimulus interval increased significantly compared to control. Other seizure parameters did not differ significantly between the two groups. In Experiment 2 scopolamine hydrobromide, 0.5 and 1.0 mg/kg i.p., was administered 1 h before each electrical stimulation until each rat showed the stage-3 seizure. PNT, ADD and BSS in the initial stage of kindling, kindling rate for the stage-3 and -5 seizures, seizure parameters at the first stage-3 and -5 were recorded and compared to the values of saline-treated, control group. Although scopolamine 1.0 mg/kg increased PNT at the 5th stimulation compared to control, no other significant changes of the seizure parameters were found by scopolamine. Together with our previous studies, these results support our finding that physiological and pharmacological characteristics of the reduction of seizure threshold, but not of the development of induced seizures in the hippocampal kindling process are quite similar to those of long-term potentiation. Clinically our present findings suggest that occurrence of the first several seizures causes a long-lasting reduction of seizure threshold which should make the disease more intractable against treatment.

Anti-somatostatin antibody enhances the rate of hippocampal kindling in rats

A somatostatin-specific antibody (Ab)(1:250) was continuously infused into the stimulated dorsal hippocampus of rats from 4 days before to 26 days after the beginning of kindling or until the first stage 5. Controls received boiled Ab. The number of stimulations to the first stage 5 were reduced by 41 ± 4% (P < 0.01,Student'st-test) in animals infused with the Ab compared to controls. The cumulative after-discharge in the stimulated hippocampus was slightly, although not significantly, reduced. Kindling was not affected when the Ab was infused only during the first 10 stimulations (stage 2). Histological analysis showed no neurotoxic effects in the hippocampus as a consequence of Ab infusion.

Are differences in dorsal hippocampal kindling related to amygdala-piriform area excitability?

It has been suggested that several structures associated with the amygdala-piriform (A-P) area are important, if not critical, for convulsive generalization of limbic seizures. In experiment 1, when examining the development of convulsive seizures kindled from the dorsal hippocampus (cornus ammonis; DH), a broad range of kindling rates was observed. Independent of electrode location within the hippocampus, kindling rates were correlated positively with both local and, more dramatically, distant excitability (measured by the duration of the primary and secondary hippocampal afterdischarges, respectively) at all stages of epileptogenesis. Because kindling rates were bimodally distributed, we bisected the distribution into ‘faster’ and ‘slower’ kindling rats. Here we examined the magnitude of both electrophysiological and behavioral differences between ‘faster’ and ‘slower’ rats and their changes over time. The ‘faster’ rats had longer primary and secondary afterdischarge (AD) durations than ‘slower’ rats throughout all stages of kindling. With the appearance of generalized convulsions, the ‘faster’ rats showed longer latencies to clonus onset, with longer clonus and AD durations than ‘slower’ rats. Also, the generalized convulsions of ‘faster’ rats appeared during a much enlarged secondary AD period, while ‘slower’ rats convulsed during primary AD. In both groups, convulsions were invariably associated with increased discharge in A-P associated structures. We interpreted the differences between ‘faster’ and ‘slower’ DH rats to reflect genetic differences in excitability in both local and A-P associated structures. If the DH kindling profile of the ‘faster’ rats differed from ‘slower’ rats largely because of naturally greater excitability in A-P associated structures, then experimentally increased excitability in those structures (via amygdala kindling) in a random sample of rats should duplicate much of the ‘faster’ DH kindling profile. In experiment 2, this outcome was observed. In conclusion, we suggest that either natural or induced differences in the excitability of A-P associated structures affect both the genesis and the profile of convulsive generalization of limbic kindled seizures.

Hippocampal kindling increases the expression of glutamate receptor-A flip and -B flip mRNA in dentate granule cells

The level of the mRNAs encoding the AMPA-selective glutamate receptors-A and -B, alternatively splice variants, Flip and Flop, was studied by in situ hybridization in the brains of rats kindled by Schaffer collateral/commissural-fiber stimulation. In comparison to control animals, the expression level of the Flip variant of both GluR-A and GluR-B mRNAs was bilaterally enhanced in the dentate granule neurons of kindled animals 24 h after last-generalized seizure, whereas no obvious alterations were observed in the GluR-A Flop and GluR-B Flop mRNA variants. In kindled animals, studied 1 month after the last seizure, GluR-A Flip and GluR-B Flip mRNA had returned to control levels. We suggest that these changes may result in an enhanced glutamate receptor sensitivity in the fascia dentata during kindling.

