Localisation of the origin of self-sustained after-discharges (SSADs) in the rat: the serrated wave (SerW) type of SSAD

The self-sustained after-discharges (SSADs) characterised by the EEG pattern of serrated waves (SerW) were induced by rhythmic low frequency electrical stimulation of thalamic nuclei and the hippocampus of Wistar albino male rats in acute experiments. We used spreading depression to eliminate functionally the cortex and the hippocampus. Suction ablation of the cortical somatosensory projection area was also used to test its involvement in the SerW SSAD induction. The hippocampal spreading depression but not the cortical one abolished the SerW SSAD induced by the stimulation of the thalamic nuclei. The animals with the suction ablation of the somatosensory projection area also produced SerW SSADs when the stimulation electrodes were placed in the thalamic ventrobasal complex (in intact animals this stimulation induces spike-and-wave SSADs but not SerW-SSAD). The crucial importance of the hippocampus in the SerW SSAD generation and its possible use as a model of partial seizures with complex symptomatology is discussed.     

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.     

Wet-dog shaking behavior in rats in hippocampal kindling

The kindling phenomenon, induced by repetitive electrical stimulation of the hippocampus of the rat is associated with the production of a particular behavior: the Wet Dog Shakes (WDS). The evolution of WDS is strongly and negatively correlated with the intensification of motor seizures. Some differences can be seen in the occurrence of WDS according to whether the stimulation is applied in dorsal or in ventral hippocampus. Although the anatomical substrate responsible for this behavior is not clearly defined, the relationship between WDS and kindling let us consider it as an index of the generalization of the epilepsy.     

Neuropeptide Y and epilepsy: varying effects according to seizure type and receptor activation

In vitro and in vivo experiments suggest antiepileptic properties for NPY. In this study, the pharmacology of these effects was examined and compared in different rat models of seizures. Agonists for Y(1), Y(2) and Y(5) receptors reduced seizure-like activity in hippocampal cultures. Intracerebral injection of NPY or Y(5) agonists reduced the expression of focal seizures produced by a single electrical stimulation of the hippocampus. Conversely, NPY agonists increased the duration of generalized convulsive seizures induced by pentylenetetrazol. These results suggest that NPY reduces seizures of hippocampal origin through activation of Y(5) receptors. They also point to probable modulatory effects of NPY in brain structures other than the hippocampus, involved in initiation, propagation or control of seizures.     

Increased levels of messenger RNAs for neurotrophic factors in the brain during kindling epileptogenesis.

Kindling, induced by repeated subconvulsive electrical or chemical stimulations leads to progressive and permanent amplification of seizure activity, culminating in generalized seizures. We report that kindling induced by electrical stimulation in the ventral hippocampus leads to a marked and transient increase in mRNA for NGF and BDNF in the dentate gyrus, the parietal cortex, and the piriform cortex. BDNF mRNA increased also in the pyramidal layer of hippocampus and in the amygdaloid complex. No change was seen in the level of HDNF/NT-3 mRNA. The increased expression of NGF and BDNF mRNAs was not influenced by pretreatment with the NMDA receptor antagonist MK801, but was partially blocked by the quisqualate, AMPA receptor antagonist NBQX. The presumed subsequent increase of the trophic factors themselves may be important for kindling-associated plasticity in specific neuronal systems in the hippocampus, which could promote hyperexcitability and contribute to the development of epileptic syndromes.     

Differences in ME-LI and VIP-LI in discrete brain regions of seizure-naive and seizure-experienced El mice

In an attempt to elucidate the relationship between endogenous methionine-enkephalin (ME) and vasoactive intestinal polypeptide (VIP) with generalized seizures, we determined regional brain levels of ME-like and VIP-like immunoreactivity (ME-LI and VIP-LI) in El mice during and after seizures induced by repeated tossing stimulation. The levels of ME-LI in the striatum and hippocampus of seizure-naive El mice (El–) were lower than those of the control ddY mice, the mother strain of El mice. Conversely, the level of VIP-LI in the medulla oblongata and pons of El– was higher than that of ddY mice. The level of ME-LI in the striatum of seizure-experienced El mice (El+) killed 96 hours after three consecutive seizures was high, while levels of VIP-LI in the striatum and hypothalamus were low, in comparison to those of El– mice. A detailed time-course study revealed that seizures in El mice caused (1) significant decreases in levels of ME-LI in the striatum and hippocampus during seizures, (2) a significant decrease of VIP-LI content in the striatum 3 hours after seizures, and (3) a significant increase in hypothalamic VIP-LI 9 hours after seizures. These observations suggest that ME and VIP may play some role in El mouse seizures.     

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.     

