Rat Pilocarpine Model Of Epilepsy Research Articles

Deficit in observational learning in experimental epilepsy.

Individuals use the observation of a conspecific to learn new behaviors and skills in many species. Whether observational learning is affected in epilepsy is not known. Using the pilocarpine rat model of epilepsy, we assessed learning by observation in a spatial task. The task involves a naive animal observing a demonstrator animal seeking a reward at a specific spatial location. After five observational sessions, the observer is allowed to explore the rewarded space and look for the reward. Although control observer rats succeed in finding the reward when allowed to explore the rewarded space, epileptic animals fail. However, epileptic animals are able to successfully learn the location of the reward through their own experience after several trial sessions. Thus, epileptic animals show a clear deficit in learning by observation. This result may be clinically relevant, in particular in children who strongly rely on observational learning.

Dynamic structural neuroplasticity during and after epileptogenesis in a pilocarpine rat model of epilepsy

BackgroundThe role of neuroplasticity in epilepsy has been widely studied in experimental models and human brain samples. However, the results are contradictory and it remains unclear if neuroplasticity is more related to the cause or the consequence of epileptic seizures. Clarifying this issue can provide insights into epilepsy therapies that target the disease mechanism and etiology rather than symptoms. Therefore, this study was aimed to investigate the dynamic changes of structural plasticity in a pilocarpine rat model of epilepsy.MethodsA single acute dose of pilocarpine (380 mg/kg, i.p.) was injected into adult male Wistar rats to induce status epilepticus (SE). Animal behavior was monitored for 2 h. Immunohistochemical staining was performed to evaluate neurogenesis in the CA3 and dentate gyrus (DG) regions of hippocampus using biomarkers Ki67 and doublecortin (DCX). The Golgi-Cox method was performed to analyze dendritic length and complexity. All experiments were performed in control rats (baseline), at 24 h after SE, on day 20 after SE (latent phase), after the first and 10th spontaneous recurrent seizures (SRS; chronic phase), and in non-epileptic rats (which did not manifest SRS 36 days after pilocarpine injection).ResultsSE significantly increased the number of Ki67 and DCX-positive cells, suggesting neurogenesis during the latent phase. The dendritic complexity monitoring showed that plasticity was altered differently during epilepsy and epileptogenesis, suggesting that the two processes are completely separate at molecular and physiological levels. The numbers of spines and mushroom-type spines were increased in the latent phase. However, the dendritogenesis and spine numbers did not increase in rats that were unable to manifest spontaneous seizures after SE.ConclusionAll parameters of structural plasticity that increase during epileptogenesis, are reduced by spontaneous seizure occurrence, which suggests that the development of epilepsy involves maladaptive plastic changes. Therefore, the maladaptive plasticity biomarkers can be used to predict epilepsy before development of SRS in the cases of serious brain injury.

Treatment with CCR2 antagonist is neuroprotective but does not alter epileptogenesis in the pilocarpine rat model of epilepsy

Neuroinflammation role on epileptogenesis has been the subject of increasing interest. Many studies showed elevation in cytokines and chemokines expression following seizures, such as, CCL2 protein (C-C motif ligand 2 chemokine) and its specific receptor, CCR2. In addition, recent studies manipulating the CCL2/CCR2 complex verified improved seizure outcome in different seizure models. In the present study, the effects of CCR2 antagonist was investigated using the pilocarpine rat model of epilepsy. Status epilepticus (SE) was induced by pilocarpine i.p. injection in adult rats. Daily oral treatment with CCR2 antagonist or vehicle was initiated 5 h following SE and lasted 5 or 10 days. Rats were euthanized 5 days after SE to evaluate neuronal damage and glial density or 30 days after SE to investigate spontaneous seizures development and seizure susceptibility to a second hit pentylenetetrazol (PTZ) test. Rats that received CCR2 antagonist presented less degenerating cells at hippocampal CA1 region. There was also a significant decrease in CA1 volume after SE that was not observed in treated rats. On the other hand, microglia cell density increased after SE regardless of CCR2 antagonist use. Treatment with CCR2 antagonist did not alter spontaneous seizure occurrence or later seizure susceptibility to PTZ in chronic rats. Additional rats were pretreated with CCR2 antagonist prior to SE induction, but this did not change SE progression. The data show that oral treatment with CCR2 antagonist is neuroprotective, but does not alter other epileptogenic factors, such as, neuroinflammation, or seizure development, after pilocarpine-induced SE in rats.

