Kainic Acid-treated Rats Research Articles

Stabilization of mitochondrial function by chlorogenic acid protects against kainic acid-induced seizures and neuronal cell death in rats

The current study investigated the effect of chlorogenic acid, a polyphenolic compound found in numerous plant products, on a kainic acid-induced seizure rat model and its potential mechanism. Rats were administered chlorogenic acid (10 and 50 mg/kg) intraperitoneally for 30 min before kainic acid (15 mg/kg) intraperitoneal administration. Pretreatment with chlorogenic acid decreased the seizure score, increased the latency to onset of the first seizure, and decreased the mortality rate. Chlorogenic acid pretreatment also resulted in a significant reduction in glutamate elevation and neuronal death in the hippocampus of kainic acid-treated rats. In addition, electron microscopy revealed that kainic acid-induced changes in hippocampal mitochondrial structure were prevented by chlorogenic acid pretreatment. Additionally, the levels of mitochondrial function-related proteins, including sirtuin 3, Complex I, glutamate dehydrogenase 1 and ATP synthase, were increased, and the level of the mitochondrial damage marker cytochrome C was decreased in the hippocampus of chlorogenic acid/kainic acid rats. Furthermore, the expression of mitochondrial biogenesis-related proteins [AMP-activated protein kinase (AMPK), sirtuin1, and peroxisome proliferator-activated receptor γ-coactivator-1α (PGC-1α)] and mitophagy-related proteins [phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), Parkin, and microtubule-associated protein 1 light chain 3 (LC3)] was decreased in the hippocampus of kainic acid-treated rats, which was reversed by chlorogenic acid pretreatment. These observations reveal the marked neuroprotective potential of chlorogenic acid against kainic acid-induced neurotoxicity and seizures through prevention of glutamate increase and preservation of AMPK/sirtuin 1/PGC-1α-mediated mitochondrial biogenesis and PINK1/Parkin-induced mitophagy to maintain adequate mitochondrial homeostasis and function.

Excitatory amino acids, possible causative agents of nodding syndrome in eastern Africa

BackgroundNodding syndrome (NS) is one type of epilepsy and a progressive disease characterized by nodding symptoms with children in sub-Saharan Africa. The burden for NS children is heavy, not only mentally but financially for themselves and their families, and yet, the cause and cure of NS remain unknown. The kainic acid-induced model in experimental animals is a well-known epilepsy model that is useful for studying human diseases. In this study, we examined similarities of clinical symptoms and histological brain changes between NS patients and kainic acid-treated rats. In addition, we argued for kainic acid agonist as one of the causes of NS.MethodsClinical signs in rats were studied after kainic acid administration, and histological lesions including the expression of tau protein and gliosis, were examined at 24 h, 8 days, and 28 days after dosing.ResultsKainic acid-induced epileptic symptoms were observed in rats, including nodding accompanied by drooling and bilateral neuronal cell death in the hippocampus and piriform cortex regions. In the regions that exhibited neuronal cell death, an increase in tau protein expression and gliosis were found immunohistochemically. The symptoms and brain histology were similar in the NS and kainic acid-induced rat models.ConclusionThe results suggest that kainic acid agonist may be one of the causative substances for NS.

Rutin prevents seizures in kainic acid-treated rats: evidence of glutamate levels, inflammation and neuronal loss modulation.