Somatostatin release is enhanced in the hippocampus of partially and fully kindled rats

The release of somatostatin (somatostatin-like immunoreactivity) from hippocampal slices during the development of hippocampal kindling in rats was measured under resting and depolarizing conditions. Preliminary experiments in naive rats showed that the spontaneous efflux of somatostatin (4.0 ± 0.3 fmol/ml every 10 min) was independent of external Ca 2+ but was reduced to 71.5 ± 6% of baseline ( P < 0.05) during 20 min incubation with 5 μM tetrodotoxin. Neuronal depolarization with 25, 50 and 100 mM KC1 induced a Ca 2+-dependent somatostatin release, respectively 4.3 ± 0.4, 16.7 ± 1.6 and 22.0 ± 1.3 times baseline ( P < 0.01). Veratridine caused a dose-dependent Ca 2+ and tetrodotoxin (5 μM) sensitive release ranging from 6.5 ± 0.1 to 13.0 ± 1.4 times baseline at 1.4μM and 50 μM respectively ( P < 0.01). One week after the last of three consecutive stage 5 seizures (full seizure expression) or 48 h after the last stage 2 stimulation (preconvulsive stage), 50 mM KC1-induced somatostatin release was significantly higher (1.8 ± 0.1, P <0.01) than in shams (animals implanted with electrodes but not stimulated) in the stimulated and contralateral hippocampus. Somatostatin release measured under resting conditions was increased by 1.5 times in the stimulated hippocampus at stage 2 ( P < 0.05) and by 2.2 and 1.7 times in both hippocampi at stage 5 ( P < 0.01). Forty-eight hours after the induction of a single afterdischarge no significant changes were found in either spontaneous or 50 mM KCl-induced release of somatostatin. The tissue concentration of somatostatin did not vary significantly in either side of the hippocampus at stage 2 compared to shams but at stage 5 increases of 56% and 71% respectively ( P < 0.05) were found in the stimulated and contralateral hippocampus. The present results provide the first evidence of enhanced neuronal release of somatostatin during kindling which suggests that this neuropeptide may play some role in epileptogenesis.

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.

Two types of neuroplasticities in the kindling phenomenon: effects of chronic MK-801 and methamphetamine

Using the low-frequency kindling technique, we studied the effects of chronic MK-801 and chronic methamphetamine (MAP) administration on hippocampal kindling seizure development. In experiment 1, MK- (0.05, 0.1 mg/kg i.p.) was administered 2 h before each electrical stimulation until kindling developed into stage-3 seizure. In experiment 2, we started daily electrical stimulations two weeks after the last injection of chronic MAP administration (6 mg/kg/day, 14 days). The number of stimulating pulses required for the triggering of epileptic afterdischarge (pulse-number threshold, PNT) was used as an indicator of the seizure threshold. PNT, afterdischarge duration (ADD) and behavioral seizure stage (BSS) of each induced seizure in the initial stage of kindling; the kindling rates for stage 3 and stage 5 seizures; seizure parameters at the completion of kindling of the drug-treated groups were recorded and compared to the values of each saline-treated control group. Our result showed that MK-801 administration prior to each electrical stimulation selectively and significantly increased PNT in the initial stage of kindling without affecting other seizure parameters. Chronic pretreatment of MAP caused a selective and significant decrease of PNT of the first two stimulations in the kindling process. Taken together with the previous studies, these results suggest that long-term potentiation plays an important role in the development of the excitability of seizure focus but not of the induced seizure's propagation in the hippocampal kindling phenomenon. Clinically MK-801 seems to be a more efficacious drug in preventing the induction of seizures than in suppressing the acquired seizures.

Hippocampal kindling leads to different changes in paired-pulse depression of local evoked field potentials in CA1 area and in fascia dentata

Monosynaptic evoked field potentials (EPs) in response to paired-pulse stimulation (20 ms interval) were recorded in area CA1 and fascial dentata of the same animal in the course of development of a kindled focus in the CA1 region. A significant reduction of paired pulse depression in response to medium and high stimulation intensity was found in CA1. A similar change was found in the fascia dentata in response to medium intensity stimulation of the angular bundle. In contrast, at high intensity, paired pulse depression was enhanced in the fascia dentata in the course of kindling. These results indicate that kindling epileptogenesis is accompanied by regionally different changes in recurrent inhibition: a reduction in CA1 and intensity dependent changes in fascia dentata.