Upregulation of hippocampal synaptophysin, GFAP and mGluR3 in a pilocarpine rat model of epilepsy with history of prolonged febrile seizure

Despite the increasing interest and efforts to prevent, treat, and control epilepsy and its clinical manifestation (seizure), the prevalence has not decreased due to incomplete understanding of its etiology. The present study aimed at determining the effect of prolonged febrile seizure (PFS) on the onset of seizure in pilocarpine rat model of temporal lobe epilepsy (TLE).After induction of PFS at postnatal day (PND) 14 in ten out of thirty pups, epilepsy was induced with 350 mg/kg of pilocarpine on PND 60 in the 10 rats with PFS and 10 from the saline group. Animals that met the criteria for TLE were then chosen for the study. Effects of PFS on status epilepticus and the development of spontaneous recurrent seizures (SRSs) were observed. The hippocampal synaptophysin, glial fibrillary acidic protein (GFAP) with metabotropic glutamate receptor 3 (mGluR3) expressions were also measured with LI Cor Tissue florescence, immunofluorescence and immunohistochemistry respectively.We found that PFS reduced the onset of status epilepticus, but had no influence on the development of SRSs. Also, there was no difference in the hippocampal expression of synaptophysin and GFAP between rats that had pilocarpine with or without background PFS. However, the mGluR3 expression was significantly higher in pilocarpine with PFS when compared to pilocarpine alone.Our result showed that PFS may lead to epilepsy. It is also obvious that epilepsy with or without PFS history affects neurochemical expression of synaptophysin, GFAP and mGluR3, hence these proteins may be good targets for drug therapy in reducing both the mortality and morbidity of the disease.

Neuroprotective effects of lovastatin in the pilocarpine rat model of epilepsy according to the expression of neurotrophic factors.

Studies have suggested that neurotrophic factors (NTFs) are involved in the status epilepticus development. This indicates their essential role in mediating acquired epileptic conditions. Therefore, modulating the expression of NTFs may inhibit seizure-induced cell loss in the epileptic lesions. In this study, we examined the anti-apoptotic, anti-necrotic and regulatory effects of lovastatin on the expression of NTFs in the pilocarpine rat model of temporal lobe epilepsy (TLE). A total of 32 male Wistar rats were divided into 4 groups (n = 8 per group): i) normal; ii) non-treated epileptic group [intraperitoneal (i.p.) administration of 350-400mg/kg pilocarpine]; iii) treatment group (pilocarpine-treated rats treated followed by 5mg/kg lovastatin); and iv) vehicle epileptic rats treated with Carboxymethyl cellulose (CMC). Animals that had a behavioral score of 4-5 according to the Racine scale were selected for study participation. Three days after the first seizure, pilocarpine-treated rats received i.p. injections of lovastatin for 14days. The rats were killed and prepared for histopathologic analysis as well as real-time RT-PCR 17days after the first seizure. The results of this study showed increased mRNA expression of glial cell line-derived neurotrophic factor (GDNF) and Ciliary neurotrophic factor (CNTF) and decreased expressions of Brain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), and Neurotrophin-4 (NT-4) mRNA in the epileptic rats treated with lovastatin. Histological analysis of neurodegeneration in the brain sections showed that the number of hippocampal apoptotic and necrotic cells significantly decreased in the treatment groups. Furthermore, numerical density of neurons per area was significantly higher in the treated groups compared with the untreated groups. Collectively, the results of this study have shown that lovastatin could attenuate seizure-induced expression of neurotrophic factors and consequently reduce hippocampal cell death in the pilocarpine rat model of TLE.