Rutin, a naturally derived flavonoid molecule with known neuroprotective properties, has been demonstrated to have anticonvulsive potential, but the mechanism of this effect is still unclear. The current study aimed to investigate the probable antiseizure mechanisms of rutin in rats using the kainic acid (KA) seizure model. Rutin (50 and 100 mg kg-1) and carbamazepine (100 mg kg-1) were administered daily by oral gavage for 7 days before KA (15 mg kg-1) intraperitoneal (i.p.) injection. Seizure behavior, neuronal cell death, glutamate concentration, excitatory amino acid transporters (EAATs), glutamine synthetase (GS), glutaminase, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, N-methyl-D-aspartate (NMDA) receptor subunits GluN2A and GluN2B, activated astrocytes, and inflammatory and anti-inflammatory molecules in the hippocampus were evaluated. Supplementation with rutin attenuated seizure severity in KA-treated rats and reversed KA-induced neuronal loss and glutamate elevation in the hippocampus. Decreased glutaminase and GluN2B, and increased EAATs, GS, GluA1, GluA2 and GluN2A were observed with rutin administration. Rutin pretreatment also suppressed activated astrocytes, downregulated the protein levels of inflammatory molecules [interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), high mobility group Box 1 (HMGB1), interleukin-1 receptor 1 (IL-1R1), and Toll-like receptor-4 (TLR-4)] and upregulated anti-inflammatory molecule interleukin-10 (IL-10) protein expression. Taken together, the results indicate that the preventive treatment of rats with rutin attenuated KA-induced seizures and neuronal loss by decreasing glutamatergic hyperactivity and suppressing the IL-1R1/TLR4-related neuroinflammatory cascade.

Allopregnanolone and Pregnanolone Are Reduced in the Hippocampus of Epileptic Rats, but Only Allopregnanolone Correlates with Seizure Frequency

Background: Neurosteroids modulate epileptic activity by interacting with the γ-aminobutyric acid type A receptor, but their brain levels are still undetermined. Objectives: We aimed to establish neurosteroid levels in the neocortex and hippocampus by liquid chromatography/mass spectrometry in epileptic rats. Methods: Kainic acid-treated rats were continuously monitored up to 9 weeks to determine seizure frequency by video electrocorticography (n = 23) and compared to age-matched controls monitored in the same manner (n = 11). Results: Decreased allopregnanolone (–50%; p < 0.05, Mann-Whitney test) and pregnanolone levels (–64%; p < 0.01) were found in the hippocampus, whereas pregnenolone sulfate, pregnenolone, progesterone, and 5α-dihydroprogesterone were nonsignificantly reduced. No changes were found in the neocortex. Moreover, allopregnanolone (but not pregnanolone) levels were positively correlated with seizure frequency (r² = 0.4606, p < 0.01). Conclusion: These findings indicate a selective reduction in hippocampal levels of 3α-reduced neurosteroids. This reduction was partially mitigated by seizures in the case of allopregnanolone.

A role for astrocyte-derived amyloid β peptides in the degeneration of neurons in an animal model of temporal lobe epilepsy.

Kainic acid, an analogue of the excitatory neurotransmitter glutamate, can trigger seizures and neurotoxicity in the hippocampus and other limbic structures in a manner that mirrors the neuropathology of human temporal lobe epilepsy (TLE). However, the underlying mechanisms associated with the neurotoxicity remain unclear. Since amyloid-β (Aβ) peptides, which are critical in the development of Alzheimer's disease, can mediate toxicity by activating glutamatergic NMDA receptors, it is likely that the enhanced glutamatergic transmission that renders hippocampal neurons vulnerable to kainic acid treatment may involve Aβ peptides. Thus, we seek to establish what role Aβ plays in kainic acid-induced toxicity using in vivo and in vitro paradigms. Our results show that systemic injection of kainic acid to adult rats triggers seizures, gliosis and loss of hippocampal neurons, along with increased levels/processing of amyloid precursor protein (APP), resulting in the enhanced production of Aβ-related peptides. The changes in APP levels/processing were evident primarily in activated astrocytes, implying a role for astrocytic Aβ in kainic acid-induced toxicity. Accordingly, we showed that treating rat primary cultured astrocytes with kainic acid can lead to increased Aβ production/secretion without any compromise in cell viability. Additionally, we revealed that kainic acid reduces neuronal viability more in neuronal/astrocyte co-cultures than in pure neuronal culture, and this is attenuated by precluding Aβ production. Collectively, these results indicate that increased production/secretion of Aβ-related peptides from activated astrocytes can contribute to neurotoxicity in kainic acid-treated rats. Since kainic acid administration can lead to neuropathological changes resembling TLE, it is likely that APP/Aβ peptides derived from astrocytes may have a role in TLE pathogenesis.

