Pilocarpine Model Research Articles

Anticonvulsant effect of phytol in a pilocarpine model in mice

The present study investigated the effects of phytol in pilocarpine-induced seizures. The latency for development of convulsions and mortality rate was recorded in this model using mice. The results revealed that phytol (25, 50 and 75mg/kg, i.p.) increased latency to first seizure and decreased percentage of these seizures. Moreover, phytol also protected the animals against status epilepticus induced by pilocarpine, and decreased the mortality rate. Mice treated with pilocarpine (n=24) presented 100% of mortality during the first hour of observation. In turn, phytol-pretreated animals within 30min before the administration of pilocarpine (400mg/kg) remained alive during the first hour of observation. On the other hand, 6–8h after administration of pilocarpine it was observed that 10 (41.66%), 8 (33.33%) and 4 (16.66%) animals died (respectively). Thus, the pretreatment with phytol was able to block mortality rate during the first hour in acute phase of seizures, and significantly reduced this rate in a dose-dependent manner (p<0.05), suggesting an anticonvulsant effect. In addition, none of the phytol effects was blocked by pre-treatment with flumazenil, an antagonist of benzodiazepine receptors. In conclusion, phytol exhibits anticonvulsant activity by modulating of neurotransmitter systems, but further investigations are in progress to confirm this pharmacological property.

Do proconvulsants modify or halt epileptogenesis? Pentylenetetrazole is ineffective in two rat models of temporal lobe epilepsy

In patients at risk of developing epilepsy after an initial precipitating injury to the brain, the epileptogenic latent period may offer a window of opportunity for initiating potential antiepileptogenic therapy in an attempt to prevent epilepsy from developing. One potential target for antiepileptogenesis is the development of neuronal hyperexcitability during the latent period. Surprisingly, some recent studies in models of temporal lobe epilepsy (TLE) have suggested that proconvulsant drugs could have favourable effects on epileptogenesis, resulting in the proposal of pursuing proconvulsant prophylaxis for epileptogenesis. In the present study, we evaluated this provocative hypothesis by experiments with the GABA(A) receptor antagonist pentylenetetrazole (PTZ) in two TLE models, the intrahippocampal kainate model and the lithium-pilocarpine model in rats. First, we repeatedly determined the PTZ seizure threshold by i.v. infusion of the convulsant during the latent period following intrahippocampal kainate. In line with recent experiments in the lithium-pilocarpine model, the PTZ seizure threshold was significantly decreased over several days following status epilepticus. We then studied whether prolonged infusion of a proconvulsant dose of PTZ at different times after kainate or pilocarpine affected the development of epilepsy. PTZ did not prevent the development of spontaneous recurrent seizures and did not decrease their frequency or severity, but exerted only a moderate disease-modifying effect in that spontaneous seizures in the kainate model were significantly shortened. These data indicate that administration of proconvulsant drugs such as PTZ during the latent period following SE is not a promising strategy for preventing epilepsy.

Manganese-enhanced magnetic resonance imaging in the acute phase of the pilocarpine-induced model of epilepsy

Magnetic resonance images are useful in the study of experimental models of temporal lobe epilepsy. The manganese-enhanced MRI (MEMRI) technique is of interest since it combines the effects caused by manganese on the increased contrast in activated cell populations, when competing with calcium in synaptic transmission. Thus, the purpose of this study was to investigate the temporal evolution of the contrast related to manganese in the acute phase of temporal lobe epilepsy induced by systemic pilocarpine and compare it to the expression of the c-Fos protein. During this phase, the intensity of the MEMRI signal was analyzed at three different time points (5, 15 or 30 minutes) after the onset of status epilepticus (SE). The group that was maintained in status epilepticus for 30 minutes showed a decrease in intensity of the signal in CA1 and the dentate gyrus (DG). There were no differences between the control group and the other groups treated with pilocarpine. The expression of the protein, c-Fos, in the same animals showed that even in the short-duration status epilepticus (5 minutes), there was already maximal cellular activation in subregions of the hippocampus (DG, CA1 and CA3). Under the experimental conditions tested, our data suggest that the MEMRI signal was not sensitive for the identification of detectable variations of cell activation in the acute phase of the pilocarpine model. Our findings are not consistent with the idea that manganese contrast reflects primarily alterations in cellular activity during SE when other signal-modifying elements can act.

