Amygdala Kindling Stimulation Research Articles

Sialyltransferase ST3Gal IV deletion protects against temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) often becomes refractory, and patients with TLE show a high incidence of psychiatric symptoms, including anxiety and depression. Therefore, it is necessary to identify molecules that were previously unknown to contribute to epilepsy and its associated disorders. We previously found that the sialyltransferase ST3Gal IV is up-regulated within the neural circuits through which amygdala-kindling stimulation propagates epileptic seizures. In contrast, this study demonstrated that kindling stimulation failed to evoke epileptic seizures in ST3Gal IV-deficient mice. Furthermore, approximately 80% of these mice failed to show tonic-clonic seizures with stimulation, whereas all littermate wild-type mice showed tonic-clonic seizures. This indicates that the loss of ST3Gal IV does not cause TLE in mice. Meanwhile, ST3Gal IV-deficient mice exhibited decreased acclimation in the open field test, increased immobility in the forced swim test, enhanced freezing during delay auditory fear conditioning, and sleep disturbances. Thus, the loss of ST3Gal IV modulates anxiety-related behaviors. These findings indicate that ST3Gal IV is a key molecule in the mechanisms underlying anxiety - a side effect of TLE - and may therefore also be an effective target for treating epilepsy, acting through the same circuits.

Effect of vagus nerve stimulation on electrical kindling in different stages of seizure severity in freely moving cats

Vagus nerve stimulation (VNS) is an adjunctive therapy for treating pharmacoresistant epilepsy. The present study analyze the effect of VNS on the epileptic activity of amygdala kindling (AK) in different seizure severity stages in freely moving cats. Fourteen adult male cats were used and were stereotaxically implanted in both amygdalae, in thalamic reticular nuclei and in prefrontal cortices. AK was developed by the application of 60Hz pulse trains that were one second in duration. VNS was applied the following day after the first stages were reached. This stimulation consisted of 10 pulse trains in the one-hour period (1min on/5min off) prior to AK. AK stimulation continued until all animals reached stage VI. The behavioral changes induced by VNS were transient and bearable. The animals showed relaxation of the nictitating membrane, ipsilateral anisocoria, swallowing and licking. Intermittent VNS application in stage I induced a delay in AK progression. The effect of VNS on the amygdala afterdischarge duration (AD) did not change progressively. VNS in stages II, III, and IV does not have an inhibitory effect on AK, and the AD further exhibited a progressive development. At the end of the generalized seizures, the animals presented with synchronized bilateral discharges of the spike-wave type (3Hz) and a behavioral "staring spell". Our results show that VNS applied during the different stages of seizure severity exerts an anti-epileptogenic effect in stage I but no anti-epileptogenic effect in stages II, III, and IV. These results suggest that VNS applied at stage I of kindling induces a delay of generalized convulsive activity.

Carbamazepine, but not valproate, displays pharmacoresistance in lamotrigine-resistant amygdala kindled rats

The voltage gated sodium channel (VGSC) blocker lamotrigine (LTG), when administered during kindling acquisition, leads to the development of resistance to LTG. The present study aimed to assess whether LTG-resistant amygdala-kindled rats display subsequent resistance to the VGSC blocker carbamazepine (CBZ) and the broad-spectrum antiepileptic drug (AED) sodium valproate (VPA). Two groups of male Sprague Dawley rats received either 0.5% methylcellulose (MC) or LTG (5mg/kg, i.p.) 1h before each amygdala kindling stimulation. Treatments were stopped once both the groups were fully kindled. Two days later, both groups were challenged with a higher dose of LTG (15mg/kg, i.p.) to verify LTG-resistance in the experimental group (i.e., LTG-pretreated rats). The efficacy of CBZ and VPA was then evaluated in both groups. A higher dose of LTG blocked fully kindled seizures in the vehicle-treated rats but not seizures in the LTG-treated group. The mean seizure score, of the control group (1.2±0.3) was significantly lower (P<.05) than that of the LTG-treated population (3.5±0.7; n=8). A lower percent of the population in the control group was observed to display a generalized stage 4-5 seizure compared to the experimental group (i.e., those that received LTG during kindling acquisition) (28.5% vs. 62%, respectively). Interestingly, CBZ (10, 20, and 40mg/kg) displayed a dose-dependent anticonvulsant effect in the vehicle-kindled group, but was less effective in LTG-treated animals. In contrast, VPA (300mg/kg) effectively blocked the behavioral seizure and decreased the afterdischarge duration (ADD) in both vehicle and LTG groups. These findings suggest that the LTG-resistant, amygdala-kindled rat may represent a novel model of pharmacoresistant epilepsy.