Myoinositol Attenuates the Cell Loss and Biochemical Changes Induced by Kainic Acid Status Epilepticus.

Identification of compounds preventing or modifying the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action are of great importance. We have previously shown that myoinositol (MI) daily treatment for 28 days prevents certain biochemical changes that are triggered by kainic acid (KA) induced status epilepticus (SE). However in these studies we have not detected any effects of MI on the first day after SE. In the present study we broadened our research and focused on other molecular and morphological changes at the early stages of SE induced by KA and effects of MI treatment on these changes. The increase in the amount of voltage-dependent anionic channel-1 (VDAC-1), cofilin, and caspase-3 activity was observed in the hippocampus of KA treated rats. Administration of MI 4 hours later after KA treatment abolishes these changes, whereas diazepam treatment by the same time schedule has no significant influence. The number of neuronal cells in CA1 and CA3 subfields of hippocampus is decreased after KA induced SE and MI posttreatment significantly attenuates this reduction. No significant changes are observed in the neocortex. Obtained results indicate that MI posttreatment after KA induced SE could successfully target the biochemical processes involved in apoptosis, reduces cell loss, and can be successfully used in the future for translational research.

Increased levels and activity of cathepsins B and D in kainate-induced toxicity

Administration of kainic acid induces acute seizures that result in the loss of neurons, gliosis and reorganization of mossy fiber pathways in the hippocampus resembling those observed in human temporal lobe epilepsy. Although these structural changes have been well characterized, the mechanisms underlying the degeneration of neurons following administration of kainic acid remain unclear. Since the lysosomal enzymes, cathepsins B and D, are known to be involved in the loss of neurons and clearance of degenerative materials in a variety of experimental conditions, we evaluated their potential roles in kainic acid-treated rats. In parallel, we also measured the levels and expression of insulin-like growth factor-II/mannose 6-phosphate (IGF-II/M6P) receptors, which mediate the intracellular trafficking of these enzymes, in kainic acid-treated rats. Our results showed that systemic administration of kainic acid evoked severe loss of neurons along with hypertrophy of astrocytes and microglia in the hippocampus of the adult rat brain. The levels and activity of cathepsins B and D increased with time in the hippocampus of kainic acid-treated rats compared to the saline-injected control animals. The expression of both cathepsins B and D, as evident by immunolabeling studies, was also markedly increased in activated astrocytes and microglia of the kainic acid-treated rats. Additionally, cytosolic levels of the cathepsins were enhanced along with cytochrome c and to some extent Bax in the hippocampus in kainic acid-treated rats. These changes were accompanied by appearance of cleaved caspase-3-positive neurons in the hippocampus of kainic acid-treated animals. The levels of IGF-II/M6P receptors, on the other hand, were not significantly altered, but these receptors were found to be present in a subset of reactive astrocytes following administration of kainic acid. These results, taken together, suggest that enhanced levels/expression and activity of lysosomal enzymes may have a role in the loss of neurons and/or clearance of degenerative materials observed in kainic acid-treated rats.

Excitatory amino acid transporter 2 downregulation correlates with thalamic neuronal death following kainic acid-induced status epilepticus in rat.

Recurrent seizures without interictal resumption (status epilepticus) have been reported to induce neuronal death in the midline thalamic region that has functional roles in memory and decision-making; however, the pathogenesis underlying status epilepticus-induced thalamic neuronal death is yet to be determined. We performed histological and immunohistochemical studies as well as cerebral blood flow measurement using 4.7 tesla magnetic resonance imaging spectrometer on midline thalamic region in Sprague-Dawley rats (n = 75, male, 7 weeks after birth, body weight 250-300 g) treated with intraperitoneal injection of kainic acid (10 mg/kg) to induce status epilepticus (n = 55) or normal saline solution (n = 20). Histological study using paraffin-embedded specimens revealed neuronal death showing ischemic-like changes and Fluoro-Jade C positivity with calcium deposition in the midline thalamic region of epileptic rats. The distribution of neuronal death was associated with focal loss of immunoreactivity for excitatory amino acid transporter 2 (EAAT2), stronger immunoreaction for glutamate and increase in number of Iba-1-positive microglial cells showing swollen cytoplasm and long processes. Double immunofluorescence study demonstrated co-expression of interleukin-1 beta (IL-1β) and inducible nitric oxide synthase (iNOS) within microglial cells, and loss of EAAT2 immunoreactivity in reactive astrocytes. These microglial alterations and astrocytic EAAT2 downregulation were also observed in tissue without obvious neuronal death in kainic acid-treated rats. These results suggest the possible role of glutamate excitotoxicity in neuronal death in the midline thalamic region following kainic acid-induced status epilepticus due to astrocytic EAAT2 downregulation following microglial activation showing upregulation of IL-1β and iNOS.