Manganese-enhanced magnetic resonance imaging detects mossy fiber sprouting in the pilocarpine model of epilepsy.

Mossy fiber sprouting (MFS) is a frequent finding following status epilepticus (SE). The present study aimed to test the feasibility of using manganese-enhanced magnetic resonance imaging (MEMRI) to detect MFS in the chronic phase of the well-established pilocarpine (Pilo) rat model of temporal lobe epilepsy (TLE). To modulate MFS, cycloheximide (CHX), a protein synthesis inhibitor, was coadministered with Pilo in a subgroup of animals. In vivo MEMRI was performed 3 months after induction of SE and compared to the neo-Timm histologic labeling of zinc mossy fiber terminals in the dentate gyrus (DG). Chronically epileptic rats displaying MFS as detected by neo-Timm histology had a hyperintense MEMRI signal in the DG, whereas chronically epileptic animals that did not display MFS had minimal MEMRI signal enhancement compared to nonepileptic control animals. A strong correlation (r = 0.81, p < 0.001) was found between MEMRI signal enhancement and MFS. This study shows that MEMRI is an attractive noninvasive method for detection of mossy fiber sprouting in vivo and can be used as an evaluation tool in testing therapeutic approaches to manage chronic epilepsy.

Time-Dependent Modulation of Mitogen Activated Protein Kinases and AKT in Rat Hippocampus and Cortex in the Pilocarpine Model of Epilepsy

The epileptogenesis may involve a variety of signaling events that culminate with synaptic reorganization. Mitogen-activated protein kinases (MAPKs) and AKT may be activated by diverse stimulus including neurotransmitter, oxidative stress, growth factors and cytokines and are involved in synaptic plasticity in the hippocampus and cerebral cortex. The pilocarpine model in rodents reproduces the main features of mesial temporal lobe epilepsy related to hippocampus sclerosis (MTLE-HS) in humans. We analyze the phosphorylation profile of MAPKs (ERK1/2, p38(MAPK), JNK1/2/3) and AKT by western blotting in the hippocampus (Hip) and cortex (Ctx) of male adult wistar rats in different periods, after pilocarpine induced status epilepticus (Pilo-SE) and compared with control animals. Biochemical analysis were done in the Hip and Ctx at 1, 3, 12h (acute period), 5days (latent period) and 50days (chronic period) after Pilo-SE onset. Hence, the main findings include increased phosphorylation of ERK1 and p38(MAPK) in the Hip and Ctx 1 and 12h after the Pilo-SE onset. The JNK2/3 isoform (54kDa) phosphorylation was decreased at 3h after the Pilo-SE onset and in the chronic period in the Hip and Ctx. The AKT phosphorylation increased only in the Hip during the latent period. Our study demonstrates, in a systematic manner, the profile of MAPKs and AKT modulation in the hippocampus and cerebral cortex in response to pilocarpine. Based in the role of each signaling enzyme is possible that these changes may be related, at least partially, to modifications in the intrinsic neuronal physiology and epileptogenic synaptic network that appears in the MTLE-HS.

