"Wet dog shakes" during olfactory bulb kindling

Kindling phenomenon provides an experimental model of limbic secondarily generalized epilepsy. It can be easily obtained by stimulation of the olfactory bulb (OB) which is strongly connected to limbic structures. The after-discharge induced by subconvulsant electrical stimulations, is followed by a behavioral phenomenon, named Wet Dog Shakes (WDS). In the course of the Rat OB kindling, the development of WDS is biphasic: 1) an ascendant phase during the first three stages, and 2) a descendant phase in the stages 4 and 5, that may give evidence of the installation and the intensification of the kindling process. The significance of this behavior in seizure generalization is discussed.     

Studies of wet-dog shake behavior induced by septohippocampal stimulation in the rat

Stimulation of the septum and the hippocampus were found to elicit a great number of "wet-dog" shakes (WDS). Their occurrence is strongly related to the evocation and to the time course of the afterdischarges elicited by the stimulation. Morphine, apomorphine, diazepam, and antiserotoninergic drugs greatly reduce the incidence of these WDS but do not alter the afterdischarge duration. Based on electroencephalographic and pharmacological data we propose that WDS induced by stimulation of the septohippocampal system may share some common mechanisms with many other models inducing WDS and offer a useful method to study further the neuroanatomical substrate of this behavior.     

Somatostatin release in the hippocampus in the kindling model of epilepsy: a microdialysis study

Somatostatin biosynthesis in the hippocampus is activated during and following kindling epileptogenesis. The aim of this study was to investigate whether this phenomenon is associated with enhanced somatostatin release in vivo. Experiments have been run in awake, freely moving rats, implanted with a bipolar electrode in the right amygdala (for kindling stimulation), and with a recording electrode and a microdialysis probe in the left hippocampus. Basal somatostatin-like immunoreactivity (-LI) release was significantly greater in kindled than naive rats. In naive rats, a 2-min perfusion with 100 mM K(+) did not affect behavior and EEG recordings and nonsignificantly increased somatostatin-LI release; a 10-min K(+) perfusion evoked numerous wet dog shakes, electrical seizures (class 0; latency congruent with 8 min, duration congruent with 8 min), and somatostatin-LI release ( congruent with 350% of basal); and a single kindling after-discharge (4 +/- 3-s duration in the hippocampus) also evoked somatostatin-LI release ( congruent with 200% of basal). In kindled rats, a 2-min 100 mM K(+) perfusion evoked hippocampal discharges in three of seven animals (latency congruent with 2 min, mean duration congruent with 1.5 min) and increased somatostatin-LI release ( congruent with 250% of basal); a 10-min K(+) perfusion evoked behavioral seizures (class 1 to 5, latency congruent with 4 min, mean duration congruent with 12 min) with numerous wet dog shakes and robust somatostatin-LI release ( congruent with 350% of basal); and a kindling stimulation evoked generalized seizures (class 4 or 5, 77 +/- 15-s duration in the hippocampus) with remarkable somatostatin-LI release ( congruent with 300% of basal). These data demonstrate that hippocampal somatostatin release is increased in the kindling model in vivo.     

Accumulation of 7S SNARE complexes in hippocampal synaptosomes from chronically kindled rats

Kindling is a model of complex partial epilepsy wherein periodic application of an initially subconvulsive stimulus leads to first limbic and then generalized tonic-clonic seizures. Several laboratories have reported that augmented neurotransmitter release of l-glutamate is associated with the chronically kindled state. Neurotransmitter release requires membrane proteins called SNAREs, which form transmembrane complexes that participate in vesicle docking and are required for membrane fusion. We show here that kindling by entorhinal stimulation is associated with an accumulation of 7S SNARE complexes in the ipsilateral hippocampus. This increase of 7S SNARE complexes appears to begin early in the kindling process, achieves a peak with full kindling, and remains at this level for at least a month following cessation of further kindling stimuli. The increase is focal and permanently limited to the ipsilateral hippocampus despite progression to generalized electrographic and behavioral seizures. It is not seen in animals that receive electroconvulsive seizures, suggesting it is related to the kindling process itself. The duration and focality of increased 7S SNARE complexes with entorhinal kindling suggest that this is an altered molecular process associated with epileptogenesis.     