Auricular electroacupuncture reduced inflammation-related epilepsy accompanied by altered TRPA1, pPKCα, pPKCε, and pERk1/2 signaling pathways in kainic acid-treated rats.

Background. Inflammation is often considered to play a crucial role in epilepsy by affecting iron status and metabolism. In this study, we investigated the curative effect of auricular acupuncture and somatic acupuncture on kainic acid- (KA-) induced epilepsy in rats. Methods. We established an epileptic seizure model in rats by KA (12 mg, ip). The 2 Hz electroacupuncture (EA) was applied at auricular and applied at Zusanli and Shangjuxu (ST36-ST37) acupoints for 20 min for 3 days/week for 6 weeks beginning on the day following the KA injection. Results. The electrophysiological results indicated that neuron overexcitation occurred in the KA-treated rats. This phenomenon could be reversed among either the auricular EA or ST36-ST37 EA treatment, but not in the sham-control rats. The Western blot results revealed that TRPA1, but not TRPV4, was upregulated by injecting KA and could be attenuated by administering auricular or ST36-ST37 EA, but not in the sham group. In addition, potentiation of TRPA1 was accompanied by increased PKCα and reduced PKCε. Furthermore, pERK1/2, which is indicated in inflammation, was also increased by KA. Furthermore, the aforementioned mechanisms could be reversed by administering auricular EA and could be partially reversed by ST36-ST37 EA. Conclusions. These results indicate a novel mechanism for treating inflammation-associated epilepsy and can be translated into clinical therapy.

In situ demonstration of Fluoro-Turquoise conjugated gelatin for visualizing brain vasculature and endothelial cells and their characterization in normal and kainic acid exposed animals

The present study describes a new method for the visualization of the vasculature lumen and endothelial cells and characterizes their morphology in the brains of normal and kainic acid (KA) treated rats. Herein, labeling was accomplished using Fluoro-Turquoise (FT), a novel reactive blue fluorochrome conjugated to gelatin. Strong blue fluorescence was observed throughout the brain vasculature following intra-cardiac perfusion with FT-gel in normal animals. However, in the brains of KA treated rats (hippocampus, midline and ventral thalamus, piriform cortex), the vascular lumen was typically constricted, sclerotic and only faintly stained. The advantages of FT-gel over other markers can be attributed to its unique chemical and spectral properties. Specifically, Fluoro-Turquoise is a very bright blue UV excitable dye that does not bleed through when visualized using other filters, making it ideal for multiple immunofluorescent labeling studies. Its brightness at low magnification also makes it ideal for low magnification whole brain imaging. Compared to alternative techniques for visualizing blood vessels, such as India ink, fluorescent dye-conjugated dextran, the corrosion technique, endothelial cell markers and lectins, the present method results in excellent visualization of blood vessels.