Ionotropic excitatory amino acid receptors in discrete brain regions of kindled rats

A study was performed to examine the specific binding of excitatory amino acid (EAA) receptor subtypes in 5 brain regions of rats kindled from the amygdala or hippocampus, using extensively washed and Triton X-100-treated membranes. Seven days after the last amygdala kindled seizure, [ 3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate ([ 3H]MK-801) binding, which labels N- methyl- D-aspartate (NMDA)-sensitive receptor-linked cation channels, decreased significantly only in the amygdala of kindled rats compared to that of controls under equilibrium assay conditions. There was no significant change in [ 3H]MK-801 binding in the amygdala or hippocampus 7 days after the last hippocampal kindled seizure, or 28 days after the last amygdala kindled seizure. Nor was there a significant change in NMDA-sensitive [ 3H]glutamate, strychnine-insensitive [ 3H]glycine, [ 3H]spermidine, [ 3H]kainate or [ 3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([ 3H]AMPA) binding in any brain region 7 days after the last amygdala kindled seizure, or in the hippocampus 28 days after the last amygdala kindled seizure. These results indicate that [ 3H]MK-801 binding sites labeling NMDA-sensitive receptor-linked cation channels in the amygdala undergo downregulation only transiently, but that none of the subcomponents of the NMDA receptor macromolecular complex exhibit enduring changes at steady state following the completion of amygdala kindling.

Role of serotonin receptor subtype in seizures kindled from the feline hippocampus

This study assessed the effects of a serotonin (5-HT)1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT2 agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and a 5-HT2 antagonist ketanserin on fully kindled seizures from the hippocampus. 8-OH-DPAT produced a marked suppression in hippocampal kindled seizures, while producing behavioral signs similar to those seen in the 5-HT syndrome. Although DOI and ketanserin did not affect kindled focal epileptic activity, DOI shortened and ketanserin prolonged the latency to onset of generalized convulsions. Our data suggest that 5-HT1A receptors play an inhibitory role in the generation of hippocampal seizures, whereas 5-HT2 receptors may participate in kindled seizure generalization from this region.

Hippocampal kindling induced paired-pulse depression in the dentate gyrus and paired-pulse facilitation in CA3

Rats received high frequency (100 Hz, 1 s) hippocampal stimulations that evoked afterdischarges (ADs) in a partial kindling model of epilepsy. Kindled rats were given 15 ADs over 3 days. Control rats received the same stimulation pulses at 0.17 Hz (low frequency stimulation, LFSs). Subsequent to in vivo stimulation, hippocampal slices were obtained from the rats and extracellular field responses were recorded in the CA3 cell layer following CA1 stratum radiatum stimulation, and in the granule cell layer of the dentate gyrus (DG) following perforant path stimulation. At the DG, the paired-pulse facilitation of the population spike was depressed in the kindled than the control group, on 1 day or 23 days post ADs/LFSs. The excitatory postsynaptic potential (EPSP) responses in DG following single or paired pulses were not different between kindled and control slices. At CA3, paired-pulsefacilitation of both the population spike and the population EPSP was increased in the kindled compared to the control group.

Positive transfer of audiogenic kindling to electrical hippocampal kindling in rats

Audiogenic seizures in genetically susceptible rodents are provoked by intense acoustic stimulations which result in a tonic seizure associated with a short flattening of the EEG. These seizures have been shown to involve primarily brainstem structures. Daily exposure to sound for 30–40 days produced a permanent change in the evoked seizure with development of facial myoclonias, rearing and falling, or of tonic-clonic seizures accompanied by high amplitude cortical spike-and-wave discharges. Kindled audiogenic seizures appear similar to seizures kindled from amygdala or hippocampus, suggesting that repeated auditory stimulations cause a progressive propagation of the epileptic discharge toward limbic structures. To verify this hypothesis, the behavioral and EEG development of electrical hippocampal kindling has been studied in 7 non epileptic controls (NE), 8 acoustic susceptible (AS), and 8 audiogenic kindled rats (KAS). The behavioral and EEG development of the electrical hippocampal kindling was similar in the AS and the NE rats. However, 2 animals in the AS group but no controls exhibited behavioral running and bouncing during the course of hippocampal kindling. In the KAS group, the hippocampal kindling was clearly facilitated as compared to NE and AS: behavioral stage ⩾ 5 was reached in a mean of 4 stimulations in KAS versus 30 and 22 stimulations respectively in NE and AS groups. This positive transfer phenomenon suggests that during kindling of audiogenic seizures, epileptic discharge spreads from the brainstem to the forebrain and progressively involves the hippocampus.

Regional Forebrain Noradrenalin Release in Response to Focal and Generalized Seizures Induced by Hippocampal Kindling Stimulation.