Repeated 6-Hz Corneal Stimulation Progressively Increases FosB/ΔFosB Levels in the Lateral Amygdala and Induces Seizure Generalization to the Hippocampus

Exposure to repetitive seizures is known to promote convulsions which depend on specific patterns of network activity. We aimed at evaluating the changes in seizure phenotype and neuronal network activation caused by a modified 6-Hz corneal stimulation model of psychomotor seizures. Mice received up to 4 sessions of 6-Hz corneal stimulation with fixed current amplitude of 32 mA and inter-stimulation interval of 72 h. Video-electroencephalography showed that evoked seizures were characterized by a motor component and a non-motor component. Seizures always appeared in frontal cortex, but only at the fourth stimulation they involved the hippocampus, suggesting the establishment of an epileptogenic process. Duration of seizure non-motor component progressively decreased after the second session, whereas convulsive seizures remained unchanged. In addition, a more severe seizure phenotype, consisting of tonic-clonic generalized convulsions, was predominant after the second session. Immunohistochemistry and double immunofluorescence experiments revealed a significant increase in neuronal activity occurring in the lateral amygdala after the fourth session, most likely due to activity of principal cells. These findings indicate a predominant role of amygdala in promoting progressively more severe convulsions as well as the late recruitment of the hippocampus in the seizure spread. We propose that the repeated 6-Hz corneal stimulation model may be used to investigate some mechanisms of epileptogenesis and to test putative antiepileptogenic drugs.     

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.     

局部高频电刺激海马对海人酸致痫大鼠海马细胞外谷氨酸和γ-氨基丁酸释放的影响

目的：通过高频电刺激海人酸癫痫模型大鼠海马,观察海马细胞外谷氨酸（Glu）和γ-氨基丁酸（GABA）的动态变化。方法：将SD大鼠分成4大组（n=10）：①空白组;②海人酸组;③假刺激组：植入刺激电极未予电刺激;④电刺激组：海人酸注射后予130 Hz电刺激。利用微透析技术收集不同时段海马细胞外液,应用高效液相-荧光检测法测定收集液Glu、GABA的浓度。结果：注射海人酸后Glu明显升高,并持续至第14天,电刺激使Glu明显下降;而注射海人酸后GABA呈短暂性升高,后逐渐下降于第4天后保持稍高于正常水平,电刺激并无明显改变GABA的水平。结论：海马细胞外Glu下降在海马电刺激治疗癫痫中起到重要作用;高频电刺激海马选择性地减少谷氨酸能神经元活动,但不影响GABA的释放。     

Involvement of glutamatergic system of amygdala in generalized seizures induced by soman: comparison with the hippocampus]

During seizures induced by soman, an organophosphorus compound, irreversible inhibitor of acetylcholinesterase, the intra-amygdaloid microdialysis of extracellular glutamate, an excitatory amino-acid, showed a sustained increase, more rapid than in hippocampus. This result suggests an early involvement of the amygdala in the development of soman-induced seizures. Moreover, the ex vivo, study by quantitative autoradiography of the binding of tritiated TCP (thienyl-phencyclidine) does not reveal an opening of ionic channels linked to N-methyl-D-aspartate (NMDA) sensitive receptors of glutamate, during seizures, unlike in the hippocampus. This difference could indicate, according to other experimental models, that in amygdala the release of glutamate could occur massively without repeated stimuli as in the hippocampus.     

Electroconvulsive shock causes neuronal death in the mouse hippocampus: correlation of neurodegeneration and convulsive activity

Relations between seizures induced by repeated electroshock (ES) and structural changes in the hippocampus were investigated in Balb/C mice. Brain sections of the animals 2 or 7 days after the last ES were stained for Nissl or TUNEL (apoptotic nuclei). Direct measurement of caspase-3 activity (a key enzyme of apoptosis) in brain regions was performed immediately after the last ES. Statistically significant, albeit moderate cell loss was demonstrated in the CA1 field and dentate gyrus, but not in the CA3 field of the hippocampus. The number of neurons in these fields inversely correlated with seizure severity. No apoptotic nuclei could be revealed either in hippocampus or in other brain regions. Caspase-3 in the hippocampus decreased after ES. The data obtained support the results from other groups showing prominent functional changes in neurons induced by repeated ES and extend this concept directly testifying for a moderate (within 10%), albeit statistically significant neuronal death in selected hippocampal fields. The inverse correlation of cell number with severity of seizures suggest that these are seizures inducing neuronal death.     

The role of the opiate mechanisms of the hippocampus and substantia nigra in the behavioral and convulsive disorders in picrotoxin-induced kindling]

It was shown in the experiments on rats that the repeated picrotoxin administration resulted in the kindling of generalized seizures. Generalized convulsions were followed by the development of either postictal depression or explosiveness. The injection of mu-opiate agonist met-enkephalin into hippocampus of kindled rats resulted in the increase in the severity of seizure reactions which were induced by picrotoxin and also in the increase in the number of animals with postictal explosiveness. The injection of dynorphin-A-1-13 (kappa-opiate agonist) into substantia nigra reticulata induced the locomotor depression which was like one in postictal period and resulted in the decrease of picrotoxin-induced seizures severity. It was concluded that mu-opiate system of hippocampus took part in the formation of generator of pathologically enhanced excitation in the structure during kindling and the development of seizure syndrome, providing also the postictal explosiveness. Kappa-opiate system of substantia nigra plays an important role in the activation of the antiepileptic system, limitation of seizures and the development of postictal depression.     