Effects of L-Arginine on Seizure Behavior and Expression of GFAP in Kainic Acid-Treated Rats

Rats i.p. injected once with 10 mg/kg kainic acid exhibited clear seizure behavior (“wet-dog shakes,” rearing on the hindlimbs, and bilateral clonus). Pretreatment with L-arginine (L-Arg twice a day for 5 days) significantly decreased these manifestations. The medium dose of L-Arg (40 mg/kg) was found to be close to optimum; 10 and 160 mg/kg L-Arg provided much smaller positive effects. In KA-treated rats, a much higher density of GFAP-positive astrocytes was found in the hilus of the dorsal hippocampus, while 40 mg/kg L-Arg+KA-treated rats demonstrated noticeably weaker GFAP overexpression. The results of Western blotting analysis were fully comparable with those obtained in the immunostaining experiments.

Antiepileptic Effect ofUncaria rhynchophyllaandRhynchophyllineInvolved in the Initiation of c-Jun N-Terminal Kinase Phosphorylation of MAPK Signal Pathways in Acute Seizures of Kainic Acid-Treated Rats

Seizures cause inflammation of the central nervous system. The extent of the inflammation is related to the severity and recurrence of the seizures. Cell surface receptors are stimulated by stimulators such as kainic acid (KA), which causes intracellular mitogen-activated protein kinase (MAPK) signal pathway transmission to coordinate a response. It is known that Uncaria rhynchophylla (UR) and rhynchophylline (RP) have anticonvulsive effects, although the mechanisms remain unclear. Therefore, the purpose of this study is to develop a novel strategy for treating epilepsy by investigating how UR and RP initiate their anticonvulsive mechanisms. Sprague-Dawley rats were administered KA (12 mg/kg, i.p.) to induce seizure before being sacrificed. The brain was removed 3 h after KA administration. The results indicate that pretreatment with UR (1.0 g/kg), RP (0.25 mg/kg), and valproic acid (VA, 250 mg/kg) for 3 d could reduce epileptic seizures and could also reduce the expression of c-Jun aminoterminal kinase phosphorylation (JNKp) of MAPK signal pathways in the cerebral cortex and hippocampus brain tissues. Proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α remain unchanged, indicating that the anticonvulsive effect of UR and RP is initially involved in the JNKp MAPK signal pathway during the KA-induced acute seizure period.

Effects of oxcarbazepine on monoamines content in hippocampus and head and body shakes and sleep patterns in kainic acid-treated rats

The aim of this work was to analyze the effect of oxcarbazepine (OXC) on sleep patterns, "head and body shakes" and monoamine neurotransmitters level in a model of kainic-induced seizures. Adult Wistar rats were administered kainic acid (KA), OXC or OXC + KA. A polysomnographic study showed that KA induced animals to stay awake for the whole initial 10h. OXC administration 30min prior to KA diminished the effect of KA on the sleep parameters. As a measure of the effects of the drug treatments on behavior, head and body shakes were visually recorded for 4h after administration of KA, OXC + KA or saline. The presence of OXC diminished the shakes frequency. 4h after drug application, the hippocampus was dissected out, and the content of monoamines was analyzed. The presence of OXC still more increased serotonin, 5-hidroxyindole acetic acid, dopamine, and homovanilic acid, induced by KA.

Kainic acid and 3-Nitropropionic acid induced expression of laminin in vascular elements of the rat brain