The mTOR pathway is activated in glial cells in mesial temporal sclerosis

Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates basic cellular processes, including development and growth. Mutations in mTOR cause tuberous sclerosis complex (TSC), a condition that is characterized by developmental brain malformations (cortical tubers) and epilepsy. Although considerable insight has been gained recently into the pathologic dysfunction of mTOR in tubers in TSC-related epilepsy, data on the mTOR cascade in mesial temporal lobe epilepsy (MTLE) are lacking. Immunohistochemical investigation with confocal microscopy was performed to evaluate mTOR cascade and to correlate its activity with cellular alterations observed in surgically resected samples of human neocortex and hippocampus in MTLE. We compared results in human tissue to findings in the rat pilocarpine model of sclerotic MTLE. In nonsclerotic and control hippocampus, many neurons in the CA1 subfield expressed high levels of phospho-S6 (p-S6), a reliable marker of mTOR activation. In nonsclerotic and control hippocampus, as well as in magnetic resonance imaging (MRI) normal human neocortex, protoplasmic astrocytes did not express p-S6. In contrast, in sclerotic hippocampus, prominent p-S6 immunostaining was observed mainly in astrocytes and microglia located in the areas of neuronal loss and astrogliosis, whereas neurons in preserved areas of CA1 expressed significantly lower levels of p-S6 immunopositivity than neurons in nonsclerotic or control CA1 subfields. In surgically resected neocortex with chronic astroglial scar tissue, only microglia revealed moderate p-S6 immunoreactivity. Different from human sclerotic epileptic hippocampus, astrogliosis in the chronic rat pilocarpine model of epilepsy was not characterized by glial cells with mTOR activation. The mTOR cascade is activated in astroglial cells in sclerotic MTLE, but not in astrocytes in chronic neocortical scarring or in the pilocarpine model of MTLE. These findings suggest that the astroglial "scar" in sclerotic MTLE has active, ongoing cellular changes. Targeting mTOR in MTLE may provide new pathways for the medical therapy of epilepsy.

Evaluation of the antiepileptic effect of curcumin and Nigella sativa oil in the pilocarpine model of epilepsy in comparison with valproate

The present study aimed to investigate the effect of curcumin and Nigella sativa oil (NSO) on amino acid neurotransmitter alterations and the histological changes induced by pilocarpine in the hippocampus and cortex of rats. Epilepsy was induced by i.p. injection of pilocarpine, and the animals were left for 22days to establish spontaneous recurrent seizures. They were then treated with curcumin, NSO or valproate for 21days. Pilocarpine induced a significant increase in hippocampal aspartate and a significant decrease in glycine and taurine levels. In the cortex, a significant increase in aspartate, glutamate, GABA, glycine, and taurine levels was obtained after pilocarpine injection. Treatment of pilocarpinized rats with curcumin and valproate ameliorated most of the changes in amino acid concentrations and reduced the histopathological abnormalities induced by pilocarpine. N. sativa oil failed to improve the pilocarpine-induced abnormalities. This may explain the antiepileptic effect of curcumin and suggest its use as an anticonvulsant.

The levels of renin–angiotensin related components are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy

We previously showed that patients with temporal lobe epilepsy (TLE) present an increased expression of angiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulation of renin–angiotensin system (RAS) in this disease.This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels of the peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1–7 (Ang 1–7), were detected by HPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin converting enzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampus of Wistar rats by real time PCR, during acute (n=10), silent (n=10) and chronic (n=10) phases of pilocarpine-induced epilepsy.We observed an increased peptide level of Ang1–7 into acute and silent phases, decreasing importantly (p⩽0.05) in the chronic phase, suggesting that AngI may be converted into Ang 1–7 by NEP, which is present in high levels in these periods.Our results also showed increased peptide level of AngII in the chronic phase of this model. In contraposition, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngI may be cleaved to AngII by tonin, which showed increased expression in all phases. We found changes in AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1–7 could act at Mas receptor during the silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides, their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-induced epilepsy.