Preconditioning with toll-like receptor agonists attenuates seizure activity and neuronal hyperexcitability in the pilocarpine rat model of epilepsy

Neuroinflammation plays an important role in epileptic disorders. Toll-like receptors (TLRs) are the key signal transduction tools by which neuroinflammation may promote epileptogenesis. Depending on the stimulus nature, TLRs may engage a distinct signaling pathway. We examined the impact of early minor activation of TLR4 and TLR2 on the severity of seizure in the pilocarpine rat model of temporal lobe epilepsy (TLE). One μg of Lipopolysaccharides (LPS), Monophosphoryl lipid A (MPL), Pam3Cysor or vehicles were microinjected into the right lateral ventricle of the male Wistar rats. 24 h later, seizures were induced by intraperitoneal injection of pilocarpine, and seizure-related behaviors were monitored. 24 h after seizure induction, the hippocampal level of pro/anti-inflammatory mediators and electrophysiological properties of the dentate gyrus (DG) granular cells were investigated by western blot and whole cell patch clamp techniques, respectively. Pretreatment with TLR ligands resulted in decreased seizure severity, lower hippocampal pro-inflammatory (IL-1β and IL-6) cytokines and higher anti-inflammatory (IL-10 and TGF- β) mediators in the pilocarpine-treated rats. Pilocarpine induced profound hyperexcitability in the DG granule cells accompanied by potentiated excitatory postsynaptic currents (EPSCs) and dampened inhibitory postsynaptic currents (IPSCs), in contrast to the control group. However, pretreatment with TLR ligands preserved almost normal excitability and synaptic transmission against the pilocarpine. In conclusion, early activation of TLR4 and TLR2, probably through preserving normal hippocampal cytokine profile and neuronal function attenuates seizure severity in the rat model of TLE.

Static magnetic fields reduce epileptiform activity in anesthetized rat and monkey

Increasing evidence indicates that static magnetic fields (SMF) reduce cortical activity in both human and animal models. The aim of this work was to investigate the effect of SMF on epileptiform cortical activity, a condition related to an abnormal increase in neuronal excitability. The first experimental block included a Pilocarpine rat model of epilepsy, in which a magnetic neodymium nickel-plated cylinder, a magnetic field of 0.5 T, or “sham” were placed over the skull. In the second experimental block, we recorded epileptic-like activity in the visual cortex of a monkey (Macaca mulatta) under control conditions and in the presence of the magnet. Between 15 and 30 minutes after the second dose of Pilocarpine, EEG changes compatible with seizure like events induced by Pilocarpine were clearly observed in the control animals (sham stimulation). Similar effects were visible in the animals exposed to the real magnet after 1–2 hours. In the monkey, SMF over the cortical focus clearly reduced abnormal activity: the intensity threshold for visual induction increased and the severity and duration decreased. These results reinforce the view that static magnets modulate cortical activity and open the door to the future therapeutic use of SMF in epilepsy as a complement to current pharmacological treatments.

Imaging correlates of behavioral impairments: An experimental PET study in the rat pilocarpine epilepsy model