Neuropeptide Y Y5 receptors inhibit kindling acquisition in rats

Neuropeptide Y inhibits neuronal excitability and seizures in various experimental models. This peptide delays kindling epileptogenesis but the receptors involved in this action are unknown. We have studied the role of Y5 receptors in kindling using the selective antagonist GW438014A (IC50=210 nM), a small heterocycle molecule that crosses the blood-brain barrier, and the selective peptide agonist Ala31Aib34 NPY (IC50=6.0 nM). Intraperitoneal injection of GW438014A (10 mg/kg), 30 min before the beginning of a rapid-kindling protocol, significantly accelerated the rate of kindling acquisition as compared to vehicle-injected rats. Thus, the number of electrical stimuli required to reach stages 3 and 4-5 of kindling were reduced by 50% and 25%, respectively. The average afterdischarge duration in the stimulated hippocampus was prolonged by 2-fold. Conversely, kindling rate was delayed by intracerebroventricular administration of 24 nmol Ala31Aib32 NPY. Thus, the number of stimuli necessary to reach stages 2 and 3 of kindling was increased by 3- and 4-fold, respectively. During the stimulation protocol (40 stimuli) none of the rats treated with the Y5 agonist showed stages 4-5 seizures. Twenty-four hours after the last kindling stimulation, thus during the re-test session, Y5 agonist- or antagonist-treated rats had stages 4-5 seizures as their controls. In rats treated with both the antagonist and the agonist, kindling rate was similar to vehicle-injected rats. These data indicate that Y5 receptors mediate inhibitory effects of NPY in kindling and display anticonvulsant rather then antiepileptogenic effects upon agonist stimulation.     

Effect of Acute Stress on the Development of Seizures in a Kindling Model in Rats

We studied the effect of acute stress induced by nociceptive stimulation of the limbs on the duration of ECoG epileptiform activity and manifestation of generalized motor convulsive reactions under conditions of a kindling model of epilepsy in rats. Two and four weeks after termination of the kindling procedure, test stimulations of the hippocampus evoked intense attacks of epileptic activity. Short-lasting pain-inducing stimulation (intense electrical stimulation of the limbs) resulted in noticeable limitation of both ECoG and motor behavioral manifestations of epileptic activity determined by the formation of an epileptogenic nidus. The antiepileptic effect of acute stress was limited in time; manifestations of this effect reached their maximum about 3 h after painful stimulation, while about 6 h after such stimulation they became smoothed to a considerable extent.     

Changes in benzodiazepine receptors in the limbic system following kindling of the olfactory bulb in rats

Repeated electrical stimulations of the olfactory bulb led to the progressive development of a generalized epilepsy (kindling effect). One week after the last stimulation eliciting a stage 5 seizure, diazepam-(3H) binding was studied in olfactory bulb-kindled rats. Numbers of benzodiazepine receptors were increased in kindled olfactory bulb and amygdala. No significant change was observed in hippocampus. This modification could be considered as a response of the inhibitory mechanisms to repeated seizures which is insufficient to counteract the installation of the kindling effect.     

Peculiarities of the Effects of Stimulation of Emotiogenic Central Structures under Conditions of a Kindling Model of Epilepsy

In our study, we tried, first, to elucidate whether induction of emotional behavior resulting from stimulation of the dorsomedial hypothalamus (DMH) influences the development of seizure activity in the course of epileptogenesis within the framework of fast kindling (stimulation of the hippocampus) and, second, to estimate if such stimulation is capable of modulating manifestations of generalized seizures under conditions of the pre-formed “full” epileptic syndrome. Stimulation of the DMH in the above two experimental situations resulted in significant suppression of both electrographic and behavioral manifestations of seizure activity. We hypothesize that the respective emotional reactions can be interpreted as phenomena of instinctive behavior having an adaptive defensive significance. These reactions are related to inhibitory processes providing protection from the development of seizure activity.     

Relationships between the hippocampus and medial septal region and their alterations during epileptogenesis

EEGs of the hippocampus and the medial septal region (MSR) in the control conditions and during repeated stimulation of the perforant path were simultaneously recorded in awake guinea pigs. Changes in correlation of activity of these structures during seizures provoked by the stimulation (model of acute epilepsy) and in the process of epileptogenesis induced by the kindling (model of chronic epilepsy) were analyzed. A high correlation of the baseline activities of the hippocampus and MSR observed in the control sharply decreased during acute and chronic seizures. Kindling led to emergence of the MSR capability of hippocampus-independent generation of the field seizure discharges. In the process of kindling the progressive disintegration of activities of the hippocampus and the MSR was revealed being indicative of disorders in functioning of the septohippocampal network during epileptogenesis.     