Cannabidiol exerts anti-convulsant effects in animal models of temporal lobe and partial seizures

Cannabis sativa has been associated with contradictory effects upon seizure states despite its medicinal use by numerous people with epilepsy. We have recently shown that the phytocannabinoid cannabidiol (CBD) reduces seizure severity and lethality in the well-established in vivo model of pentylenetetrazole-induced generalised seizures, suggesting that earlier, small-scale clinical trials examining CBD effects in people with epilepsy warrant renewed attention. Here, we report the effects of pure CBD (1, 10 and 100mg/kg) in two other established rodent seizure models, the acute pilocarpine model of temporal lobe seizure and the penicillin model of partial seizure. Seizure activity was video recorded and scored offline using model-specific seizure severity scales. In the pilocarpine model CBD (all doses) significantly reduced the percentage of animals experiencing the most severe seizures. In the penicillin model, CBD (≥10mg/kg) significantly decreased the percentage mortality as a result of seizures; CBD (all doses) also decreased the percentage of animals experiencing the most severe tonic–clonic seizures. These results extend the anti-convulsant profile of CBD; when combined with a reported absence of psychoactive effects, this evidence strongly supports CBD as a therapeutic candidate for a diverse range of human epilepsies.

Statistical parametric mapping reveals regional alterations in cannabinoid CB1 receptor distribution and G‐protein activation in the 3D reconstructed epileptic rat brain

The endocannabinoid system is known to modulate seizure activity in several in vivo and in vitro models, and CB(1) -receptor activation is anticonvulsant in the rat pilocarpine model of acquired epilepsy (AE). In these epileptic rats, a unique redistribution of the CB(1) receptor occurs within the hippocampus; however, an anatomically inclusive analysis of the effect of status epilepticus (SE)-induced AE on CB(1) receptors has not been thoroughly evaluated. Therefore, statistical parametric mapping (SPM), a whole-brain unbiased approach, was used to study the long-term effect of pilocarpine-induced SE on CB(1) -receptor binding and G-protein activation in rats with AE. Serial coronal sections from control and epileptic rats were cut at equal intervals throughout the neuraxis and processed for [(3) H]WIN55,212-2 (WIN) autoradiography, WIN-stimulated [(35) S]GTPγS autoradiography, and CB(1) -receptor immunohistochemistry (IHC). The autoradiographic techniques were evaluated with both region of interest (ROI) and SPM analyses. In rats with AE, regionally specific increases in CB(1) -receptor binding and activity were detected in cortex, discrete thalamic nuclei, and other regions including caudate-putamen and septum, and confirmed by IHC. However, CB(1) receptors were unaltered in several brain regions, including substantia nigra and cerebellum, and did not exhibit regional decreases in rats with AE. This study provides the first comprehensive evaluation of the regional distribution of changes in CB(1) -receptor expression, binding, and G-protein activation in the rat pilocarpine model of AE. These regions may ultimately serve as targets for cannabinomimetic compounds or manipulation of the endocannabinoid system in epileptic brain.

Increased Gαo activity in C57Bl/6J mice enhances sensitivity to a model of epilepsy

Despite a large number of anti‐epileptic drugs on the market, many patients suffering from partial complex epilepsy with secondary generalizations (PCE) are refractory to current medications. Thus identification of new therapeutic targets is required. This work focused on two targets, Gαo and Gαi2. We hypothesized that increasing activity of these proteins would be protective from seizures as inhibition of these and related proteins by pertussis toxin enhances seizure susceptibility in a pilocarpine model. Mouse lines were created with increased activity of Gαo (Gαo RGSi) or Gαi2 (Gαi2 RGSi). Both mouse lines were exposed to a chemical kindling model with relevance to PCE using repeated injections of pentylenetetrazol (35mg/kg, i.p.) three times per week. Gαo +/RGSi mice have a faster onset to seizure on the C57Bl/6J background while Gαi2 +/RGSi mice have no change compared to littermate controls. The enhanced kindling in Gαo +/RGSi mutant mice is background dependent, whereby Gαo +/RGSi mice on the 129SvImJ background kindle no faster than their littermate controls. Furthermore, a 129SvImJ modifier locus that we previously identified on Chromosome 17 that reduces early lethality of Gαo +/RGSi mice also reduces the rate at which Gαo +/RGSi mice kindle on the C57Bl/6J background. (Supported by NIH R01 GM036592‐23 and a fellowship from the PhRMA Foundation)

Upregulation of dysbindin in temporal lobe epileptic foci of human and experimental animals