Laminin is a glycoprotein component of the basement membrane and has been reported to be found in different areas of the nervous system including brain endothelial cells, Schwann cells and peripheral nerves. Although the in-vitro studies suggest that laminin plays an important role in growth and neurite extension of cultured neurons, localization of laminin in the brain has been controversial and inconsistent results have been reported. Recently, laminin immunoreactivity has been used as a marker for vascular elements in the brain. In this study, we have investigated the effect of two mechanistically different neurotoxins, kainic acid (KA), an NMDA agonist and 3-Nitropropionic acid (3-NPA), an inhibitor of mitochondrial respiration, on brain vascular elements revealed by laminin immunolabeling. We also explored whether administration of these two neurotoxic drugs correlate with the neuronal degeneration observed after neurotoxic insult by staining with Fluoro-Jade C dye. We have employed single immunolabeling to localize laminin in the brains. In KA treated rats, most of the laminin immunoreactivity is present in the piriform cortex, corpus callosum (myelinated tracts) amygdala, hippocampus, ventral thalamus and tenia tacta. In 3-NPA treated animals, laminin immunoreactivity was confined mostly to the striatum. In contrast, saline treated rats showed very little laminin immunolabeling around capillaries, arteries and in the meningeal membranes. To determine the effects of these neurotoxins on the integrity of the blood brain barrier (BBB), endothelial brain barrier antigen (EBA) immunolabeling was also performed. In addition, we performed CD11b immunolabeling to evaluate the effect of 3-NPA and KA on the activation of microglia in the brain. CD11b was dramatically increased in KA and 3-NPA treated animals. We have also combined laminin immunolabeling with Fluoro-Jade C labeling to evaluate the spatio-temporal association of degenerating neurons and the expression of laminin containing microvessels. Areas which showed intense laminin immunolabeling following KA or 3-NPA exposure correlated with those exhibiting the greatest number of degenerating neurons observed after Fluoro-Jade C staining. EBA-laminin double immunolabeling demonstrated that the expressions of laminin were predominantly localized in the areas (cortex, thalamus and hippocampus) where EBA has been either reduced or is absent. Our results from these experiments demonstrate that vascular laminin expression increases after treatment with KA or 3-NPA, suggesting the occurrence of neovascularization. Microglia may also contribute to the neurotoxic induced neovascularization and neurodegeneration.

The microtubule interacting drug candidate NAP protects against kainic acid toxicity in a rat model of epilepsy

NAP (NAPVSIPQ, generic name, davunetide), a neuroprotective peptide in clinical development for neuroprotection against Alzheimer's disease and other neurodegenerative indications, has been recently shown to provide protection against kainic acid excitotoxicity in hippocampal neuronal cultures. In vivo, kainic acid toxicity models status epilepticus that is associated with hippocampal cell death. Kainic acid toxicity has been previously suggested to involve the microtubule cytoskeleton and NAP is a microtubule-interacting drug candidate. In the current study, kainic acid-treated rats showed epileptic seizures and neuronal death. Injection of NAP into the dentate gyrus partially protected against kainic acid-induced CA3 neuron death. Microarray analysis (composed of > 31 000 probe sets, analyzing over 30 000 transcripts and variants from over 25 000 well-substantiated rat genes) in the kainic acid-injured rat brain revealed multiple changes in gene expression, which were prevented, in part, by NAP treatment. Selected transcripts were further verified by reverse transcription coupled with quantitative real-time polymerase chain reaction. Importantly, among the transcripts regulated by NAP were key genes associated with proconvulsant properties and with long-lasting changes that underlie the epileptic state, including activin A receptor (associated with apoptosis), neurotensin (associated with proper neurotransmission) and the Wolfram syndrome 1 homolog (human, associated with neurodegeneration). These data suggest that NAP may provide neuroprotection in one of the most serious neurological conditions, epilepsy.

Are nociceptin(1‐13)NH2and its structural analogue [ORN9]nociceptin(1‐13)NH2able to affect brain antioxidant status in control and kainic acid‐treated rats?

In-vivo effects of nociceptin (N/OFQ(1-13)NH(2)) on the levels of lipid peroxidation and cell enzyme (superoxide dismutase, glutathione peroxidase and glutathione reductase) and non-enzyme (glutathione) antioxidants in brain of control and kainic acid-treated rats were studied. N/OFQ(1-13)NH(2) effects were compared with those of its structural analogue [Orn(9)]N/OFQ(1-13)NH(2). Kainic acid (25 microg, i.c.v) increased the lipid peroxidation (4 and 24 h after kainic acid treatment) and decreased the glutathione level (1 h after kainic acid injection). We failed to find, any changes in antioxidant enzyme activities, independently of the time of kainic acid treatment. At the background of kainic acid-effects, N/OFQ(1-13)NH(2) and [Orn(9)] N/OFQ(1-13)NH(2), injected 30 min before kainic acid, had no effects on all parameters, tested in brain. In addition, the neuropeptides did not change the antioxidant status in brain of control animals. It might be concluded that N/OFQ(1-13)NH(2) and [Orn(9)]N/OFQ(1-13)NH(2) have neither pro- nor anti-oxidant activity.