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.     

A study of the action of anticonvulsant drugs on an experimental model of epilepsy

Repeated electrical low intensity stimulation of various regions of the brain has been shown to induce epileptic seizure activity. This experimental model of epilepsy has been used in our laboratory in rats. Male rats, anaesthetized with nembutal (20 mg/kg, i.p.) and ketalar (60 mg/kg, i.p.) were implanted stereotaxically with electrodes in the dorsal hippocampus and neocortex and received 2 hours stimulation sessions via the hippocampal electrodes (1 sec, 60 Hz, 200-800 microA) one stimulus per minute, during which electrographical and behavioral seizures were induced. The effect of anticonvulsant drugs was tested on this model: phenobarbital (40 mg/kg) reduced the quantity of epileptic electrographic activity and abolished the behavioral aspects of the seizures; diphenylhydantoin (20 mg/kg) reduced the quantity of epileptic electrographic activity, but had no effect on the behavioral component of the seizures; diazepam (6 mg/kg) only blocked the behavioral component of the seizures leaving the animal stuporous and immobile, but the electrographical component was unaffected; carbamazepine (10 mg/kg) had no effect on the electrographical epileptic seizures and reduced the behavioral aspects, but to a lesser extent than diazepam.     

Medial septal region as a target for modulation of seizure discharges in the hippocampus in a model of acute temporal lobe epilepsy

Investigation of changes in the hippocampal EEG produced by GABAergic and cholinergic substances delivered into the medial septum region was performed in awake rabbits. Changes in the threshold of seizure discharges in the hippocampus evoked by perforant path stimulation (model of acute epilepsy) were also examined. Injections of GABAA receptor antagonist picrotoxin or agonist of cholinergic receptors carbacholine in low doses induced an increase in the power of delta- and theta modulation and appearance of 7-12-Hz oscillations. The threshold of hippocampal seizure afterdischarges decreased. In higher doses, these substances evoked 7-15-Hz oscillations followed by seizures. GABAA receptor agonist muscimol and muscarinic receptor antagonist scopolamine decreased the power of the theta rhythm and increased the seizure threshold. Picrotoxin or carbacholine injected after muscimol or scopolamine, respectively, did not evoke seizures. Thus, we have shown the possibility to control hippocampal activity by local changes in the GABAergic and cholinergic systems of the medial septum region.     

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.     

Cyclic Nucleotide Response of the Hippocampal Formation to Septal Stimulation in Naive and Kindled Rats

Rats were kindled through nonmagnetic electrodes stereotaxically implanted into the medial septum. Concentrations of cyclic AMP and cyclic GMP were measured by radioimmunoassay in seven brain regions after microwave fixation during the development and expression of kindled seizures. Hippocampal concentrations were similar to untreated controls (cyclic GMP level in the left and right hippocampus, 0.66 +/- 0.04 and 0.68 +/- 0.07 pmol/mg of protein, respectively; cyclic AMP, 9.4 +/- 0.9 and 9.6 +/- 0.8 pmol/mg of protein, respectively), in kindled animals that were not stimulated, and in naive animals in response to septal stimulation, in spite of the presence in the latter group of bilateral hippocampal afterdischarges. Animals that failed to develop kindling and kindled animals that failed to have a seizure in response to stimulation also showed no change in cyclic nucleotide concentrations in any brain region. Kindled animals that developed a seizure following stimulation showed significant elevations in levels of both cyclic GMP and cyclic AMP in hippocampus and in several other brain regions. A single naive animal that had a seizure in response to its first stimulation also appeared to have elevated concentrations of both cyclic nucleotides in hippocampus. These data suggest that the elevation in levels of both cyclic GMP and cyclic AMP during kindled seizures is associated with seizure development rather than with the generation of afterdischarges or with the kindling engram.     

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.     

Mouse Brain c-fos mRNA Distribution Following a Single Electroconvulsive Shock

The regional distribution of c-fos mRNA in the mouse brain has been investigated by in situ hybridization autoradiography after seizures induced by an acute electroconvulsive shock (ECS). ECS led to a widespread induction of the proto-oncogene c-fos in the brain, with highest concentrations in discrete areas within the limbic system and also in the hypothalamus and cerebellum. The mild stress of sham treatment in earclipped animals induced a weaker and qualitatively different pattern of c-fos mRNA expression involving the cortex, hippocampus, and cerebellum. These data suggest the usefulness of c-fos in situ hybridization as a marker of neuronal stimulation and in mapping a range of effects from a mild stress to the robust changes of an electroconvulsive seizure.