The gene encoding dystrobrevin-binding-protein-1 (dysbindin) is expressed in many areas of the central nervous system and plays a role in intracellular vesicle trafficking, synaptic vesicle trafficking, and neurotransmitter release. At a cellular level, dysbindin is thought to mediate presynaptic glutamatergic transmission. Using Western blotting and immunofluorescence, we investigated dysbindin expression in brain tissues of the patients with temporal lobe epilepsy (TLE) and rats with TLE (lithium chloride pilocarpine model) to explore its possible role in epileptogenesis. Twenty-five samples of temporal neocortex from patients undergoing surgery for drug-refractory TLE epilepsy and 10 histologically normal temporal lobes tissues from control subjects were used in our study. We also examined dysbindin expression in the hippocampus and adjacent cortex from experimental Sprague-Dawley rats. Dysbindin was expressed in the cytoplasm of neurons from epileptic specimens, and levels of dysbindin proteins were significantly increased in patients with TLE. Dysbindin was also expressed in the neurons of the hippocampus and adjacent cortex from experimental and control rats. Western blotting of rat brain tissue showed that dysbindin was upregulated gradually from 6 h after kindling. Maximal expression was seen around 2 months in chronic epileptic phase. These results demonstrated that the increased expression of dysbindin might play a role in the pathogenesis of drug-refractory TLE.

Variations in elemental compositions of rat hippocampal formation between acute and latent phases of pilocarpine-induced epilepsy: an X-ray fluorescence microscopy study

There is growing experimental evidence that tracing the elements involved in brain hyperexcitability, excitotoxicity, and/or subsequent neurodegeneration could be a valuable source of data on the molecular mechanisms triggering or promoting further development of epilepsy. The most frequently used experimental model of the temporal lobe epilepsy observed in clinical practice is the one based on pilocarpine-induced seizures. In the frame of this study, the elemental anomalies occurring for the rat hippocampal tissue in acute and silent periods after injection of pilocarpine in rats were compared. X-ray fluorescence microscopy was applied for the topographic and quantitative elemental analysis. The differences in the levels of elements such as P, S, K, Ca, Fe, Cu, and Zn between the rats 3 days (SE72) and 6 h (SE6) after pilocarpine injection as well as naive controls were examined. Comparison of SE72 and control groups showed, for specific areas of the hippocampal formation, lower levels of P, K, Cu, and Zn, and an increase in Ca accumulation. These results as well as further analysis of the differences between the SE72 and SE6 groups confirmed that seizure-induced excitotoxicity as well as mossy fiber sprouting are the mechanisms involved in the neurodegenerative processes which may finally lead to spontaneous seizures in the chronic period of the pilocarpine model. Moreover, in the light of the results obtained, Cu seems to play a very important role in the pathogenesis of epilepsy in this animal model. For all areas analyzed, the levels of this element recorded in the latent period were not only lower than those for controls but were even lower than the levels found in the acute period. The decreased hippocampal accumulation of Cu in the phase of behavior and EEG stabilization, a possible inhibitory effect of this element on excitatory amino acid receptors, and enhanced seizure susceptibility in Menkes disease (an inherited Cu transport disorder leading to Cu deficiency in the brain) suggest a neuroprotective role rather than neurodegenerative and proconvulsive roles of Cu in pilocarpine-induced epilepsy.

Hyperpolarization-activated cation current Ih of dentate gyrus granule cells is upregulated in human and rat temporal lobe epilepsy