Neuro-protective effects of carbamazepine on sleep patterns and head and body shakes in kainic acid-treated rats

The aim of this work was to analyze the effect of carbamazepine (CBZ) on sleep patterns and on “head and body shakes” and to determine the role of serotonin (5-HT) in a model of kainic-induced seizures. Thirty male Wistar rats (280–300 g) were used for polygraphic sleep recording. After a basal recording, the rats were allocated into three groups: kainic acid-treated animals (KA; 10 mg/kg; n = 10), carbamazepine-treated animals (CBZ; 30 mg/kg; n = 10) and animals injected with KA 30 min after pretreatment with CBZ (CBZ + KA; n = 10). Polygraphic recordings were performed for 10 h for 3 days, with the exception of the CBZ group, which were observed for 1 day. In order to measure the head and body shakes that occurred over that time, a behavioral assessment was performed in two additional groups of KA ( n = 10) and CBZ + KA ( n = 10) animals. After 10 h of behavioral assessment, the rats were sacrificed, and the levels of 5-HT and 5-hydroxy-indol-acetic acid (5-HIAA) were analyzed. We compared these findings with the results from a group of rats without pharmacological intervention ( n = 10). All of the recordings were performed from 08:00 to 18:00 h. Data analysis: the electrographic parameters, head and body shake counting and monoamine concentrations were analyzed by an ANOVA test. Differences of * p ≤ 0.01 and ** p ≤ 0.001 were considered statistically significant. Our results showed that CBZ exerted a protective effect on sleep pattern alterations induced by KA, which when administered alone caused a complete inhibition of sleep for the first 10 h after administration. Although there was a reduction in the amount of sleep after the administration of KA in CBZ-pretreated animals, sleep inhibition was incomplete. In addition, CBZ decreased the frequency of head and body shakes by 60% as compared to KA. The 5-HT and 5-HIAA levels in the pons were increased in the KA and KA + CBZ groups. Our conclusion is that in addition to decreasing seizure intensity, CBZ facilitates the partial recovery of sleep. These results suggest that CBZ provides neuro-protective effects on sleep and against seizures.

Gene profiling the response to kainic acid induced seizures

Kainic acid activates non- N-methyl- d-aspartate (NMDA) glutamate receptors where it increases synaptic activity resulting in seizures, neurodegeneration, and remodeling. We performed microarray analysis on rat hippocampal tissue following kainic acid treatment in order to study the signaling mechanisms underlying these diverse processes in an attempt to increase our current understanding of mechanisms contributing to such fundamental processes as neuronal protection and neuronal plasticity. The kainic acid-treated rats used in our array experiments demonstrated severe seizure behavior that was also accompanied by neuronal degeneration which is suggested by fluoro-jade B staining and anti-caspase-3 immunohistochemistry. The gene profile revealed 36 novel kainic acid regulated genes along with additional genes previously reported. The functional roles of these novel genes are discussed. These genes mainly have roles in transcription and to a lesser extent have roles in cell death, extracellular matrix remodeling, cell cycle progression, neuroprotection, angiogenesis, and synaptic signaling. Gene regulation was confirmed via quantitative real time polymerase chain reaction and in situ hybridization.

Ultrastructural features of sprouted mossy fiber synapses in kindled and kainic acid-treated rats.