Altered synapsin I immunoreactivity and fear behavior in male and female rats subjected to long-term amygdala kindling

Long-term amygdala kindling dramatically increases fearful behavior in both male and female rats. In this experiment, we studied the relation between sex, kindled fear behavior, and synapsin I immunoreactivity in various brain regions. Male and female adult Long-Evans rats received either 99 left amygdala kindling stimulations or sham stimulations. One day after the final stimulation, fear behavior was assessed in each rat by measuring exploration and thigmotaxia in an unfamiliar open field, as well as resistance to capture from the open field. Four hours after the behavioral testing, the rats were sacrificed and their brains were processed for immunohistochemical determination of synapsin I expression. As expected, kindling significantly increased fear behavior in both male and female rats. It also increased synapsin I immunoreactivity bilaterally in most hippocampal subfields, but not in the caudate nucleus, sensorimotor cortices, or piriform cortex. Interestingly, kindling decreased synapsin I immunoreactivity bilaterally in the central and basolateral amygdala of male rats but not female rats. Correlational analyses revealed that in male rats, fearful behavior was positively correlated with synapsin I immunoreactivity in hippocampal brain regions located ipsilateral to the site of stimulation (i.e., the CA1 and CA3 subfields of the hippocampus, the dentate gyrus and the hilus) and negatively correlated with synapsin I immunoreactivity bilaterally in the basolateral and central amygdala. In female rats, fear behavior was positively correlated with synapsin I immunoreactivity in the ipsilateral CA1 and CA3 subfields only. These results suggest that altered synaptic plasticity in specific brain regions might be involved in the exaggerated fearfulness produced by long-term amygdala kindling, especially in male rats.

Ictogenesis

Ictogenesis

Low frequency stimulation modifies receptor binding in rat brain

Experiments were designed to reproduce the antiepileptic effects of low frequency stimulation (LFS) during the amygdala kindling process and to examine LFS-induced changes in receptor binding levels of different neurotransmitters in normal brain. Male Wistar rats were stereotactically implanted in the right amygdala with a bipolar electrode. Rats ( n=14) received twice daily LFS (15 min train of 1 Hz, 0.1 ms at an intensity of 100 to 400 μA) immediately after amygdala kindling stimulation (1 s train of 60 Hz biphasic square waves, each 1 ms at amplitude of 200–500 μA) during 20 days. The LFS suppressed epileptogenesis (full attainment of stage V kindling) but not the presence of partial seizures (lower stages of kindling) in 85.7% of the rats. Thereafter, normal rats ( n=7) received amygdala LFS twice daily for 40 trials. Animals were sacrificed 24 h after last stimulation and their brain used for labeling μ opioid, benzodiazepine (BZD), α 1-adrenergic, and adenylyl cyclase binding. Autoradiography experiments revealed increased BZD receptor binding in basolateral amygdala (20.5%) and thalamus (29.3%) ipsilateral to the place of stimulation and in contralateral temporal cortex (18%) as well as decreased values in ipsilateral frontal cortex (24.2%). Concerning μ receptors, LFS decreased binding values in ipsilateral sensorimotor (7.2%) and temporal (5.6%) cortices, dentate gyrus (5.8% ipsi and 6.8% contralateral, respectively), and contralateral CA1 area of dorsal hippocampus (5.5%). LFS did not modify α 1 receptor and adenylyl cyclase binding values. These findings suggest that the antiepileptic effects of LFS may involve activation of GABA–BZD and endogenous opioid systems.