The hyperpolarization-activated cation current Ih is an important regulator of neuronal excitability and may contribute to the properties of the dentate gyrus granule (DGG) cells, which constitute the input site of the canonical hippocampal circuit. Here, we investigated changes in Ih in DGG cells in human temporal lobe epilepsy (TLE) and the rat pilocarpine model of TLE using the patch-clamp technique. Messenger-RNA (mRNA) expression of Ih-conducting HCN1, 2 and 4 isoforms was determined using semi-quantitative in-situ hybridization. Ih density was ∼1.8-fold greater in DGG cells of TLE patients with Ammon’s horn sclerosis (AHS) as compared to patients without AHS. The magnitude of somatodendritic Ih was enhanced also in DGG cells in epileptic rats, most robustly during the latent phase after status epilepticus and prior to the occurrence of spontaneous epileptic seizures. During the chronic phase, Ih was increased ∼1.7-fold. This increase of Ih was paralleled by an increase in HCN1 and HCN4 mRNA expression, whereas HCN2 expression was unchanged. Our data demonstrate an epilepsy-associated upregulation of Ih likely due to increased HCN1 and HCN4 expression, which indicate plasticity of Ih during epileptogenesis and which may contribute to a compensatory decrease in neuronal excitability of DGG cells.

Social behavior impairment in offspring exposed to maternal seizures in utero

Human and animal models have demonstrated that maternal seizures in utero could be deleterious to the development of the offspring. This study focused on the social behavior of offspring exposed to seizures in utero. A pilocarpine model of temporal lobe epilepsy was induced in female Wistar rats that were mated after the first spontaneous seizure. Early after birth, pups from an epileptic mother were reared by a control mother. To evaluate the influence of the adoption process, two other groups were added: rat pups from control mothers cross-fostered with other control mothers, and rat pups reared by their birth mother. Animals exposed to seizures in utero showed impaired social behavior with no signs of anxiety-like behavior. This study demonstrated that epileptic seizures during pregnancy could be harmful to brain development and may increase the risk of developing neurodevelopmental disorders. The mechanisms underlying the abnormalities of social behavior are not well understood, and further studies in this field are warranted.

Prolonged increase in rat hippocampal chemokine signalling after status epilepticus

Temporal lobe epilepsy (TLE) is one of the most common focal epilepsy syndromes. In a genome-wide expression study of the human TLE hippocampus we previously showed up-regulation of genes involved in chemokine signalling. Here we investigate in the rat pilocarpine model for TLE, whether changes in chemokine signalling occur during epileptogenesis and are persistent. Therefore we analysed hippocampal protein expression and cellular localisation of CCL2, CCL4, CCR1 and CCR5 after status epilepticus. We found increased CCL4 (but not CCL2) expression in specific populations of hilar astrocytes at 2 and 19weeks after SE concomitant with a persistent up-regulation of its receptor CCR5. Our results show an early and persistent up-regulation of CCL4/CCR5 signalling during epileptogenesis and suggest that CCL4 signalling, rather than CCL2 signalling, could have a role in the epileptogenic process.

Involvement of GABAergic and glutamatergic systems in the anticonvulsant activity of 3-alkynyl selenophene in 21 day-old rats

In this study, we investigated the role of GABAergic and glutamatergic systems in the anticonvulsant action of 3-alkynyl selenophene (3-ASP) in a pilocarpine (PC) model of seizures. To this purpose, 21 day-old rats were administered with an anticonvulsant dose of 3-ASP (50 mg/kg, per oral, p.o.), and [(3)H]γ-aminobutyric acid (GABA) and [(3)H]glutamate uptakes were carried out in slices of cerebral cortex and hippocampus. [(3)H]GABA uptake was decreased in cerebral cortex (64%) and hippocampus (58%) slices of 21 day-old rats treated with 3-ASP. In contrast, no alteration was observed in [(3)H]glutamate uptake in cerebral cortex and hippocampus slices of 21 day-old rats that received 3-ASP. Considering the drugs that increase synaptic GABA levels, by inhibiting its uptake or catabolism, are effective anticonvulsants, we further investigated the possible interaction between sub-effective doses of 3-ASP and GABA uptake or GABA transaminase (GABA-T) inhibitors in PC-induced seizures in 21 day-old rats. For this end, sub-effective doses of 3-ASP (10 mg/kg, p.o.) and DL-2,4-diamino-n-butyric acid hydrochloride (DABA, an inhibitor of GABA uptake--2 mg/kg, intraperitoneally; i.p.) or aminooxyacetic acid hemihydrochloride (AOAA; a GABA-T inhibitor--10 mg/kg, i.p.) were co-administrated to 21 day-old rats before PC (400 mg/kg; i.p.) treatment, and the appearance of seizures was recorded. Results demonstrated that treatment with AOAA and 3-ASP or DABA and 3-ASP significantly abolished the number of convulsing animals induced by PC. The present study indicates that 3-ASP reduced [(3)H]GABA uptake, suggesting that its anticonvulsant action is related to an increase in inhibitory tonus.