Psychiatric comorbidities are prevalent in patients with epilepsy and greatly contribute to the overall burden of disease. The availability of reliable biomarkers to diagnose epilepsy-associated comorbidities would allow for effective treatment and improved disease management. Due to their non-invasive nature, molecular imaging techniques such as positron emission tomography (PET) are ideal tools to measure pathologic changes. In the current study we investigated the potential of [18F]fluoro-2-deoxy-d-glucose ([18F]FDG) and 2′-methoxyphenyl-(N-2′-pyridinyl)-p-18F-fluoro-benzamidoethylpiperazine ([18F]MPPF) as imaging correlates of neurobehavioral comorbidities in the pilocarpine rat model of epilepsy. Findings from rats with epilepsy revealed a regional reduction in [18F]FDG uptake indicating thalamic hypometabolism. In addition, an increase in septal [18F]MPPF binding was observed in rats with spontaneous recurrent seizures. Both thalamic [18F]FDG and septal [18F]MPPF data proved to correlate with behavioral alterations including decreases in luxury behavior such as burrowing and social interaction, and changes in behavioral patterns in anxiety tests. A correlation with seizure frequency was confirmed for thalamic [18F]FDG data. Moreover, thalamic [18F]FDG and septal [18F]MPPF data exhibited a correlation with brain-derived neurotrophic factor (BDNF) serum concentrations, which were lowered in rats with epilepsy.In conclusion, μPET data from rats with pilocarpine-induced epileptogenesis indicate altered septal 5-HT1A receptor binding. Further research is necessary assessing whether septal 5-HT1A receptor binding may serve as an imaging correlate of neuropsychiatric comorbidities in epilepsy patients and for severity assessment in rodent epilepsy models. In contrast, we obtained evidence that [18F]FDG uptake also reflects the severity of epilepsy and, thus, might not constitute a biomarker with sufficient specificity for psychiatric comorbidities. Evidence has been obtained that BDNF might serve as a peripheral circulatory biomarker. Further experimental and clinical assessment is necessary for validation of the marker candidates.

Quantitative longitudinal imaging of activated microglia as a marker of inflammation in the pilocarpine rat model of epilepsy using [11C]-(R)-PK11195 PET and MRI

Inflammation may play a role in the development of epilepsy after brain insults. [11C]-(R)-PK11195 binds to TSPO, expressed by activated microglia. We quantified [11C]-(R)-PK11195 binding during epileptogenesis after pilocarpine-induced status epilepticus (SE), a model of temporal lobe epilepsy. Nine male rats were studied thrice (D0-1, D0 + 6, D0 + 35, D0 = SE induction). In the same session, 7T T2-weighted images and DTI for mean diffusivity (MD) and fractional anisotropy (FA) maps were acquired, followed by dynamic PET/CT. On D0 + 35, femoral arterial blood was sampled for rat-specific metabolite-corrected arterial plasma input functions (AIFs). In multiple MR-derived ROIs, we assessed four kinetic models (two with AIFs; two using a reference region), standard uptake values (SUVs), and a model with a mean AIF. All models showed large (up to two-fold) and significant TSPO binding increases in regions expected to be affected, and comparatively little change in the brainstem, at D0 + 6. Some individuals showed increases at D0 + 35. AIF models yielded more consistent increases at D0 + 6. FA values were decreased at D0 + 6 and had recovered by D0 + 35. MD was increased at D0 + 6 and more so at D0 + 35. [11C]-(R)-PK11195 PET binding and MR biomarker changes could be detected with only nine rats, highlighting the potential of longitudinal imaging studies.

Persistent Hyperactivity of Hippocampal Dentate Interneurons After a Silent Period in the Rat Pilocarpine Model of Epilepsy.

Profile of GABAergic interneuron activity after pilocarpine-induced status epilepticus (SE) was examined in the rat hippocampal dentate gyrus by analyzing immediate early gene expression and recording spontaneous firing at near resting membrane potential (REM). SE for exact 2 h or more than 2 h was induced in the male Sprague-Dawley rats by an intraperitoneal injection of pilocarpine. Expression of immediate early genes (IEGs) was examined at 1 h, 1 week, 2 weeks or more than 10 weeks after SE. For animals to be examined at 1 h after SE, SE lasted for exact 2 h was terminated by an intraperitoneal injection of diazepam. Spontaneous firing at near the REM was recorded in interneurons located along the border between the granule cell layer and the hilus more than 10 weeks after SE. Results showed that both c-fos and activity-regulated cytoskeleton associated protein (Arc) in hilar GABAergic interneurons were up-regulated after SE in a biphasic manner; they were increased at 1 h and more than 2 weeks, but not at 1 week after SE. Ten weeks after SE, nearly 60% of hilar GABAergic cells expressed c-fos. With the exception of calretinin (CR)-positive cells, percentages of hilar neuronal nitric oxide synthase (nNOS)-, neuropeptide Y (NPY)-, parvalbumin (PV)-, and somatostatin (SOM)-positive cells with c-fos expression are significantly higher than those of controls more than 10 weeks after SE. Without the REM to be more depolarizing and changed threshold potential level in SE-induced rats, cell-attached recording revealed that nearly 90% of hilar interneurons fired spontaneously at near the REM while only 22% of the same cell population did so in the controls. In conclusion, pilocarpine-induced SE eventually leads to a state in which surviving dentate GABAergic interneurons become hyperactive with a subtype-dependent manner; this implies that a fragile balance between excitation and inhibition exists in the dentate gyrus and in addition, the activity-dependent up-regulation of IEGs may underlie plastic changes seen in some types of GABAergic cells in the pilocarpine model of epilepsy.