The mossy fiber pathway in the dentate gyrus undergoes sprouting and synaptic reorganization in response to seizures. The types of new synapses, their location and number, and the identity of their postsynaptic targets determine the functional properties of the reorganized circuitry. The goal of this study was to characterize the types and proportions of sprouted mossy fiber synapses in kindled and kainic acid-treated rats. In normal rats, synapses labeled by Timm histochemistry or dynorphin immunohistochemistry were rarely observed in the supragranular region of the inner molecular layer when examined by electron microscopy. In epileptic rats, sprouted mossy fiber synaptic terminals were frequently observed. The ultrastructural analysis of the types of sprouted synapses revealed that 1) in the supragranular region, labeled synaptic profiles were more frequently axospinous than axodendritic, and many axospinous synapses were perforated; 2) sprouted mossy fiber synaptic terminals formed exclusively asymmetric, putatively excitatory synapses with dendritic spines and shafts in the supragranular region and with the soma of granule cells in the granule cell layer; 3) in contrast to the large sprouted mossy fiber synapses in resected human epileptic hippocampus, the synapses formed by sprouted mossy fibers in rats were smaller; and 4) in several cases, the postsynaptic targets of sprouted synapses were identified as granule cells, but, in one case, a sprouted synaptic terminal formed a synapse with an inhibitory interneuron. The results demonstrate that axospinous asymmetric synapses are the most common type of synapse formed by sprouted mossy fiber terminals, supporting the viewpoint that most sprouted mossy fibers contribute to recurrent excitation in epilepsy.

Gap Junctions and Fast Oscillations: A Role in Seizures and Epileptogenesis?

A Possible Role for Gap Junctions in Generation of Very Fast EEG Oscillations Preceding the Onset of and Perhaps Initiating, Seizures We propose an experimentally and clinically testable hypothesis, concerning the origin of very fast (> approximately 70 Hz) EEG oscillations that sometimes precede the onset of focal seizures. These oscillations are important, as they may play a causal role in the initiation of seizures. Subdural EEG recordings were obtained from children with focal cortical dysplasias and intractable seizures. Intra- and extracellular recordings were performed in rat hippocampal slices, with induction of population activity, as follows: (a) bath-applied tetramethylamine (an intracellular alkalinizing agent, that opens gap junctions); (b) bath-applied carbachol, a cholinergic agonist; and (c) focal pressure ejection of hypertonic K+ solution. Detailed network simulations were performed, the better to understand the cellular mechanisms underlying oscillations. A major feature of the simulations was inclusion of axon-axon gap junctions between principal neurons, as supported by recent experimental data. Very fast oscillations were found in children before seizure onset, but also superimposed on bursts during the seizure, and on interictal bursts. In slice experiments, very fast oscillations had previously been seen on interictal-like bursts; we now show such oscillations before, between, and after epileptiform bursts. Very fast oscillations were also seen superimposed on gamma (30–70 Hz) oscillations induced by carbachol or hypertonic K+, and in the latter case, very fast oscillations became continuous when chemical synapses were blocked. Simulations replicate these data, when axonal gap junctions are included. Electrical coupling between principal neurons, perhaps via axonal gap junctions, could underlie very fast population oscillations, in seizure-prone brain, but possibly also in normal brain. The anticonvulsant potential of gap-junction blockers such as carbenoxolone, now in clinical use for treatment of ulcer disease, should be considered. Aperiodic high-frequency oscillations (>100 Hz) reflect a short-term synchronization of neuronal electrical activity. It has been shown in the epileptic brain that spontaneous oscillations in the frequency range of 250–600 Hz reflect action potential population bursts of synchronously discharging neuronal clusters. These oscillations occur in the early stages of epileptogenesis in areas adjacent to the brain lesion and may trigger the formation of seizure-generating neuronal networks. We studied the extent of the area generating oscillations in the frequency range of 250–600 Hz [fast ripples (FRs)] in intrahippocampal kainic acid-treated rats with spontaneous seizures, by analyzing voltage versus depth profiles of FRs in hippocampal and parahippocampal areas in freely moving animals and by spatial mapping in hippocampal slice preparations in vitro. The strength of inhibition was compared in areas with and without FRs using a paired-pulse paradigm. The extent of the areas generating FRs did not exceed 1 mm(3). The areas generating FRs became broader after the application of the GABA(A) receptor antagonist bicuculline. Paired-pulse fast inhibition at 15–30 msec intervals was similar in areas generating FRs and areas not generating FRs. Our data illustrate that hypothesized clusters of highly interconnected neurons are capable of overcoming interneuron feedback inhibition, resulting in generation of epileptiform bursts, eventually leading to seizure activity.