Altered neurotransmitter release, vesicle recycling and presynaptic structure in the pilocarpine model of temporal lobe epilepsy

In searching for persistent seizure-induced alterations in brain function that might be causally related to epilepsy, presynaptic transmitter release has relatively been neglected. To measure directly the long-term effects of pilocarpine-induced status epilepticus on vesicular release and recycling in hippocampal mossy fibre presynaptic boutons, we used (i) two-photon imaging of FM1-43 vesicular release in rat hippocampal slices; and (ii) transgenic mice expressing the genetically encoded pH-sensitive fluorescent reporter synaptopHluorin preferentially at glutamatergic synapses. In this study we found that, 1-2 months after pilocarpine-induced status epilepticus, there were significant increases in mossy fibre bouton size, faster rates of action potential-driven vesicular release and endocytosis. We also analysed the ultrastructure of rat mossy fibre boutons using transmission electron microscopy. Pilocarpine-induced status epilepticus led to a significant increase in the number of release sites, active zone length, postsynaptic density area and number of vesicles in the readily releasable and recycling pools, all correlated with increased release probability. Our data show that presynaptic release machinery is persistently altered in structure and function by status epilepticus, which could contribute to the development of the chronic epileptic state and may represent a potential new target for antiepileptic therapies.

Reduced ictogenic potential of 4-aminopyridine in the hippocampal region in the pilocarpine model of epilepsy

It was previously shown that the ictogenic potential of 4-aminopyridine (4-AP) was reduced in the parahippocampal region of kainate treated chronic epileptic rats. In the actual study we investigated the potential of 4-aminopyridine (50 and 100μM) to induce seizure like events (SLEs) in combined entorhinal cortex hippocampal slices from Wistar rats following pilocarpine induced status epilepticus. The potential of 4-AP to induce SLEs in the entorhinal cortex was reduced in the latent period and in slices of chronic epileptic animals with a high seizure incidence in vivo (>2seizures/24h). 4-AP induced SLEs in slices from animals with a low incidence of seizures in vivo (<2seizures/24h) in a similar manner as compared to controls. The hippocampal formation displayed no SLEs, instead short recurrent epileptiform discharges (REDs) were evoked by application of 4-AP in areas CA3 and CA1. The incidence of REDs was largest in slices from control animals. This study shows that the reduced ictogenic potential of 4-AP is not restricted to kainate treated chronic epileptic animals as it can be found in the pilocarpine model as well. The underlying mechanisms may relate to altered expression and editing of voltage gated potassium channels.

Corticotropin releasing factor (CRF) in the hippocampus of the mouse pilocarpine model of status epilepticus

We investigated the cellular localization and progressive changes of corticotropin releasing factor (CRF) in the mouse hippocampus, during and after pilocarpine induced status epilepticus (PISE) and subsequent epileptogenesis. We found that CRF gene expression was up-regulated significantly at 2h during and 1d after PISE in comparison to control mice. Immunohistochemical analysis showed that the number of CRF and Fos immunoreactive cells was increased significantly in the strata oriens and pyramidale of CA1 area and in the stratum pyramidale of CA3 area at 2h during and 1d after PISE. CRF was induced in calbindin (CB) or calretinin (CR) immunoreactive interneurons in stratum oriens at 2h during PISE. It suggests that induced CRF may be related to the over excitation of hippocampal neurons and occurrence of status epilepticus. It may also cause excitoneurotoxicity and delayed loss of CA3 and CA1 pyramidal neurons, leading to the onset of epilepsy.