Time- and Region-Specific Variations in Somatostatin Release Following Amygdala Kindling in the Rat

Abstract: Somatostatin biosynthesis is activated during and following kindling epileptogenesis. The aim of this study was to investigate whether this phenomenon translates into enhanced release of the peptide and whether it is involved in kindling maintenance. A marked increase in somatostatin-like immunoreactivity (somatostatin-LI) was observed in hilar interneurons of the hippocampus and in their presumed projections to the outer molecular layer 1 week, but not 1 month, after the last kindled seizure. No overt changes were observed in the striatum or in the cortex. Compared with sham-stimulated controls, (a) in the hippocampus, high-K⁺-evoked somatostatin-LI release was unchanged in synaptosomes taken from rats killed 7 days after the last kindled seizure but was bilaterally reduced after 30 days; (b) in the striatum, it was increased (mainly ipsilaterally to stimulation) 7, but not 30, days after the last seizure; and (c) in the cortex, somatostatin-LI release was bilaterally increased in synaptosomes taken from kindled rats 30, but not 7, days after the last seizure. This study shows that distinct changes occur in synaptosomal somatostatin-LI release after kindling acquisition, depending on the brain area analyzed and on the time elapsed from the last generalized seizure.     

Morphofunctional features of microtubule ultrastructure and MAP2 protein phosphorylation in hippocampal neurons of rats with predisposition to audiogenic epilepsy

It has been shown that modification of microtubule (MT) ultrastructure are accompanied by functional changes in microtubule-associated protein MAP2 in the hippocampus of Krushinsky--Molodkina rats (KM), which are prone to autogenic seizures. The morphogenetic analysis revealed that contrary to Wistar rats, which are insensitive to sound stimulation, in KM the middle length of microtubule fragments in the apical dendrites of pyramidal neurons in CA3 hippocampal area was reduced. Using immunoblot and autoradiography methods, we found that the level of MAP2 and the rate of its cAMP = and Ca(2+)-calmodulin-dependent phosphorylation were increased in hippocampus of KM, in comparison with Wistar rats. Daily repeated sound stimulation for 20 days (audiogenic kindling) induced a further decrease in length of MT fragments, and an increase of their density in the proximal part of apical dendrites of KM. Moreover, audiogenic kindling induced additional increase in MAP2 phosphorylation state, but did not change the level of MAP2 in KM hippocampus. We suppose that the obtained alteration of MAP2 phosphorylation state exerted influence on kinetic parameters of microtubule assembly, serving as part of genetically determined predisposition of KM to audiogenic epilepsy.     

Amygdala Kindling Modifies Extracellular Opioid Peptide Content in Rat Hippocampus Measured by Microdialysis

Abstract: 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.     

Variations of Rat Brain Calmodulin Content in Dark and Light Phases: Effect of Pentylenetetrazol-Induced Kindling

Calmodulin (CaM) through activation of CaM-kinase II may be involved in the molecular mechanisms underlying the epileptogenic processes. Some evidence suggests that kindling responses change across the day-night cycle. In order to test if kindling stimulation modifies CaM content, we measured CaM concentrations in amygdala, hippocampus and hypothalamus obtained from control and kindled rats during light and darkness. Male Wistar rats (250–300 g), were injected i.p. with Pentylenetetrazol (PTZ) (35 mg/kg/24 h). Once chemical kindling was established, rats were sacrificed by decapitation at 10:30 a.m. and 01:30 a.m. The brains were obtained, and the amygdala, hippocampus and hypothalamus dissected. CaM content was measured in the cytosol and membrane fractions by radioimmunoassay. We found a significant increase in CaM content in cytosol and membrane fractions of both control and kindled rats during the dark phase. No significant differences in CaM concentrations were observed between control and experimental rats, whether during the light or the dark phase. The data suggest a well defined photoperiodic variation in CaM concentrations in limbic structures, despite the neuronal excitability produced by kindling. In addition, the observed CaM increases during the dark time may be related to a protective mechanism against enhanced sensitivity to seizures observed during the night.     

Effects of L-dopa and transcranial magnetic stimulation on behavioral reactions in kindled rats

In acute experiment in rats, the chronic epileptogenesis was reproduced in the form of pharmacological kindling induced via repeated picrotoxin administrations (1.0-1.2 mg/kg, intraperitoneally). A reduction of exploratory behavior was shown in the early period of kindling (24 h as of the moment of the last epileptogen administration). The marked alleviation of these disturbances was registered in two weeks from the moment of cessation of kindled irritations. L-DOPA (100 mg/kg, intraperitoneally) and transcranial magnetic stimulation (20 impulses with an induction at the height of their development of 1.5 TI) was followed by the net increasing exploratory, sexual and eating behavior. This is in favour of regarding the activation of dopaminergic system as a mechanism of action of transcranial magnetic stimulation upon kindling-induced behavioral deterioration.     