Neuronal synchrony and the transition to spontaneous seizures

The role of inhibitory neuronal activity in the transition to seizure is unclear. On the one hand, seizures are associated with excessive neuronal activity that can spread across the brain, suggesting run-away excitation. On the other hand, recent in vitro studies suggest substantial activity of inhibitory interneurons prior to the onset of evoked seizure-like activity. Yet, little is known about the behavior of interneurons before and during spontaneous seizures in chronic temporal lobe epilepsy. Here, we examined the relationship between the on-going local field potential (LFP) and the activity of populations of hippocampal neurons during the transition to spontaneous seizures in the pilocarpine rat model of epilepsy. Pilocarpine treated rats that exhibited spontaneous seizures were implanted with drivable tetrodes including an LFP electrode and recordings were obtained from the CA3 region. For each recorded seizure, identified single units were classified into putative interneurons or pyramidal cells based on average firing rate, autocorrelation activity and waveform morphology. The onset of sustained ictal spiking, a consistent seizure event that occurred within seconds after the clinically defined seizure onset time, was used to align data from each seizure to a common reference point. Ictal spiking, in this paper, refers to spiking activity in the low-pass filtered LFP during seizures and not the neuronal action potentials. Results show that beginning minutes before the onset of sustained ictal spiking in the local field, subpopulations of putative interneurons displayed a sequence of synchronous behaviors. This includes progressive synchrony with local field oscillations at theta, gamma, and finally ictal spiking frequencies, and an increased firing rate seconds before the onset of ictal spiking. Conversely, putative pyramidal cells did not exhibit increased synchrony or firing rate until after ictal spiking had begun. Our data suggest that the transition to spontaneous seizure in this network is not mediated by increasing excitatory activity, but by distinct changes in the dynamical state of putative interneurons. While these states are not unique for seizure onset, they suggest a series of state transitions that continuously increase the likelihood of a seizure. These data help to interpret the link between in vitro studies demonstrating interneuron activation at the transition to seizure, and human studies demonstrating heterogeneous neuronal firing at this time.

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.

PAF receptor activation in hippocampal plasticity induced by SE

One of the defining characteristics of epilepsy is abberant mossy fiber sprouting (MFS) in the dentate gyrus. These axonal projections within the granule cell layer of the dentate gyrus create a hyperexcitable network and lead to spontaneous seizures. Growth‐associated protein 43 (GAP‐43), a presynaptic marker that is expressed during development and axonal growth, has been preveiously implicated in the abnormal MFS found in epileptic models. The purpose of our study is to investigate the role of platelet activating factor (PAF) receptor‐mediated signaling in abnormal MFS by evaulating GAP‐43 immunoreactivity. PAF is a mediator of inflammation, a potent gene expression inducer and a modulator of long term potentiation (LTP), synaptic plasticity, and memory formation. PAF abnormally accumulates in the brain during seizures, or injuries, contributing to neuroinflammation and neurotoxicity. Using the novel PAF receptor antagonist, LAU‐0901, in the rat pilocarpine model of epilepsy, we found a positive correlation between a decrease in spontenous seizure activity and an increase GAP‐43 immunoreacitivy in our treated group, suggesting that PAF receptor activation is involved in the abberant plasticity associated with epilepsy.Source of support: This work was supported by NIH/NCRR grant P20 RR016816.