Environmental Enrichment Facilitates Amygdala Kindling but Reduces Kindling-Induced Fear in Male Rats.

The purpose of this experiment was to determine the effect of prior environmental enrichment on the acquisition of kindling and the expression of kindling-induced fear. Sixty male rats were housed either in an enriched environment or in isolation, starting immediately after weaning. As adults, they were subjected to either 50 amygdala-kindling stimulations or sham stimulations, followed by testing in an unfamiliar open field. The kindled-enriched rats acquired the kindled state more quickly than did the kindled-isolated rats, but they also showed less fear in the open field than did the kindled-isolated rats. These results suggest that environmental enrichment has differential effects on kindling acquisition and its behavioral consequences.

Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions

Amygdala kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides an excellent model for studying the nature and neural mechanisms of fear sensitization. In the present experiment, we studied whether the development of kindling-induced fear is related to changes in glucocorticoid receptor (GR) mRNA expression in various brain regions. Rats received 20, 60 or 100 amygdala kindling stimulations or 100 sham stimulations. One day after the final stimulation, their fearful behavior was assessed in an unfamiliar open field. Then, the rats were sacrificed and their brains were processed for in situ hybridization of GR mRNA expression. We found that compared with the sham-stimulated rats, the rats that received 60 or 100 kindling stimulations were significantly more fearful in the open field and also had significantly less GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Importantly, the changes in fearful behavior were significantly correlated with the changes in GR mRNA expression. These results suggest that alterations in GR mRNA expression in hippocampal regions may play a role in the development of kindling-induced fear.

Amygdala kindling modifies extracellular opioid peptide content in rat hippocampus measured by microdialysis.

Opioid peptide release in the hippocampus was shown to be increased immediately following amygdala kindling stimulation in freely moving rats using microdialysis combined with a universal opioid peptide radioimmunoassay (RIA). Extracellular opioid peptide levels were elevated (55% above basal levels) within the first 10 min after electrical stimulation-induced partial seizures in previously nonkindled animals. Fully kindled rats showed lower extracellular opioid peptide levels (40% reduction) during the interictal period [16 +/- 2.1 days (mean +/- SEM) after the last stage V seizure], in comparison with values obtained from the sham-kindled group under basal conditions. However, opioid peptide release in fully kindled rats increased above 152% of interictal levels within the first 20 min after onset of fully kindled seizures, attaining peak levels equal to that of the partial kindled group and returning to prestimulation conditions 40-60 min following the ictal events. The majority of the immunoreactive material recovered from the hippocampus within the first 20 min following partial and generalized kindled seizures coeluted with dynorphin-A (1-6), dynorphin-A (1-8), and Leu-enkephalin by HPLC/RIA analysis. It is proposed that the enhanced opioid peptide release in hippocampus induced by amygdala kindling stimulation might be associated with either enhanced excitability or seizure suppression as seizure susceptibility fluctuates. The reduced interictal opioid peptide levels may also underlie some interictal behavioral disturbances.

Relation of the enhancement of entorhinal tetanic responses by 50-Hz amygdala stimulation to the progression of kindling in the rat