Effects of destruction and activation of limbic structures on formation of convulsive and emotional disorders during picrotoxin kindling

In the experiments on rats it was shown that the picrotoxin kindling, which consists of the progressive increasing of convulsive reactions during daily systemic administration of picrotoxin in subconvulsive dosages results also in the development of the pathologically enhanced defensive reactions. The destruction of hippocampal structures by kainic acid prevented the seizure syndrome, while their activation due to blood injection in hippocampus promoted its development; under these conditions the kindling of pathologically enhanced defensive reactions was not significantly changed. Bilateral amygdalar destruction significantly attenuated the development of pathologically increased defensive behavior; under these conditions the seizure syndrome was not significantly changed. The data are discussed on the theory of generator, and systemic mechanisms of neuropathologic syndromes and show that picrotoxin kindling results in the formation of two different pathologic systems which cause the development of two mentioned syndromes: seizure syndrome and syndrome of pathologically enhanced defensive behavior.     

Excitability cycles of epileptic after-discharges in rats

An epileptic seizure is regularly followed by a postictal depression and then by a phase of increased excitability. The time course of these two phases was described for two types of epileptic after-discharges induced by stimulation of the hippocampus and/or the thalamus in acute experiments in rats. Using hippocampal stimulation, an interval of 10 min was necessary for induction of the second self-sustained after-discharge (SSAD) of the same duration as the first one. Significant prolongation of the second SSAD appeared with a 30-min interstimulation interval. The spike-and-wave rhythm induced by stimulation of thalamic nuclei exhibited a shorter refractory phase - up to 5 min - and also the facilitation took place sooner: with 15-min intervals a significant increase in duration of SSAD was recovered. The results are discussed in connection with the kindling model of epilepsy.     

Failure to kindle seizures after repeated intracerebral administration of arginine vasopressin

In an attempt to kindle seizures with arginine-vasopressin (AVP), we injected AVP into the amygdala or hippocampus of rats. Although behavioral and electrographic alterations were sometimes observed, seizures failed to develop, even in rats that had previously been kindled with electrical stimulation. This and previous failures to kindle seizures by intraventricular injections of AVP call into question the possibility of AVP kindling.     

Z944, a Novel Selective T-Type Calcium Channel Antagonist Delays the Progression of Seizures in the Amygdala Kindling Model

Temporal lobe epilepsy (TLE) is the most common form of drug resistant epilepsy. Current treatment is symptomatic, suppressing seizures, but has no disease modifying effect on epileptogenesis. We examined the effects of Z944, a potent T-type calcium channel antagonist, as an anti-seizure agent and against the progression of kindling in the amygdala kindling model of TLE. The anti-seizure efficacy of Z944 (5mg/kg, 10mg/kg, 30mg/kg and 100mg/kg) was assessed in fully kindled rats (5 class V seizures) as compared to vehicle, ethosuximide (ETX, 100mg/kg) and carbamazepine (30mg/kg). Each animal received the seven treatments in a randomised manner. Seizure class and duration elicited by six post-drug stimulations was determined. To investigate for effects in delaying the progression of kindling, naive animals received Z944 (30mg/kg), ETX (100mg/kg) or vehicle 30-minutes prior to each kindling stimulation up to a maximum of 30 stimulations, with seizure class and duration recorded after each stimulation. At the completion of drug treatment, Ca_V3.1, Ca_V3.2 and Ca_V3.3 mRNA expression levels were assessed in the hippocampus and amygdala using qPCR. Z944 was not effective at suppressing seizures in fully kindled rats compared to vehicle. Animals receiving Z944 required significantly more stimulations to evoke a class III (p<0.05), IV (p<0.01) or V (p<0.0001) seizure, and to reach a fully kindled state (p<0.01), than animals receiving vehicle. There was no significant difference in the mRNA expression of the T-type Ca²⁺ channels in the hippocampus or amygdala. Our results show that selectively targeting T-type Ca²⁺ channels with Z944 inhibits the progression of amygdala kindling. This could be a potential for a new therapeutic intervention to mitigate the development and progression of epilepsy.     

A Possible Mechanism of Blocking of Limbic Motor Seizure Reactions Induced by Activation of the Thalamic Reticular Nucleus

Experiments were directed toward elucidation of the role of the thalamic reticular nucleus (R) in the modulation of generalized seizure reactions under kindling conditions and of the mechanisms mediating the effects of stimulation of the above nucleus on seizure activity. It was shown that activation of the thalamic R in rats limits generalization of the seizure reactions both in the course of development of seizures of limbic genesis (evoked by stimulation of the hippocampus) and under conditions of the existence of a pre-formed epileptic nidus. Tetanic stimulation of the R in cats under conditions of acute experiments induced significant facilitation of IPSPs in thalamo-cortical neurons of the ventrolateral thalamic nucleus. This effect is rather long-lasting and may be considered a mechanism providing blocking of generalized seizures under kindling conditions. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 352–361, July–August, 2005.