Funny Team Member Makes Key Plays, but Leaves the Dendritic Field When Hit Hard

Progressive Dendritic HCN Channelopathy during Epileptogenesis in the Rat Pilocarpine Model of Epilepsy. Jung S, Jones TD, Lugo JN Jr, Sheerin AH, Miller JW, D'Ambrosio R, Anderson AE, Poolos NP. J Neurosci 2007;27(47):13012–13021. Ion channelopathy plays an important role in human epilepsy with a genetic cause and has been hypothesized to occur in epilepsy after acquired insults to the CNS as well. Acquired alterations of ion channel function occur after induction of status epilepticus (SE) in animal models of epilepsy, but it is unclear how they correlate with the onset of spontaneous seizures. We examined the properties of hyperpolarization-activated cation (HCN) channels in CA1 hippocampal pyramidal neurons in conjunction with video-EEG (VEEG) recordings to monitor the development of spontaneous seizures in the rat pilocarpine model of epilepsy. Our results showed that dendritic HCN channels were significantly downregulated at an acute time point 1 week postpilocarpine, with loss of channel expression and hyperpolarization of voltage-dependent activation. This downregulation progressively increased when epilepsy was established in the chronic period. Surprisingly, VEEG recordings during the acute period showed that a substantial fraction of animals were already experiencing recurrent seizures. Suppression of these seizures with phenobarbital reversed the change in the voltage dependence of Ih, the current produced by HCN channels, but did not affect the loss of HCN channel expression. These results suggest two mechanisms of HCN channel downregulation after SE, one dependent on and one independent of recurrent seizures. This early and progressive downregulation of dendritic HCN channel function increases neuronal excitability and may be associated with both the process of epileptogenesis and maintenance of the epileptic state.

Progressive Dendritic HCN Channelopathy during Epileptogenesis in the Rat Pilocarpine Model of Epilepsy

Ion channelopathy plays an important role in human epilepsy with a genetic cause and has been hypothesized to occur in epilepsy after acquired insults to the CNS as well. Acquired alterations of ion channel function occur after induction of status epilepticus (SE) in animal models of epilepsy, but it is unclear how they correlate with the onset of spontaneous seizures. We examined the properties of hyperpolarization-activated cation (HCN) channels in CA1 hippocampal pyramidal neurons in conjunction with video-EEG (VEEG) recordings to monitor the development of spontaneous seizures in the rat pilocarpine model of epilepsy. Our results showed that dendritic HCN channels were significantly downregulated at an acute time point 1 week postpilocarpine, with loss of channel expression and hyperpolarization of voltage-dependent activation. This downregulation progressively increased when epilepsy was established in the chronic period. Surprisingly, VEEG recordings during the acute period showed that a substantial fraction of animals were already experiencing recurrent seizures. Suppression of these seizures with phenobarbital reversed the change in the voltage dependence of I(h), the current produced by HCN channels, but did not affect the loss of HCN channel expression. These results suggest two mechanisms of HCN channel downregulation after SE, one dependent on and one independent of recurrent seizures. This early and progressive downregulation of dendritic HCN channel function increases neuronal excitability and may be associated with both the process of epileptogenesis and maintenance of the epileptic state.

N-methyl- d-aspartic acid-induced and Ca-dependent neuronal swelling and its retardation by brain-derived neurotrophic factor in the epileptic hippocampus