We recorded entorhinal tetanic responses to 50-Hz kindling stimulations applied at the amygdala in conscious rats, which produced facilitation and depression during the train pulses, in order to analyze the relationship of the changes in the tetanic responses to the development of both after-discharges (ADs) and behavioral seizures. Facilitation was always produced in the earlier tetanic responses and was followed by depression which reached a quasi-steady level in the later tetanic responses during each kindling stimulation. To estimate the changes in magnitude of the excitatory synaptic activation in the tetanic responses, with reference to the development of seizure stages, tetanic responses which produced the same behavioral seizure stage in each rat were averaged and the area between the negative (excitatory) potentials and the baseline of the averaged tetanic response was measured in terms of mV x ms. Magnitudes of the averaged negative components were significantly enhanced with an increase in the order of seizure stages in eight rats ( P < 0.01). In addition, the mean magnitude of the averaged negative components had a linear correlation ( r = 0.95, P < 0.05) with the mean AD duration with reference to the order of seizure stages in the eight rats. The magnitude of the positive (inhibitory) component in the averaged tetanic responses was also measured and found to decrease with an increase in the seizure stages ( P < 0.01). The magnitude of the negative component in the test responses to single (0.3 Hz) stimuli just before kindling stimulations also increased with an increase in the order of seizure stages, indicating long term potentiation of the responses by kindling stimulations. We concluded from the results that the enhancement of facilitation of the excitatory synaptic activation and the reduction of the inhibitory synaptic activation in entorhinal tetanic responses by 50-Hz amygdala kindling stimulation is involved in the electrophysiological source of the progression of kindling epilepsy.

Effects of Pentobarbital on Entorhinal Tetanic Responses and the Progression of Afterdischarges During the Early Course of Amygdala Kindling in Rats

We investigated the relationship between the progress of afterdischarges (AD) and the development of facilitated entorhinal tetanic responses by amygdala kindling stimulations in conscious and pentobarbital (PB)-treated rats. The entorhinal responses consisted of deep negative components and the following shallow positive components. The negative potential (mean +/- SE) reflecting excitatory synaptic activation in the test response evoked by a single stimulation (600 microA) before kindling stimulations was greater in PB-treated rats (1.3 +/- 0.21 mV, n = 6) than in conscious rats (0.5 +/- 0.08 mV, n = 9). The positive potential reflecting inhibitory synaptic activation in the test response was also greater in PB-treated rats (0.6 +/- 0.14 mV, n = 6) than in conscious rats (0.2 +/- 0.04 mV, n = 9). The magnitude of the tetanic response was estimated as the area between the excitatory negative potential and the baseline in the averaged tetanic response during each kindling stimulation (10 Hz, 100 pulses). The magnitude of the tetanic response was significantly enhanced in association with the prolongation of AD duration in the conscious rats. In the PB-treated (50 mg/kg intraperitoneally, i.p.) rats, enhancement of tetanic response was very slight and the progress of AD duration was prevented. There was a linear correlation (r = 0.9) between the magnitude of tetanic response and AD duration. These findings indicate that PB suppresses kindling-induced enhancement of excitatory synaptic activation in tetanic responses and that this enhancement is intimately related to the development of AD.

Effects of an [formula omitted] antagonist and a GABAergic antagonist on entorhinal tetanic responses during the early stages of amygdala kindling in rats

Changes in synaptic potentials during each train stimulation (tetanic responses) have been suggested to intimately relate to the development of kindling. We examined the effects of an NMDA antagonist, carboxypiperazinephosphonate (CPP), and a GABAergic antagonist, picrotoxin, on entorhinal tetanic responses evoked by train stimuli (10 Hz, 100 pulses) at the developmental stage (seizure stage; 0–2) of amygdala kindling in conscious rats, to clarify the significance of facilitation in tetanic responses and the roles of NMDA and GABA receptors in the development of kindling. Facilitation of tetanic responses was noted as a progressive increase in both amplitude and duration of negative potentials in the tetanic responses, especially during the later half of train pulses (51–100). The negative potential area (mV × ms) of the averaged tetanic responses was used as an estimate of the magnitudes of excitatory synaptic activity in the tetanic responses, and correlated significantly ( P < 0.001) with the duration of afterdischarges (AD). CPP (10 mg/kg) reversibly blocked AD in association with a significant decrease ( P < 0.05) in the negative potential area. The CPP-sensitive component consisted of a slow negative potential with a duration longer than 60 ms and was greater in the later tetanic responses (51–100) than the earlier ones (1–50). Picrotoxin (2–3 mg/kg), which did not produce convulsions, significantly ( P < 0.005) increased the negative potential area in the tetanic responses in association with a reversible decrease in the AD threshold. Although positive potentials ascribable to inhibitory synaptic activity were often negligible in the tetanic responses in controls, picrotoxin further decreased the positive potentials of tetanic responses, if any. We suggest from these results that facilitation of entorhinal tetanic responses during amygdala kindling stimulation is enhanced by NMDA-receptor activation and reduction of GABAergic inhibition, and that both NMDA and GABA receptors are responsible for the electrophysiological origin of the development of kindling.