In vivo microdialysis was used to monitor noradrenalin (NA) release in the rat hippocampus, sensorimotor cortex and amygdala in response to seizures induced by electrical kindling stimulation in the hippocampus. Generalized seizures increased NA output in the hippocampus five-fold above baseline level (as assessed with 2-min sampling periods). The peak value was seen 2 - 4 min after onset of seizure activity and baseline was reached after another 6 - 8 min. In the sensorimotor cortex, there was a seven-fold increase showing a similar time-course. Focal hippocampal seizures gave rise to three-fold and 80% increases above baseline in the hippocampus and sensorimotor cortex, respectively. A unilateral knife transection of the dorsal noradrenergic bundle reduced hippocampal NA release induced by focal seizures by 53%. In animals subjected to 30 stimulus-evoked seizures with 5-min intervals ('rapid kindling'), maximal NA output was observed after the third seizure in both hippocampus (237% increase) and amygdala (122% increase). NA levels tapered off with repeated stimulation and reached baseline after nine stimulations in the hippocampus; in the amygdala, the NA output was still slightly elevated at the end of the stimulation period. These results indicate that there is a general activation of the locus coeruleus system during focal as well as generalized seizures, as evidenced by marked increases in transmitter release from noradrenergic terminals in all forebrain areas studied. NA output in areas exhibiting seizure activity is dependent on impulse flow in locus coeruleus neurons and probably also on local regulatory mechanisms active at the noradrenergic terminal level. The increase in inhibitory noradrenergic transmission in both epileptic and non-epileptic brain regions may dampen ongoing seizure activity as well as lessen its spread and generalization.

Increase in the number of axospinous synapses with segmented postsynaptic densities following hippocampal kindling

Kindling results from intermittent electrical stimulation of a local brain region and leads to a virtually permanent augmentation of synaptic responsiveness in the stimulated circuit. It has been hypothesized that an increase in the number of synapses may represent a structural basis for the enduring expression of synaptic plasticity following kindling, but such an alteration has not been demonstrated unequivocally. The present report provides evidence that hippocampal kindling is indeed accompanied by an increase in synaptic numbers. Young adult rats were kindled via medial perforant path stimulation and sacrified 4 weeks after reaching a criterion of 5 generalized seizures. Stimulated but not kindled and implanted but not stimulated rats served as controls. Synapses were analyzed in the middle (MML) and inner (IML) molecular layer of the hippocampal dentate gyrus. Using the stereological disector technique, unbiased estimates of the number of synapses per neuron were differentially obtained for 3 morphological subtypes of perforated exospinous synapses characterized by a fenestrated, horseshoe-shaped or segmented postsynaptic density (PSD). A significant increase in synaptic numbers was found to selectively involve only those perforated synapses which are distinguished by a segmented PSD consisting of 2–5 discrete plates. This structural modification was restricted to the terminal synaptic field of stimulated axons (MML), but was not observed in an immediately adjacent synaptic field (IML) which was not directly stimulated during kindling. Since synapses distinguished by a segmented PSD may represent specialized synaptic contacts of an unusually high efficacy, a selective increase in their numbers is likely to provide a structural substrate of the augmented synaptic gain associated with kindling.

Bicuculline-induced blockade of neocortical rapid kindling suggesting facilitative GABAergic action on seizure development

To investigate how GABAergic function affects seizure development, the effects of a GABA antagonist, bicuculline, on neocortical and hippocampal kindling were examined in chronically prepared rabbits. Kindling-inducing stimulations consisted of stimulus trains repeated at 5-min interstimulus intervals to produce so-called "rapid kindling". The changes in after-discharge (AD) durations induced by each of 15 trials of stimulus trains per session were compared before and 30 min after i.p. injection of bicuculline solution (2 mg/kg) in each of three kindling groups consisting of 5 rabbits each, i.e. visual cortical, motor cortical and hippocampal kindling groups. In the visual cortex and to a less extent, the motor cortex kindling groups, the AD durations were shortened after bicuculline injection and did not show the progressive prolongation seen before the injection. In contrast, the hippocampal kindling group showed a further marked prolongation of the AD durations after the injection. The bicuculline-induced blockade of neocortical kindling suggests facilitative GABAergic action on seizure development, while the drug-induced enhancement of hippocampal kindling reflects the known inhibitory GABAergic action.

Serotonergic inhibition of limbic and thalamic seizures in cats.

To clarify the role of the serotonergic system in limbic and thalamic epileptic activity, we examined the effects of 5-hydroxytryptophan (5-HTP, 20 and 40 mg/kg) on fully kindled seizures from the hippocampus and lateral geniculate nucleus. The intraperitoneal administration of 20 mg/kg 5-HTP exerted no anticonvulsant effect on hippocampal kindled seizures, and a higher dose (40 mg/kg) produced a significant reduction in the behavioral seizure stage. 5-HTP at 20 mg/kg displayed no suppressive effect on lateral geniculate seizures, and 5-HTP at 40 mg/kg significantly reduced both the seizure stage and afterdischarge duration. Our data suggest that serotonergic mechanisms play an inhibitory role in seizures kindled from these brain regions.