Dentate granule cell (DGC) swelling was studied by imaging changes in light transmittance from hippocampal slices in the rat pilocarpine model of epilepsy and human epileptic specimens. Brief bath-application of N-methyl- d-aspartic acid (NMDA) induced swelling in the control rat DGC (physiological swelling). Physiological swelling was short-lasting, and rapidly recovered upon removal of NMDA. In contrast, the swelling induced in the pilocarpine-treated rat hippocampus and human epileptic hippocampus (epileptic swelling) was long-lasting, and often recovered slowly over an hour. Both types of swelling were blocked by the NMDA receptor (NMDAR) antagonist, D-APV, suggesting that they shared the same induction mechanism. However, the swellings differed in their sensitivity to a calcium chelator, 1.2-bis(2-aminophenoxy)ethane- N, N, N, N-tetra-acetate (BAPTA), and an endoplasmic reticulum (ER) Ca 2+-ATPase inhibitor, thapsigargin (TG). BAPTA and TG affected only epileptic swelling, and physiological swelling was spared. This suggested that the NMDAR-induced epileptic swelling might involve an additional mechanism for its maintenance, likely recruiting ER Ca 2+ stores. Brain-derived neurotrophic factor (BDNF) slightly attenuated physiological swelling, and blocked epileptic swelling. The present study suggests a functional link between the activation of NMDAR and a release of Ca 2+ from internal stores during the induction of epileptic swelling, and a neuroprotective role of BDNF on the NMDAR-induced swelling in the epileptic hippocampus.

The endogenous cannabinoid system regulates seizure frequency and duration in a model of temporal lobe epilepsy.

Several lines of evidence suggest that cannabinoid compounds are anticonvulsant. However, the anticonvulsant potential of cannabinoids and, moreover, the role of the endogenous cannabinoid system in regulating seizure activity has not been tested in an in vivo model of epilepsy that is characterized by spontaneous, recurrent seizures. Here, using the rat pilocarpine model of epilepsy, we show that the marijuana extract Delta9-tetrahydrocannabinol (10 mg/kg) as well as the cannabimimetic, 4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one [R(+)WIN55,212 (5 mg/kg)], completely abolished spontaneous epileptic seizures. Conversely, application of the cannabinoid CB1 receptor (CB1) antagonist, N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A), significantly increased both seizure duration and frequency. In some animals, CB1 receptor antagonism resulted in seizure durations that were protracted to a level consistent with the clinical condition status epilepticus. Furthermore, we determined that during an short-term pilocarpine-induced seizure, levels of the endogenous CB1 ligand 2-arachidonylglycerol increased significantly within the hippocampal brain region. These data indicate not only anticonvulsant activity of exogenously applied cannabinoids but also suggest that endogenous cannabinoid tone modulates seizure termination and duration through activation of the CB1 receptor. Furthermore, Western blot and immunohistochemical analyses revealed that CB1 receptor protein expression was significantly increased throughout the CA regions of epileptic hippocampi. By demonstrating a role for the endogenous cannabinoid system in regulating seizure activity, these studies define a role for the endogenous cannabinoid system in modulating neuroexcitation and suggest that plasticity of the CB1 receptor occurs with epilepsy.

Chronic inhibition of Ca 2+/calmodulin kinase II activity in the pilocarpine model of epilepsy

The development of symptomatic epilepsy is a model of long-term plasticity changes in the central nervous system. The rat pilocarpine model of epilepsy was utilized to study persistent alterations in calcium/calmodulin-dependent kinase II (CaM kinase II) activity associated with epileptogenesis. CaM kinase II-dependent substrate phosphorylation and autophosphorylation were significantly inhibited for up to 6 weeks following epileptogenesis in both the cortex and hippocampus, but not in the cerebellum. The net decrease in CaM kinase II autophosphorylation and substrate phosphorylation was shown to be due to decreased kinase activity and not due to increased phosphatase activity. The inhibition in CaM kinase II activity and the development of epilepsy were blocked by pretreating seizure rats with MK-801 indicating that the long-lasting decrease in CaM kinase II activity was dependent on N-methyl- d-aspartate receptor activation. In addition, the inhibition of CaM kinase II activity was associated in time and regional localization with the development of spontaneous recurrent seizure activity. The decrease in enzyme activity was not attributed to a decrease in the α or β kinase subunit protein expression level. Thus, the significant inhibition of the enzyme occurred without changes in kinase protein expression, suggesting a long-lasting, post-translational modification of the enzyme. This is the first published report of a persistent, post-translational alteration of CaM kinase II activity in a model of epilepsy characterized by spontaneous recurrent seizure activity.