Chronic pretreatment with naloxone modifies benzodiazepine receptor binding in amygdaloid kindled rats

Male Sprague-Dawley rats received either naloxone (75 μg/h) or saline (0.5 μ/h) s.c. for 14 days delivered with osmotic minipumps. Two days after termination of either treatment, daily amygdala kindling stimulation was applied until the animals experienced stage V kindled seizures. Benzodiazepine (BDZ) binding sites were labeled with [ 3H]flunitrazepam (2 nM) and changes in specific brain areas were determined by in vitro quantitative autoradiography. Twenty-four hours after the last electrical stimulation, the saline pretreated fully kindled rats showed enhanced BDZ receptor binding in dentate gyrus and decreased binding in cingulate cortex ipsilateral to the stimulation compared to saline controls. Twenty-eight days after the last stage V kindled seizure, the significant alterations were no longer evident. In agreement with a previous study, we found that naloxone pretreated amygdala kindled rats showed stage V kindled seizures followed by intervals of 3–5 days in which the same electrical stimulation failed to induce any behavioral and EEG alterations. In comparison with the saline pretreated kindled and saline control groups, the naloxone pretreated kindled rats had significantly higher BDZ binding in different cortical areas, amygdala complex, hippocampus, substantia nigra and periaqueductal gray, 24 h after the last electrical stimulation. The present study indicates that previous chronic exposure to naloxone increases BDZ receptor binding in kindled rats, and suggests that this effect may be associated with the enhanced seizure suppression observed in these animals.

Effect of N-methyl-D-aspartate antagonist on entorhinal responses by amygdala kindling stimulations.

Effect of N-methyl-D-aspartate antagonist on entorhinal responses by amygdala kindling stimulations.

Amygdala kindling and associated changes of entorhinal responses in suckling rats

Entorhinal field potential with amygdala stimulation in suckling (16–18 days old) and adult rats was recorded with a tungsten wire electrode (tip diameter 2–5 μm) to study the developmental changes in behavioral seizures and long-term potentiation (LTP) in the responses to amygdala kindling stimulations. Stimulating (twisted enamel-coated wires) and recording electrodes were implanted in anesthetized rats 2–3 days before kindling. The mean amplitude of the responses to test pulses (600 μA, 0.3 Hz) in the sucklings (0.58 mV) was smaller than in the adults (1.32 mV), and latency was about 3.3 ms longer. Kindling stimulations consisted of 0.5-ms monophasic rectangular pulses of 10 Hz with a 10-s train duration; the intensity was the afterdischarge (AD) threshold. Kindling stimulation in the sucklings usually increased the amplitude of the test responses evoked 10 min or 1 h after the kindling stimulation. The increased amplitude persisted for at least 24 h, showing LTP in the synaptic transmission. The LTP was especially prominent in the first kindling stimulation, and the LTP gradually increased with successive stimulations, with gradual progression of AD and the behavioral seizure stage as well. The mean number of kindling stimulations to cause generalized seizures in the suckling rats (10.5) was less than that for adults (12.5), and the continued evolution of LTP over the course of kindling was more or less easier in the sucklings than in the adults. The LTP in kindled suckling rats was observed for all intensities of test pulses and the input—output (I/O) curve in the kindled rats was significantly higher (P < 0.01) than the control I/O curve for the rats which received low frequency (0.3 Hz) stimuli only. However, the mean amplitude of the field potentials enhanced by the LTP in kindled sucklings did not reach the mean amplitude of control responses without kindling stimulations in the adults. The initial strong LTP in the monosynaptic entorhinal responses with amygdala kindling may contribute to the emergence of epileptogenesis by kindling from the pathway, and the easier evolution of LTP over the course of kindling in suckling rats may contribute to the earlier acquisition of kindling phenomena in suckling rats than in adult rats.

Relationship between the changes of field potential and initiation of afterdischarge (AD) in the entorhinal cortex to amygdala kindling stimulations in rats

Relationship between the changes of field potential and initiation of afterdischarge (AD) in the entorhinal cortex to amygdala kindling stimulations in rats

Inferior collicular interactions with limbic seizure activity.

Because under certain conditions, seizure activity electrically elicited from layers III and IV of the inferior collicular cortex can spread into forebrain regions, potential inferior collicular interactions with the amygdala were studied. Following acute electrical initiation of seizure activity from the inferior colliculus, no changes in amygdala EEG activity were noted. Following repetitive stimulation of the inferior colliculus, however, postictal spiking activity was noted in the amygdala, similar to interictal spiking reported for amygdala kindling, but this abnormal EEG activity did not coincide with any observable behavioral change. Conversely, the course of amygdala kindling in animals previously stimulated in the inferior colliculus progressed quite differently in comparison to control animals. Those animals repetitively stimulated in the inferior colliculus required a significantly greater number of amygdala kindling stimulations to reach class 5 seizure activity than did animals that received no inferior collicular stimulation and, unlike the controls, the chronic inferior collicular stimulation group usually regressed to class 2 or 3 seizure activity after the first class 5 seizure. Furthermore, the chronic inferior collicular stimulation group all exhibited wet-dog shakes during the amygdaloid kindling stimulation, whereas at no time did controls exhibit wet-dog shake behaviors. Finally, the seizure generalization from the inferior colliculus appears to be mediated by a mechanism distinct from the acute seizure activity, because a dose of diazepam (0.4 mg/kg), or carbamazepine (10 mg/kg), which had no effect on the wild running seizure, blocked behaviors indicative of seizure generalization. The significance of these results to epilepsy is discussed.

The substantia nigra is an important site for the containment of seizure generalization in the kindling model of epilepsy.

To investigate the role of the substantia nigra (SN) in kindling, electrical stimulation of the SN was delivered at various times before or after stimulation of the amygdala (AM) or pyriform cortex during or after kindling in rats. The results were as follows: Ipsilateral SN stimulation delivered prior to each AM kindling stimulation for 14 days significantly retarded the appearance of Stage 4 and 5 seizures and shortened the afterdischarge (AD) duration. Bilateral SN prestimulation blocked seizure generalization in some AM- or pyriform cortex-kindled animals, prolonged the latency to bilateral forelimb clonus in others, and shortened the AD duration of the kindled seizure in a current intensity-dependent fashion. These effects were only partially antagonized by haloperidol, but were completely abolished by picrotoxin. The picrotoxin alone significantly reduced the latency. Almost no effect was found when the SN stimulation was delivered after the onset of bilateral forelimb clonus. We conclude that the SN might be an important mediator of the early aspects of seizure generalization from limbic epileptic foci. The relative involvements of GABAergic gamma-aminobutyric acid and dopaminergic systems of the SN in this inhibitory function are discussed.

Kindled seizure induction alters and is altered by zinc absorption

Amygdala kindling stimulation produced significant changes in plasma zinc levels in cats otherwise unaffected by zinc loading or deprivation. While a normal diet had no effect, moderate zinc loading was accompanied by a marked increase in plasma zinc during kindling. Conversely, plasma zinc sharply declined in animals fed a zinc-deficient diet. Corresponding differences were obtained in the development of generalized seizures with kindling such that loading delayed and deprivation accelerated this process.